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Sample records for toxin biosynthesis gene

  1. Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5

    Directory of Open Access Journals (Sweden)

    Neilan Brett A

    2009-03-01

    Full Text Available Abstract Background Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. Results We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. Conclusion The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved

  2. Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5.

    Science.gov (United States)

    Mihali, Troco K; Kellmann, Ralf; Neilan, Brett A

    2009-03-30

    Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of

  3. Role of the pathotype-specific ACRTS1 gene encoding a hydroxylase involved in the biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata.

    Science.gov (United States)

    Izumi, Yuriko; Kamei, Eri; Miyamoto, Yoko; Ohtani, Kouhei; Masunaka, Akira; Fukumoto, Takeshi; Gomi, Kenji; Tada, Yasuomi; Ichimura, Kazuya; Peever, Tobin L; Akimitsu, Kazuya

    2012-08-01

    The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of the rootstock species rough lemon (Citrus jambhiri) and Rangpur lime (C. limonia). Genes controlling toxin production were localized to a 1.5-Mb chromosome carrying the ACR-toxin biosynthesis gene cluster (ACRT) in the genome of the rough lemon pathotype. A genomic BAC clone containing a portion of the ACRT cluster was sequenced which allowed identification of three open reading frames present only in the genomes of ACR-toxin producing isolates. We studied the functional role of one of these open reading frames, ACRTS1 encoding a putative hydroxylase, in ACR-toxin production by homologous recombination-mediated gene disruption. There are at least three copies of ACRTS1 gene in the genome and disruption of two copies of this gene significantly reduced ACR-toxin production as well as pathogenicity; however, transcription of ACRTS1 and production of ACR-toxin were not completely eliminated due to remaining functional copies of the gene. RNA-silencing was used to knock down the remaining ACRTS1 transcripts to levels undetectable by reverse transcription-polymerase chain reaction. The silenced transformants did not produce detectable ACR-toxin and were not pathogenic. These results indicate that ACRTS1 is an essential gene in ACR-toxin biosynthesis in the rough lemon pathotype of A. alternata and is required for full virulence of this fungus.

  4. Biosynthesis of cylindrospermopsin and 7-epicylindrospermopsin in Oscillatoria sp. strain PCC 6506: identification of the cyr gene cluster and toxin analysis.

    Science.gov (United States)

    Mazmouz, Rabia; Chapuis-Hugon, Florence; Mann, Stéphane; Pichon, Valérie; Méjean, Annick; Ploux, Olivier

    2010-08-01

    Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 microg/mg and 0.29 microg/mg, respectively) and cylindrospermopsin (0.10 microg/mg and 0.11 microg/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 +/- 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA.

  5. Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview.

    Science.gov (United States)

    Wang, Da-Zhi; Zhang, Shu-Fei; Zhang, Yong; Lin, Lin

    2016-03-01

    Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in

  6. (vitamin B1) biosynthesis genes

    African Journals Online (AJOL)

    In this study, the gene transcripts of first two enzymes in thiamine biosynthesis pathway, THIC and THI1/THI4 were identified and amplified from oil palm tissues. Primers were designed based on sequence comparison of the genes from Arabidopsis thaliana, Zea mays, Oryza sativa and Alnus glutinosa. Oil palm's responses ...

  7. PR Toxin - Biosynthesis, Genetic Regulation, Toxicological Potential, Prevention and Control Measures: Overview and Challenges.

    Science.gov (United States)

    Dubey, Manish K; Aamir, Mohd; Kaushik, Manish S; Khare, Saumya; Meena, Mukesh; Singh, Surendra; Upadhyay, Ram S

    2018-01-01

    Out of the various mycotoxigenic food and feed contaminant, the fungal species belonging to Penicillium genera, particularly Penicillium roqueforti is of great economic importance, and well known for its crucial role in the manufacturing of Roquefort and Gorgonzola cheese. The mycotoxicosis effect of this mold is due to secretion of several metabolites, of which PR toxin is of considerable importance, with regard to food quality and safety challenges issues. The food products and silages enriched with PR toxin could lead into damage to vital internal organs, gastrointestinal perturbations, carcinogenicity, immunotoxicity, necrosis, and enzyme inhibition. Moreover, it also has the significant mutagenic potential to disrupt/alter the crucial processes like DNA replication, transcription, and translation at the molecular level. The high genetic diversities in between the various strains of P. roqueforti persuaded their nominations with Protected Geographical Indication (PGI), accordingly to the cheese type, they have been employed. Recently, the biosynthetic mechanism and toxicogenetic studies unraveled the role of ari1 and prx gene clusters that cross-talk with the synthesis of other metabolites or involve other cross-regulatory pathways to negatively regulate/inhibit the other biosynthetic route targeted for production of a strain-specific metabolites. Interestingly, the chemical conversion that imparts toxic properties to PR toxin is the substitution/oxidation of functional hydroxyl group (-OH) to aldehyde group (-CHO). The rapid conversion of PR toxin to the other derivatives such as PR imine, PR amide, and PR acid, based on conditions available reflects their unstability and degradative aspects. Since the PR toxin-induced toxicity could not be eliminated safely, the assessment of dose-response and other pharmacological aspects for its safe consumption is indispensable. The present review describes the natural occurrences, diversity, biosynthesis, genetics

  8. PR ToxinBiosynthesis, Genetic Regulation, Toxicological Potential, Prevention and Control Measures: Overview and Challenges

    Science.gov (United States)

    Dubey, Manish K.; Aamir, Mohd; Kaushik, Manish S.; Khare, Saumya; Meena, Mukesh; Singh, Surendra; Upadhyay, Ram S.

    2018-01-01

    Out of the various mycotoxigenic food and feed contaminant, the fungal species belonging to Penicillium genera, particularly Penicillium roqueforti is of great economic importance, and well known for its crucial role in the manufacturing of Roquefort and Gorgonzola cheese. The mycotoxicosis effect of this mold is due to secretion of several metabolites, of which PR toxin is of considerable importance, with regard to food quality and safety challenges issues. The food products and silages enriched with PR toxin could lead into damage to vital internal organs, gastrointestinal perturbations, carcinogenicity, immunotoxicity, necrosis, and enzyme inhibition. Moreover, it also has the significant mutagenic potential to disrupt/alter the crucial processes like DNA replication, transcription, and translation at the molecular level. The high genetic diversities in between the various strains of P. roqueforti persuaded their nominations with Protected Geographical Indication (PGI), accordingly to the cheese type, they have been employed. Recently, the biosynthetic mechanism and toxicogenetic studies unraveled the role of ari1 and prx gene clusters that cross-talk with the synthesis of other metabolites or involve other cross-regulatory pathways to negatively regulate/inhibit the other biosynthetic route targeted for production of a strain-specific metabolites. Interestingly, the chemical conversion that imparts toxic properties to PR toxin is the substitution/oxidation of functional hydroxyl group (-OH) to aldehyde group (-CHO). The rapid conversion of PR toxin to the other derivatives such as PR imine, PR amide, and PR acid, based on conditions available reflects their unstability and degradative aspects. Since the PR toxin-induced toxicity could not be eliminated safely, the assessment of dose-response and other pharmacological aspects for its safe consumption is indispensable. The present review describes the natural occurrences, diversity, biosynthesis, genetics

  9. Two enzymes involved in biosynthesis of the host-selective phytotoxin HC-toxin

    International Nuclear Information System (INIS)

    Walton, J.D.

    1987-01-01

    Cochliobolus carbonum race 1 produces a cyclic tetrapeptide HC-toxin, which is necessary for its exceptional virulence on certain varieties of maize. Previous genetic analysis of HC-toxin production by the fungus has indicated that a single genetic locus controls HC-toxin production. Enzymes involved in the biosynthesis of HC-toxin have been sought by following the precedents established for the biosynthetic enzymes of cyclic peptide antibiotics. Two enzymatic activities from C. carbonum race 1 were found, a D-alanine- and an L-proline-dependent ATP/PP/sub i/ exchange, which by biochemical and genetic criteria were shown to be involved in the biosynthesis of HC-toxin. These two activities were present in all tested race 1 isolates of C. carbonum, which produce HC-toxin, and in none of the tested race 2 and race 3 isolates, which do not produce the toxin. In a genetic cross between two isolates of C. carbonum differing at the tox locus, all tox + progeny had both activities, and all tox - progeny lacked both activities

  10. An Acidic pH is a determinant factor for TRI genes expression and trichothecenes B biosynthesis in Fusarium graminearum

    OpenAIRE

    2010-01-01

    Abstract Reducing production of trichothecene B by Fusarium graminearum on cereals is necessary to avoid contamination leading to yields reduction and having harmful impacts on human and animal health. Understanding how trichothecenes biosynthesis is induced is essential. Effect of ambient pH on fungal growth, toxin biosynthesis and TRI genes expression was studied during in vitro liquid culture of F. graminearum on minimal medium. Fungal development stopped at day 3 after a sharp ...

  11. Gene therapy for carcinoma of the breast: Genetic toxins

    International Nuclear Information System (INIS)

    Vassaux, Georges; Lemoine, Nick R

    2000-01-01

    Gene therapy was initially envisaged as a potential treatment for genetically inherited, monogenic disorders. The applications of gene therapy have now become wider, however, and include cardiovascular diseases, vaccination and cancers in which conventional therapies have failed. With regard to oncology, various gene therapy approaches have been developed. Among them, the use of genetic toxins to kill cancer cells selectively is emerging. Two different types of genetic toxins have been developed so far: the metabolic toxins and the dominant-negative class of toxins. This review describes these two different approaches, and discusses their potential applications in cancer gene therapy

  12. A Biotin Biosynthesis Gene Restricted to Helicobacter

    Science.gov (United States)

    Bi, Hongkai; Zhu, Lei; Jia, Jia; Cronan, John E.

    2016-01-01

    In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections. PMID:26868423

  13. Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

    DEFF Research Database (Denmark)

    Schembri, Mark; Neilan, B.A.; Saint, C.P.

    2001-01-01

    , Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide...... synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYPO2OB were identified by PCR using degenerate primers based on conserved regions within each gene. Examination...... bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii....

  14. Expression profiles of genes involved in tanshinone biosynthesis of ...

    Indian Academy of Sciences (India)

    Expression profiles of genes involved in tanshinone biosynthesis of two. Salvia miltiorrhiza genotypes with different tanshinone contents. Zhenqiao Song, Jianhua Wang and Xingfeng Li. J. Genet. 95, 433–439. Table 1. S. miltiorrhiza genes and primer pairs used for qRT-PCR. Gene. GenBank accession. Primer name.

  15. Genes Involved in the Biosynthesis and Transport of Acinetobactin in

    Directory of Open Access Journals (Sweden)

    Tarik Hasan

    2015-03-01

    Full Text Available Pathogenic bacteria survive in iron-limited host environments by using several iron acquisition mechanisms. Acinetobacter baumannii, causing serious infections in compromised patients, produces an iron-chelating molecule, called acinetobactin, which is composed of equimolar quantities of 2,3-dihydroxybenzoic acid (DHBA, L-threonine, and N-hydroxyhistamine, to compete with host cells for iron. Genes that are involved in the production and transport of acinetobactin are clustered within the genome of A. baumannii. A recent study showed that entA, located outside of the acinetobactin gene cluster, plays important roles in the biosynthesis of the acinetobactin precursor DHBA and in bacterial pathogenesis. Therefore, understanding the genes that are associated with the biosynthesis and transport of acinetobactin in the bacterial genome is required. This review is intended to provide a general overview of the genes in the genome of A. baumannii that are required for acinetobactin biosynthesis and transport.

  16. Anthocyanin biosynthesis in fruit tree crops: Genes and their regulation

    African Journals Online (AJOL)

    The anthocyanin biosynthesis pathway is a little complex with branches responsible for the synthesis of a variety of metabolites. In fruit tree crops, during the past decade, many structural genes encoding enzymes in the anthocyanin biosynthetic pathway and various regulatory genes encoding transcription factors that ...

  17. Temporal expression of genes involved in the biosynthesis of ...

    African Journals Online (AJOL)

    Gibberellins (GAs) are a large family of endogenous plant growth regulators. Bioactive GAs influence nearly all processes during plant growth and development. In the present study, we cloned and identified 10 unique genes that are potentially involved in the biosynthesis of GAs, including one BpGGDP gene, two BpCPS ...

  18. Toxins

    Science.gov (United States)

    Toxins are substances created by plants and animals that are poisonous to humans. Toxins also include some medicines that are helpful in small doses, but poisonous in large amounts. Most toxins that cause problems ...

  19. Alternaria Toxins: Potential Virulence Factors and Genes Related to Pathogenesis

    Directory of Open Access Journals (Sweden)

    Mukesh Meena

    2017-08-01

    Full Text Available Alternaria is an important fungus to study due to their different life style from saprophytes to endophytes and a very successful fungal pathogen that causes diseases to a number of economically important crops. Alternaria species have been well-characterized for the production of different host-specific toxins (HSTs and non-host specific toxins (nHSTs which depend upon their physiological and morphological stages. The pathogenicity of Alternaria species depends on host susceptibility or resistance as well as quantitative production of HSTs and nHSTs. These toxins are chemically low molecular weight secondary metabolites (SMs. The effects of toxins are mainly on different parts of cells like mitochondria, chloroplast, plasma membrane, Golgi complex, nucleus, etc. Alternaria species produce several nHSTs such as brefeldin A, tenuazonic acid, tentoxin, and zinniol. HSTs that act in very low concentrations affect only certain plant varieties or genotype and play a role in determining the host range of specificity of plant pathogens. The commonly known HSTs are AAL-, AK-, AM-, AF-, ACR-, and ACT-toxins which are named by their host specificity and these toxins are classified into different family groups. The HSTs are differentiated on the basis of bio-statistical and other molecular analyses. All these toxins have different mode of action, biochemical reactions and signaling mechanisms to cause diseases. Different species of Alternaria produced toxins which reveal its biochemical and genetic effects on itself as well as on its host cells tissues. The genes responsible for the production of HSTs are found on the conditionally dispensable chromosomes (CDCs which have been well characterized. Different bio-statistical methods like basic local alignment search tool (BLAST data analysis used for the annotation of gene prediction, pathogenicity-related genes may provide surprising knowledge in present and future.

  20. Alternaria Toxins: Potential Virulence Factors and Genes Related to Pathogenesis

    Science.gov (United States)

    Meena, Mukesh; Gupta, Sanjay K.; Swapnil, Prashant; Zehra, Andleeb; Dubey, Manish K.; Upadhyay, Ram S.

    2017-01-01

    Alternaria is an important fungus to study due to their different life style from saprophytes to endophytes and a very successful fungal pathogen that causes diseases to a number of economically important crops. Alternaria species have been well-characterized for the production of different host-specific toxins (HSTs) and non-host specific toxins (nHSTs) which depend upon their physiological and morphological stages. The pathogenicity of Alternaria species depends on host susceptibility or resistance as well as quantitative production of HSTs and nHSTs. These toxins are chemically low molecular weight secondary metabolites (SMs). The effects of toxins are mainly on different parts of cells like mitochondria, chloroplast, plasma membrane, Golgi complex, nucleus, etc. Alternaria species produce several nHSTs such as brefeldin A, tenuazonic acid, tentoxin, and zinniol. HSTs that act in very low concentrations affect only certain plant varieties or genotype and play a role in determining the host range of specificity of plant pathogens. The commonly known HSTs are AAL-, AK-, AM-, AF-, ACR-, and ACT-toxins which are named by their host specificity and these toxins are classified into different family groups. The HSTs are differentiated on the basis of bio-statistical and other molecular analyses. All these toxins have different mode of action, biochemical reactions and signaling mechanisms to cause diseases. Different species of Alternaria produced toxins which reveal its biochemical and genetic effects on itself as well as on its host cells tissues. The genes responsible for the production of HSTs are found on the conditionally dispensable chromosomes (CDCs) which have been well characterized. Different bio-statistical methods like basic local alignment search tool (BLAST) data analysis used for the annotation of gene prediction, pathogenicity-related genes may provide surprising knowledge in present and future. PMID:28848500

  1. SxtA and sxtG Gene Expression and Toxin Production in the Mediterranean Alexandrium minutum (Dinophyceae

    Directory of Open Access Journals (Sweden)

    Federico Perini

    2014-10-01

    Full Text Available The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP. The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1 and Gonyautoxin-4 (GTX4. The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.

  2. An Emulsion Based Microarray Method to Detect the Toxin Genes of Toxin-Producing Organisms

    Directory of Open Access Journals (Sweden)

    Yunfei Bai

    2011-08-01

    Full Text Available Toxins produced by bacteria and fungi are one of the most important factors which may cause food contamination. The study of detection methods with high sensitivity and throughput is significant for the protection of food safety. In the present study, we coupled microarray with emulsion PCR and developed a high throughput detection method. Thirteen different gene sites which encode the common toxins of several bacteria and fungi were assayed in parallel in positive and maize samples. Conventional PCR assays were carried out for comparison. The results showed that the developed microarray method had high specificity and sensitivity. Two zearalenone-related genes were investigated in one of the ten maize samples obtained with this present method. The results indicated that the emulsion based microarray detection method was developed successfully and suggested its potential application in multiple gene site detection.

  3. Temporal expression of genes involved in the biosynthesis of ...

    African Journals Online (AJOL)

    Jane

    2011-10-10

    Oct 10, 2011 ... Gibberellins (GAs) are a large family of endogenous plant growth regulators. Bioactive GAs influence nearly all processes during plant growth and development. In the present study, we cloned and identified 10 unique genes that are potentially involved in the biosynthesis of GAs, including one. BpGGDP ...

  4. Plankton composition, biomass, phylogeny and toxin genes in Lake ...

    African Journals Online (AJOL)

    Plankton composition, biomass, phylogeny and toxin genes in Lake Big Momela, Tanzania. ... cyanobacteria during the whole year. In general, our data illustrate the presence of rich planktonic communities, including some unique and potentially endemic cyanobacteria. Keywords: cyanotoxin, limnology, plankton diversity, ...

  5. Shiga Toxin (Stx) Gene Detection and Verotoxigenic Potentials of ...

    African Journals Online (AJOL)

    Non-0157 Escherichia coli, isolated from Nono (fermented fresh cow milk) sampled from four major Nigerian cities, namely, Abuja, Benin City, Lagos and Onitsha were investigated for the presence shiga toxins (stx1 and stx2) genes using PCR technique and for their verotoxigenic potentials using tissue culture assay on ...

  6. Identification of two aflatrem biosynthesis gene loci in Aspergillus flavus and metabolic engineering of Penicillium paxilli to elucidate their function.

    Science.gov (United States)

    Nicholson, Matthew J; Koulman, Albert; Monahan, Brendon J; Pritchard, Beth L; Payne, Gary A; Scott, Barry

    2009-12-01

    Aflatrem is a potent tremorgenic toxin produced by the soil fungus Aspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search of Aspergillus complete genome sequence data identified putative aflatrem gene clusters in the genomes of A. flavus and Aspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first, ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes, atmG, atmC, and atmM, and the second, ATM2, is telomere distal on chromosome 7 and contains five genes, atmD, atmQ, atmB, atmA, and atmP. Reverse transcriptase PCR in A. flavus demonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer of atmP and atmQ into Penicillium paxilli paxP and paxQ deletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis in A. flavus based on these reconstitution experiments in P. paxilli and identification of putative intermediates in wild-type cultures of A. flavus.

  7. Arrangement of the Clostridium baratii F7 toxin gene cluster with identification of a σ factor that recognizes the botulinum toxin gene cluster promoters.

    Science.gov (United States)

    Dover, Nir; Barash, Jason R; Burke, Julianne N; Hill, Karen K; Detter, John C; Arnon, Stephen S

    2014-01-01

    Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.

  8. Bacterial Toxins for Oncoleaking Suicidal Cancer Gene Therapy.

    Science.gov (United States)

    Pahle, Jessica; Walther, Wolfgang

    For suicide gene therapy, initially prodrug-converting enzymes (gene-directed enzyme-producing therapy, GDEPT) were employed to intracellularly metabolize non-toxic prodrugs into toxic compounds, leading to the effective suicidal killing of the transfected tumor cells. In this regard, the suicide gene therapy has demonstrated its potential for efficient tumor eradication. Numerous suicide genes of viral or bacterial origin were isolated, characterized, and extensively tested in vitro and in vivo, demonstrating their therapeutic potential even in clinical trials to treat cancers of different entities. Apart from this, growing efforts are made to generate more targeted and more effective suicide gene systems for cancer gene therapy. In this regard, bacterial toxins are an alternative to the classical GDEPT strategy, which add to the broad spectrum of different suicide approaches. In this context, lytic bacterial toxins, such as streptolysin O (SLO) or the claudin-targeted Clostridium perfringens enterotoxin (CPE) represent attractive new types of suicide oncoleaking genes. They permit as pore-forming proteins rapid and also selective toxicity toward a broad range of cancers. In this chapter, we describe the generation and use of SLO as well as of CPE-based gene therapies for the effective tumor cell eradication as promising, novel suicide gene approach particularly for treatment of therapy refractory tumors.

  9. Capsular exopolysaccharide biosynthesis gene of Propionibacterium freudenreichii subsp. shermanii.

    Science.gov (United States)

    Deutsch, Stéphanie-Marie; Falentin, Hélène; Dols-Lafargue, Marguerite; Lapointe, Gisèle; Roy, Denis

    2008-07-31

    In the dairy industry, exopolysaccharides (EPS) contribute to improving the texture and viscosity of cheese and yoghurt and also receive increasing attention because of their beneficial properties for health. For lactic acid bacteria, the production of EPS is well studied. However, for dairy propionibacteria the biosynthesis of EPS is poorly documented. A polysaccharide synthase-encoding gene was identified in the genome of Propionibacterium freudenreichii subsp. shermanii TL 34 (CIP 103027). This gene best aligns with Tts, the polysaccharide synthase gene of Streptococcus pneumoniae type 37 that is responsible for the production of a beta-glucan capsular polysaccharide. PCR amplification showed the presence of an internal fragment of this gene in twelve strains of P. freudenreichii subsp. shermanii with a ropy phenotype in YEL+ medium. The gene sequence is highly conserved, as less than 1% of nucleotides differed among the 10 strains containing the complete gtf gene. The same primers failed to detect the gene in Propionibacterium acidipropionici strain TL 47, which is known to excrete exopolysaccharides in milk. The presence of (1-->3, 1-->2)-beta-d-glucan capsule was demonstrated for 7 out of 12 strains by agglutination with a S. pneumoniae-type 37-specific antiserum. The presence of mRNA corresponding to the gene was detected by RT-PCR in three strains at both exponential and stationary growth phases. This work represents the first identification of a polysaccharide synthase gene of P. freudenreichii, and further studies will be undertaken to elucidate the role of capsular EPS.

  10. Thioridazine affects transcription of genes involved in cell wall biosynthesis in methicillin-resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Bonde, Mette; Højland, Dorte Heidi; Kolmos, Hans Jørn

    2011-01-01

    have previously shown that the expression of some resistance genes is abolished after treatment with thioridazine and oxacillin. To further understand the mechanism underlying the reversal of resistance, we tested the expression of genes involved in antibiotic resistance and cell wall biosynthesis...... reversal of resistance by thioridazine relies on decreased expression of specific genes involved in cell wall biosynthesis....

  11. Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi

    Directory of Open Access Journals (Sweden)

    Daniel Cook

    2017-06-01

    Full Text Available Swainsonine—a cytotoxic fungal alkaloid and a potential cancer therapy drug—is produced by the insect pathogen and plant symbiont Metarhizium robertsii, the clover pathogen Slafractonia leguminicola, locoweed symbionts belonging to Alternaria sect. Undifilum, and a recently discovered morning glory symbiont belonging to order Chaetothyriales. Genome sequence analyses revealed that these fungi share orthologous gene clusters, designated “SWN,” which included a multifunctional swnK gene comprising predicted adenylylation and acyltransferase domains with their associated thiolation domains, a β-ketoacyl synthase domain, and two reductase domains. The role of swnK was demonstrated by inactivating it in M. robertsii through homologous gene replacement to give a ∆swnK mutant that produced no detectable swainsonine, then complementing the mutant with the wild-type gene to restore swainsonine biosynthesis. Other SWN cluster genes were predicted to encode two putative hydroxylases and two reductases, as expected to complete biosynthesis of swainsonine from the predicted SwnK product. SWN gene clusters were identified in six out of seven sequenced genomes of Metarhzium species, and in all 15 sequenced genomes of Arthrodermataceae, a family of fungi that cause athlete’s foot and ringworm diseases in humans and other mammals. Representative isolates of all of these species were cultured, and all Metarhizium spp. with SWN clusters, as well as all but one of the Arthrodermataceae, produced swainsonine. These results suggest a new biosynthetic hypothesis for this alkaloid, extending the known taxonomic breadth of swainsonine producers to at least four orders of Ascomycota, and suggest that swainsonine has roles in mutualistic symbioses and diseases of plants and animals.

  12. Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates.

    Science.gov (United States)

    Murray, Shauna A; Diwan, Rutuja; Orr, Russell J S; Kohli, Gurjeet S; John, Uwe

    2015-11-01

    A group of marine dinoflagellates (Alveolata, Eukaryota), consisting of ∼10 species of the genus Alexandrium, Gymnodinium catenatum and Pyrodinium bahamense, produce the toxin saxitoxin and its analogues (STX), which can accumulate in shellfish, leading to ecosystem and human health impacts. The genes, sxt, putatively involved in STX biosynthesis, have recently been identified, however, the evolution of these genes within dinoflagellates is not clear. There are two reasons for this: uncertainty over the phylogeny of dinoflagellates; and that the sxt genes of many species of Alexandrium and other dinoflagellate genera are not known. Here, we determined the phylogeny of STX-producing and other dinoflagellates based on a concatenated eight-gene alignment. We determined the presence, diversity and phylogeny of sxtA, domains A1 and A4 and sxtG in 52 strains of Alexandrium, and a further 43 species of dinoflagellates and thirteen other alveolates. We confirmed the presence and high sequence conservation of sxtA, domain A4, in 40 strains (35 Alexandrium, 1 Pyrodinium, 4 Gymnodinium) of 8 species of STX-producing dinoflagellates, and absence from non-producing species. We found three paralogs of sxtA, domain A1, and a widespread distribution of sxtA1 in non-STX producing dinoflagellates, indicating duplication events in the evolution of this gene. One paralog, clade 2, of sxtA1 may be particularly related to STX biosynthesis. Similarly, sxtG appears to be generally restricted to STX-producing species, while three amidinotransferase gene paralogs were found in dinoflagellates. We investigated the role of positive (diversifying) selection following duplication in sxtA1 and sxtG, and found negative selection in clades of sxtG and sxtA1, clade 2, suggesting they were functionally constrained. Significant episodic diversifying selection was found in some strains in clade 3 of sxtA1, a clade that may not be involved in STX biosynthesis, indicating pressure for diversification

  13. A Cell-Based Approach for the Biosynthesis/Screening of Cyclic Peptide Libraries against Bacterial Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R; Woo, Y; Cantor, J; Steenblock, E

    2007-10-24

    Available methods for developing and screening small drug-like molecules able to knockout toxins or pathogenic microorganisms have some limitations. In order to be useful, these new methods must provide high-throughput analysis and identify specific binders in a short period of time. To meet this need, we are developing an approach that uses living cells to generate libraries of small biomolecules, which are then screened inside the cell for activity. Our group is using this new, combined approach to find highly specific ligands capable of disabling anthrax Lethal Factor (LF) as proof of principle. Key to our approach is the development of a method for the biosynthesis of libraries of cyclic peptides, and an efficient screening process that can be carried out inside the cell.

  14. Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

    Energy Technology Data Exchange (ETDEWEB)

    Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R., E-mail: wolfgang.hess@biologie.uni-freiburg.de [Genetics and Experimental Bioinformatics, Institute of Biology 3, Faculty of Biology, University of Freiburg, Freiburg (Germany)

    2014-07-14

    In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria.

  15. Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

    International Nuclear Information System (INIS)

    Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R.

    2014-01-01

    In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria.

  16. Alkane biosynthesis genes in cyanobacteria and their transcriptional organization

    Directory of Open Access Journals (Sweden)

    Stephan eKlähn

    2014-07-01

    Full Text Available In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl-acyl carrier protein reductase (AAR and aldehyde deformylating oxygenase (ADO. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado and sll0209 (aar, that give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313 and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in

  17. Comparison of GeneXpert PCR to BD GeneOhm for detecting C. difficile toxin gene in GDH positive toxin negative samples.

    Science.gov (United States)

    Agaronov, Maksim; Karak, Soma G; Maldonado, Yanice; Tetreault, Janice; Aslanzadeh, Jaber

    2012-01-01

    The need for rapid diagnosis of Clostridium difficile (C. difficile) associated infection in a clinical microbiology laboratory has provided the stimulus for new diagnostic tests and testing protocols. A two-test algorithm has been proposed using assays such as Quik Chek Complete, which detects both C. difficile glutamate dehydrogenase (GDH) and C. difficile toxins A and B, followed by reflex testing of samples having inconclusive results (GDH positive and toxin negative) with PCR for identification of toxin gene specific DNA. The goal of this study was to compare the outcome and efficiency of PCR assays, BD GeneOhm and GeneXpert, for detecting C. difficile toxin B gene in samples that have tested indeterminate for C. difficile by the Quik Chek Complete test. Over a three-month period, a total of 41 cases tested indeterminate by Quik Chek Complete test and were retested by the aforementioned PCR assays. Out of the 41 samples, 36 had matching results in both assays; 19 negative samples and 17 positive samples. In terms of efficiency, GeneXpert was user-friendly and had a turnaround time (TAT) of 45 minutes with two-minute specimen processing compared to BD GeneOhm which had a TAT of 75 to 90 minutes.

  18. Prediction of Toxin Genes from Chinese Yellow Catfish Based on Transcriptomic and Proteomic Sequencing

    Directory of Open Access Journals (Sweden)

    Bing Xie

    2016-04-01

    Full Text Available Fish venom remains a virtually untapped resource. There are so few fish toxin sequences for reference, which increases the difficulty to study toxins from venomous fish and to develop efficient and fast methods to dig out toxin genes or proteins. Here, we utilized Chinese yellow catfish (Pelteobagrus fulvidraco as our research object, since it is a representative species in Siluriformes with its venom glands embedded in the pectoral and dorsal fins. In this study, we set up an in-house toxin database and a novel toxin-discovering protocol to dig out precise toxin genes by combination of transcriptomic and proteomic sequencing. Finally, we obtained 15 putative toxin proteins distributed in five groups, namely Veficolin, Ink toxin, Adamalysin, Za2G and CRISP toxin. It seems that we have developed a novel bioinformatics method, through which we could identify toxin proteins with high confidence. Meanwhile, these toxins can also be useful for comparative studies in other fish and development of potential drugs.

  19. Prediction of Toxin Genes from Chinese Yellow Catfish Based on Transcriptomic and Proteomic Sequencing

    Science.gov (United States)

    Xie, Bing; Li, Xiaofeng; Lin, Zhilong; Ruan, Zhiqiang; Wang, Min; Liu, Jie; Tong, Ting; Li, Jia; Huang, Yu; Wen, Bo; Sun, Ying; Shi, Qiong

    2016-01-01

    Fish venom remains a virtually untapped resource. There are so few fish toxin sequences for reference, which increases the difficulty to study toxins from venomous fish and to develop efficient and fast methods to dig out toxin genes or proteins. Here, we utilized Chinese yellow catfish (Pelteobagrus fulvidraco) as our research object, since it is a representative species in Siluriformes with its venom glands embedded in the pectoral and dorsal fins. In this study, we set up an in-house toxin database and a novel toxin-discovering protocol to dig out precise toxin genes by combination of transcriptomic and proteomic sequencing. Finally, we obtained 15 putative toxin proteins distributed in five groups, namely Veficolin, Ink toxin, Adamalysin, Za2G and CRISP toxin. It seems that we have developed a novel bioinformatics method, through which we could identify toxin proteins with high confidence. Meanwhile, these toxins can also be useful for comparative studies in other fish and development of potential drugs. PMID:27089325

  20. Biosynthesis of flavonoids in bilberry and blueberry - possibilities of the gene level information for the future

    OpenAIRE

    Jaakola, Laura

    2007-01-01

    We have studied the biosynthesis of flavonoids in various tissues of naturally growing European blueberry (bilberry) and the blueberry cultivar 'Northblue'. Focus has also been on the biosynthesis of flavonoids in developing bilberry fruits as well as on the control genes regulating fruit development.

  1. Identification of aminotransferase genes for biosynthesis of aminoglycoside antibiotics from soil DNA.

    Science.gov (United States)

    Nagaya, Atsushi; Takeyama, Satoko; Tamegai, Hideyuki

    2005-07-01

    Aminoglycoside has been known as a clinically important antibiotic for a long time, but genetic information for the biosynthesis of aminoglycoside is still insufficient. In this study, we tried to clone aminoglycoside-biosynthetic genes from soil DNA for accumulation of genetic information. We chose the genes encoding L-glutamine:(2-deoxy-)scyllo-inosose aminotransferase as the target, because it is specific for all types of aminoglycoside biosynthesis. By degenerate PCR, we obtained 33 individual clones that were homologous with aminotransferase genes in aminoglycoside biosynthesis. Phylogenetic analysis and alignment of these genes showed that horizontal gene transfer has occurred in the soil. Among these, several quite interesting genes were obtained. Some genes probably originated from non-actinomycetes, and some were far from the known homologs. These genes can be useful markers for the isolation of entire gene clusters and originating organisms.

  2. Cytolethal Distending Toxin Genes in Campylobacter jejuni and Campylobacter coli Isolates: Detection and Analysis by PCR

    OpenAIRE

    Eyigor, Aysegul; Dawson, Karl A.; Langlois, Bruce E.; Pickett, Carol L.

    1999-01-01

    Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). Knowledge of the prevalence and homogeneity of Campylobacter sp. cdt genes is incomplete. In this work, we identified four PCR primer pairs that collectively amplified cdt genes in all of the C. jejuni and Campylobacter coli strains tested. Restriction analyses of the cdt PCR products showed clear differences between the cdt genes of these two species, yet there were few heterogeneities noted between members of th...

  3. A regulatory gene (ECO-orf4) required for ECO-0501 biosynthesis in Amycolatopsis orientalis.

    Science.gov (United States)

    Shen, Yang; Huang, He; Zhu, Li; Luo, Minyu; Chen, Daijie

    2014-02-01

    ECO-0501 is a novel linear polyene antibiotic, which was discovered from Amycolatopsis orientalis. Recent study of ECO-0501 biosynthesis pathway revealed the presence of regulatory gene: ECO-orf4. The A. orientalis ECO-orf4 gene from the ECO-0501 biosynthesis cluster was analyzed, and its deduced protein (ECO-orf4) was found to have amino acid sequence homology with large ATP-binding regulators of the LuxR (LAL) family regulators. Database comparison revealed two hypothetical domains, a LuxR-type helix-turn-helix (HTH) DNA binding motif near the C-terminal and an N-terminal nucleotide triphosphate (NTP) binding motif included. Deletion of the corresponding gene (ECO-orf4) resulted in complete loss of ECO-0501 production. Complementation by one copy of intact ECO-orf4 restored the polyene biosynthesis demonstrating that ECO-orf4 is required for ECO-0501 biosynthesis. The results of overexpression ECO-orf4 on ECO-0501 production indicated that it is a positive regulatory gene. Gene expression analysis by reverse transcription PCR of the ECO-0501 gene cluster showed that the transcription of ECO-orf4 correlates with that of genes involved in polyketide biosynthesis. These results demonstrated that ECO-orf4 is a pathway-specific positive regulatory gene that is essential for ECO-0501 biosynthesis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction

    Directory of Open Access Journals (Sweden)

    Antonia Y. Tetteh

    2014-01-01

    Full Text Available Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se0, but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3 treatment and then used quantitative real-time PCR (qRT-PCR to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

  5. [Characterization of a Cl. Perfringens type D strain, isolated in the field and optimization of epsilon toxin biosynthesis in a cell culture].

    Science.gov (United States)

    Maaroufi, A; Metoui, W; Rahmouni, S; Ghram, A

    2000-01-01

    A field strain of cl. perfringens, named Dt001, was isolated from kidney of ovine enterotoemia case. The isolate characterized as Cl. perfringens, type D was based on its cultural and biochemical characters and its factors of virulence. The strain was very toxinogenic and well adapted to culture conditions of biofermentation when the parameters related to ptt, incubation time, substrat ... were optimized. Thus, the use of carbon source as polymer (destrine), the continuous control of pH allowed improvement of the rate of biosynthesis of Epsilon toxine by 10 times. The study of the immunogenicity of the isolate showed that preparations of anacultures were more immunogenic then those of anatoxine type. The fact that the two forms of epsilon antigens (protoxin and active toxin) show similar immune response in rabbits, indicates that the proteolytic action of trypsin is limited only to the toxic sites and does not affect the immunogenic epsitopes of the toxin. It also suggests a molecular organization of epsilon toxin in which the immunogenic epsitopes and the toxin sites are apart. The biotechnological performances and the immunogenicity and toxinogenical of the Dt001 isolate are in favor of its possible use as a component of an inactivated vaccine against enterotoxenia.

  6. Dysregulated gliotoxin biosynthesis attenuates the production of unrelated biosynthetic gene cluster-encoded metabolites in Aspergillus fumigatus.

    Science.gov (United States)

    Doyle, Sean; Jones, Gary W; Dolan, Stephen K

    2018-04-01

    Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus. The gliotoxin bis-thiomethyltransferase, GtmA, attenuates gliotoxin biosynthesis in A. fumigatus by conversion of dithiol gliotoxin to bis-thiomethylgliotoxin (BmGT). Here we show that disruption of dithiol gliotoxin bis-thiomethylation functionality in A. fumigatus results in significant remodelling of the A. fumigatus secondary metabolome upon extended culture. RP-HPLC and LC-MS/MS analysis revealed the reduced production of a plethora of unrelated biosynthetic gene cluster-encoded metabolites, including pseurotin A, fumagillin, fumitremorgin C and tryprostatin B, occurs in A. fumigatus ΔgtmA upon extended incubation. Parallel quantitative proteomic analysis of A. fumigatus wild-type and ΔgtmA during extended culture revealed cognate abundance alteration of proteins encoded by relevant biosynthetic gene clusters, allied to multiple alterations in hypoxia-related proteins. The data presented herein reveal a previously concealed functionality of GtmA in facilitating the biosynthesis of other BGC-encoded metabolites produced by A. fumigatus. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  7. Detection of Shiga toxins genes by Multiplex PCR in clinical samples

    Directory of Open Access Journals (Sweden)

    2013-09-01

    Full Text Available Background: Different methods have been used for detection of shiga toxins; such as,  cell culture, ELISA, and RFPLA. However, all of these methods suffer from high cost, time-consumption and relatively low sensitivity. In this study we used Multiplex PCR method for detection of genes encoding shiga toxins. Material and Methods: In this study, 63 clinical samples were obtained from positive cultures of Shigella and E. coli O157, from Bahman 1391 until Ordibehesht 1392 in Mazandaran province. Initial confirmation of shiga toxins producing bacteria was performed by biochemical and serological methods. After DNA extraction, detection of stx1 and stx2 genes was accomplished by multiplex PCR.  For confirmation of the PCR amplicon, DNA sequencing was used. Antibiotic sensitivity tests were performed by disk diffusion method. Results:  Among the positive strains, 13 strains contained stx2 genes, 4 strains contained Stx/Stx1 genes and 4 strains harbored both Stx/Stx1 and Stx2. The DNA extracted from other Gram-negative bacteria was not protected by the relevant parts of these toxins. Sequencing of the amplified fragments indicated the correct toxin sequences.  The sensitivity for identification of Stx/Stx1 gene was 1.56 pg/ µl and for Stx2 was 1.08 pg/µl. The toxin positive strains were all sensitive to Cefixime, Gentamicin, Amikacin, Ceftriaxone, and Nitrofurantoin. Conclusion: This method is fast and accurate for detection of bacteria producing shiga toxin and can be used to identify different types of shiga toxin.

  8. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

    Directory of Open Access Journals (Sweden)

    Yuepeng eHan

    2015-04-01

    Full Text Available Proanthocyanidins (PAs are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR and anthocyanidin reductase (ANR was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

  9. A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Ahrens, Peter

    2004-01-01

    Aims: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). Methods and Results......: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened...... by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. Conclusions: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more...

  10. Dynamics of plc gene transcription and α-toxin production during growth of Clostridium perfringens strains with contrasting α-toxin production

    DEFF Research Database (Denmark)

    Abildgaard, Lone; Schramm, Andreas; Rudi, Knut

    2009-01-01

    The aim of the present study was to investigate transcription dynamics of the α-toxin-encoding plc gene relative to two housekeeping genes (gyrA and rplL) in batch cultures of three Clostridium perfringens strains with low, intermediate, and high levels of α-toxin production, respectively. The plc...... transcript level was always low in the low α-toxin producing strain. For the two other strains, plc transcription showed an inducible pattern and reached a maximum level in the late exponential growth phase. The transcription levels were however inversely correlated to α-toxin production for the two strains....... We propose that this discrepancy is due to differences in plc translation rates between the strains and that strain-specific translational rates therefore must be determined before α-toxin production can be extrapolated from transcript levels in C. perfringens....

  11. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway

    Science.gov (United States)

    Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

  12. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

    Directory of Open Access Journals (Sweden)

    Praveen Guleria

    Full Text Available Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins.RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes.SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  13. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    Science.gov (United States)

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  14. Transcriptomic variation in proanthocyanidin biosynthesis pathway genes in soybean (Glycine spp.).

    Science.gov (United States)

    Ha, Jungmin; Kim, Myoyeon; Kim, Moon Young; Lee, Taeyoung; Yoon, Min Young; Lee, Jayern; Lee, Yeong-Ho; Kang, Young-Gyu; Park, Jun Seong; Lee, John Hwan; Lee, Suk-Ha

    2018-04-01

    Proanthocyanidins are oligomeric or polymeric end products of flavonoid metabolic pathways starting with the central phenylpropanoid pathway. Although soybean (Glycine spp.) seeds represent a major source of nutrients for the human diet, as well as components for the cosmetics industry as a result of their high levels of flavonoid metabolites, including isoflavonoids, anthocyanins and proanthocyanidins, the genetic regulatory mechanisms underlying proanthocyanidin biosynthesis in soybean remain unclear. We evaluated interspecific and intraspecific variability in flavonoid components in soybean using 43 cultivars, landraces and wild soybean accessions. We performed transcriptomic profiling of genes encoding enzymes involved in flavonoid biosynthesis using three soybean genotypes, Hwangkeum (elite cultivar), IT109098 (landrace) and IT182932 (wild accession), in seeds. We identified a Glycine max landrace, IT109098, with a proanthocyanidin content as high as that of wild soybean. Different homologous genes for anthocyanidin reductase, which is involved in proanthocyanidin biosynthesis, were detected as differentially expressed genes between IT109098 and IT182932 compared to Hwangkeum. We detected major differences in the transcriptional levels of genes involved in the biosynthesis of proanthocyanidin and anthocyanin among genotypes beginning at the early stage of seed development. The results of the present study provide insights into the underlying genetic variation in proanthocyanidin biosynthesis among soybean genotypes. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  15. Identification of Two Aflatrem Biosynthesis Gene Loci in Aspergillus flavus and Metabolic Engineering of Penicillium paxilli To Elucidate Their Function ▿

    Science.gov (United States)

    Nicholson, Matthew J.; Koulman, Albert; Monahan, Brendon J.; Pritchard, Beth L.; Payne, Gary A.; Scott, Barry

    2009-01-01

    Aflatrem is a potent tremorgenic toxin produced by the soil fungus Aspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search of Aspergillus complete genome sequence data identified putative aflatrem gene clusters in the genomes of A. flavus and Aspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first, ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes, atmG, atmC, and atmM, and the second, ATM2, is telomere distal on chromosome 7 and contains five genes, atmD, atmQ, atmB, atmA, and atmP. Reverse transcriptase PCR in A. flavus demonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer of atmP and atmQ into Penicillium paxilli paxP and paxQ deletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis in A. flavus based on these reconstitution experiments in P. paxilli and identification of putative intermediates in wild-type cultures of A. flavus. PMID:19801473

  16. An attempt to identify the likely sources of Escherichia coli harboring toxin genes in rainwater tanks.

    Science.gov (United States)

    Ahmed, W; Sidhu, J P S; Toze, S

    2012-05-01

    In this study, 200 Escherichia coli isolates from 22 rainwater tank samples in Southeast Queensland, Australia were tested for the presence of 10 toxin genes (i.e., stx(1), stx(2), hlyA, ehxA, LT1, ST1, cdtB, east1, cnf1, and cvaC) associated with intestinal and extraintestinal pathotypes. Among the 22 rainwater tanks tested, 5 (28%), 7 (32%), 7 (32%), and 1 (5%) tanks contained E. coli harboring ST1, east1, cdtB, and cvaC genes, respectively. Of the 200 E. coli isolates from the 22 tanks, 43 (22%) strains from 13 (59%) tanks were harboring toxin gene. An attempt was made to establish a link between bird and possum fecal contamination and the presence of these potential clinically significant E. coli strains harboring toxin genes in rainwater tanks. Among the 214 E. coli isolates tested from birds, 30 (14%), 11 (5%) and 18 (8%) strains contained east1, cdtB, and cvaC toxin genes, respectively. Similarly, among the 214 possum E. coli isolates, 74 (35%) contained only the east1 toxin gene. All E. coli strains from rainwater tanks, bird and possum fecal samples harboring toxin genes were biochemically fingerprinted. Biochemical phenotypes (BPTs) of 14 (33%) E. coli strains from 7 rainwater tanks and 9 (21%) E. coli strains from 6 rainwater tanks were identical to a number of BPTs of E. coli strains isolated from bird and possum feces suggesting that these animals may be the sources of these E. coli in rainwater tanks. as a precautionary measure, it is recommended that rainwater should be treated prior to drinking. In addition, proper maintenance of roof and gutter hygiene and elimination of overhanging tree branches and other structures where possible to prevent the movement of possums are highly recommended. © 2012 American Chemical Society

  17. Detection of Genes for Superantigen Toxins in Methicillin-Resistant Staphylococcus aureus Clinical Isolates in Karachi

    International Nuclear Information System (INIS)

    Taj, Y.; Fatima, I.; Ali, S. W.; Kazmi, S. U.

    2014-01-01

    Objective: To detect genes for enterotoxins, exfoliative and toxic shock syndrome toxins in Staphylococcus aureus (S. aureus) strains isolated from clinical specimens. Study Design: Cross-sectional observational study. Place and Duration of Study: Department of Molecular Genetics, Dr. Ziauddin Hospital, Karachi, from January to December 2010. Methodology: Two hundred and ninety eight S. aureus clinical isolates were obtained from various clinical samples received at Dr. Ziauddin Hospital, Karachi. Out of these, 115 were detected as methicillin resistant (MRSA) by cefoxitin disk diffusion test showing a prevalence rate of 38.6%. Detection of individual toxin genes was performed by Polymerase Chain Reaction (PCR) by using only one primer pair for each tube. Uniplex primers were preferred as multiplex primers are longer in base pairs and have the potential for cross reaction due to non-specific binding and increase in optimization time. Results: The possession of a single gene or more than a single gene in MRSA isolates was found in 61.73% of clinical samples; the highest number was found in pus swab, followed by sputum, blood, urethral swab, and urine. The prevalence of toxin genes was higher in MRSA as compared to methicillin sensitive (MSSA) isolates (19.12%). Conclusion: PCR detects strains possessing toxin genes independent of their expression. The possession of genes for super-antigens seems to be a frequent and habitual trait of S. aureus more so in MRSA. (author)

  18. Detection of seven virulence and toxin genes of Campylobacter jejuni isolates from Danish turkeys by PCR and cytolethal distending toxin production of the isolates

    DEFF Research Database (Denmark)

    Bang, Dang Duong; Borck, Birgitte; Nielsen, Eva Møller

    2004-01-01

    A total of 117 Campylobacter jejuni isolates from Danish turkeys were tested for the presence of seven virulence and toxin genes by PCR. One hundred seventeen (100%) isolates were positive for flaA, cadF, and ceuE gene primers. One hundred three (88%) isolates were positive for cdt gene cluster PCR...

  19. Genomic survey of bZIP transcription factor genes related to tanshinone biosynthesis in Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    2018-03-01

    Full Text Available Tanshinones are a class of bioactive components in the traditional Chinese medicine Salvia miltiorrhiza, and their biosynthesis and regulation have been widely studied. Current studies show that basic leucine zipper (bZIP proteins regulate plant secondary metabolism, growth and developmental processes. However, the bZIP transcription factors involved in tanshinone biosynthesis are unknown. Here, we conducted the first genome-wide survey of the bZIP gene family and analyzed the phylogeny, gene structure, additional conserved motifs and alternative splicing events in S. miltiorrhiza. A total of 70 SmbZIP transcription factors were identified and categorized into 11 subgroups based on their phylogenetic relationships with those in Arabidopsis. Moreover, seventeen SmbZIP genes underwent alternative splicing events. According to the transcriptomic data, the SmbZIP genes that were highly expressed in the Danshen root and periderm were selected. Based on the prediction of bZIP binding sites in the promoters and the co-expression analysis and co-induction patterns in response to Ag+ treatment via quantitative real-time polymerase chain reaction (qRT-PCR, we concluded that SmbZIP7 and SmbZIP20 potentially participate in the regulation of tanshinone biosynthesis. These results provide a foundation for further functional characterization of the candidate SmbZIP genes, which have the potential to increase tanshinone production. KEY WORDS: bZIP genes, Salvia miltiorrhiza, Phylogenetic analysis, Expression pattern analysis, Tanshinone biosynthesis

  20. A Clostridioides difficile bacteriophage genome encodes functional binary toxin-associated genes.

    Science.gov (United States)

    Riedel, Thomas; Wittmann, Johannes; Bunk, Boyke; Schober, Isabel; Spröer, Cathrin; Gronow, Sabine; Overmann, Jörg

    2017-05-20

    Pathogenic clostridia typically produce toxins as virulence factors which cause severe diseases in both humans and animals. Whereas many clostridia like e.g., Clostridium perfringens, Clostridium botulinum or Clostridium tetani were shown to contain toxin-encoding plasmids, only toxin genes located on the chromosome were detected in Clostridioides difficile so far. In this study, we determined, annotated, and analyzed the complete genome of the bacteriophage phiSemix9P1 using single-molecule real-time sequencing technology (SMRT). To our knowledge, this represents the first C. difficile-associated bacteriophage genome that carries a complete functional binary toxin locus in its genome. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... fermentation processes in which the escape of genetically modified cells would be considered highly risky....

  2. In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay

    Science.gov (United States)

    Williamson, Judy L.; Rocke, Tonie E.; Aiken, Judd M.

    1999-01-01

    A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.

  3. Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus).

    Science.gov (United States)

    Fedorov, Vadim B; Goropashnaya, Anna V; Tøien, Øivind; Stewart, Nathan C; Gracey, Andrew Y; Chang, Celia; Qin, Shizhen; Pertea, Geo; Quackenbush, John; Showe, Louise C; Showe, Michael K; Boyer, Bert B; Barnes, Brian M

    2009-04-10

    We conducted a large-scale gene expression screen using the 3,200 cDNA probe microarray developed specifically for Ursus americanus to detect expression differences in liver and skeletal muscle that occur during winter hibernation compared with animals sampled during summer. The expression of 12 genes, including RNA binding protein motif 3 (Rbm3), that are mostly involved in protein biosynthesis, was induced during hibernation in both liver and muscle. The Gene Ontology and Gene Set Enrichment analysis consistently showed a highly significant enrichment of the protein biosynthesis category by overexpressed genes in both liver and skeletal muscle during hibernation. Coordinated induction in transcriptional level of genes involved in protein biosynthesis is a distinctive feature of the transcriptome in hibernating black bears. This finding implies induction of translation and suggests an adaptive mechanism that contributes to a unique ability to reduce muscle atrophy over prolonged periods of immobility during hibernation. Comparing expression profiles in bears to small mammalian hibernators shows a general trend during hibernation of transcriptional changes that include induction of genes involved in lipid metabolism and carbohydrate synthesis as well as depression of genes involved in the urea cycle and detoxification function in liver.

  4. A Single Gene Cluster for Chalcomycins and Aldgamycins: Genetic Basis for Bifurcation of Their Biosynthesis.

    Science.gov (United States)

    Tang, Xiao-Long; Dai, Ping; Gao, Hao; Wang, Chuan-Xi; Chen, Guo-Dong; Hong, Kui; Hu, Dan; Yao, Xin-Sheng

    2016-07-01

    Aldgamycins are 16-membered macrolide antibiotics with a rare branched-chain sugar d-aldgarose or decarboxylated d-aldgarose at C-5. In our efforts to clone the gene cluster for aldgamycins from a marine-derived Streptomyces sp. HK-2006-1 capable of producing both aldgamycins and chalcomycins, we found that both are biosynthesized from a single gene cluster. Whole-genome sequencing combined with gene disruption established the entire gene cluster of aldgamycins: nine new genes are incorporated with the previously identified chalcomycin gene cluster. Functional analysis of these genes revealed that almDI/almDII, (encoding α/β subunits of pyruvate dehydrogenase) triggers the biosynthesis of aldgamycins, whereas almCI (encoding an oxidoreductase) initiates chalcomycins biosynthesis. This is the first report that aldgamycins and chalcomycins are derived from a single gene cluster and of the genetic basis for bifurcation in their biosynthesis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Detection of toxin genes and RAPD analysis of bacillus cereus isolates from different soil types

    Directory of Open Access Journals (Sweden)

    Savic Dejana

    2015-01-01

    Full Text Available The aim of this study was to detect genes for enterotoxins (hbla, entFM and bceT and for emetic toxin (cer, to determine antibiotic resistance, and to estimate intraspecies diversity in B. cereus isolates by RAPD analysis. B. cereus was identified in 12 out of 117 indigenous Bacillus spp. using the classical microbiological methods and PCR. All isolates were resistant to penicillin and ampicillin, two to tetracyclin and four to trimethoprim-sulphamethoxazole. Also, all isolates produced inducible penicillinases and β-lactamase. Toxin genes were detected with PCR. EntFM and cer genes were present in all isolates, hbla in all, but two, and bceT in none. RAPD analysis was performed with four different primers, two of them designed for this study. The intraspecies diversity revealed 10 different patterns at the 90% similarity level. Two separate clusters were formed regardless of a soil type or utilization. The detection of genes encoding toxins in all B. cereus isolates indicated these bacteria as potentially pathogenic and seriously for human health. Regardless of a soil type or utilization, the RAPD analysis showed high intraspecies heterogeneity in B. cereus isolates. To the best of our knowledge, this is the first study to analyse the presence of entero- and emetic toxin genes and genetic heterogeneity in B. cereus isolates from different soil types and different soil utilization in Serbia. [Projekat Ministarstva nauke Republike Srbije, br. TR37006

  6. Cloning and molecular characterization of the beta toxin (phospholipase C) gene of Clostridium haemolyticum.

    Science.gov (United States)

    Hauer, Paul J; Yeary, Teresa J; Rosenbusch, Ricardo F

    2004-08-01

    The phospholipase C (PLPC) gene from Clostridium haemolyticum was amplified using the polymerase chain reaction. Primers were selected from a consensus sequence of closely related clostridial PLPC genes and used to amplify an 871-base pair internal segment of the gene. The internal sequence was used to design nested primers that, together with adapter-specific primers, were used to amplify upstream and downstream sequences. The sequences of upstream and downstream segments were aligned with the internal segment to obtain the entire gene sequence. Primers were selected from the aligned sequence, and the entire gene was amplified, and the PCR product was inserted by ligatation into the pCR 2.1 plasmid. An open reading frame that encodes a 399-amino acid protein, containing a 27-amino acid signal sequence, was identified (GenBank Accession Number AF525415). The molecular weight of the active protein was 42869 Da. A 16-amino acid N-terminal sequence, determined by Edman degradation, exactly matched the putative amino acid sequence of the gene product. Together, N-terminal peptide sequencing and tryptic digestion followed by MALDI-ToF mass spectroscopy verified 48% of the amino acid sequences of the active beta toxin. Comparison of the nucleotide and amino acid sequences with Gene-bank databases demonstrated that the beta toxin of C. haemolyticum exhibits high homology with other bacterial PLPCs. The N-terminal portion of the beta toxin contains zinc-binding residues common to clostridial and other bacterial PLPCs, and it shows 34% homology to the N-terminal domain of bovine arachidonate 5-lipoxygenase. The C-terminal domain of the beta toxin protein shows considerable homology with the C-terminal domains of C. novyi type A PLPC, C. perfringens alpha toxin, C. bifermentens PLPC, although the percent identity between the N-terminal regions is much higher overall than that in the C-terminal domain.

  7. Accumulation of Charantin and Expression of Triterpenoid Biosynthesis Genes in Bitter Melon (Momordica charantia).

    Science.gov (United States)

    Cuong, Do Manh; Jeon, Jin; Morgan, Abubaker M A; Kim, Changsoo; Kim, Jae Kwang; Lee, Sook Young; Park, Sang Un

    2017-08-23

    Charantin, a natural cucurbitane type triterpenoid, has been reported to have beneficial pharmacological functions such as anticancer, antidiabetic, and antibacterial activities. However, accumulation of charantin in bitter melon has been little studied. Here, we performed a transcriptome analysis to identify genes involved in the triterpenoid biosynthesis pathway in bitter melon seedlings. A total of 88,703 transcripts with an average length of 898 bp were identified in bitter melon seedlings. On the basis of a functional annotation, we identified 15 candidate genes encoding enzymes related to triterpenoid biosynthesis and analyzed their expression in different organs of mature plants. Most genes were highly expressed in flowers and/or fruit from the ripening stages. An HPLC analysis confirmed that the accumulation of charantin was highest in fruits from the ripening stage, followed by male flowers. The accumulation patterns of charantin coincide with the expression pattern of McSE and McCAS1, indicating that these genes play important roles in charantin biosynthesis in bitter melon. We also investigated optimum light conditions for enhancing charantin biosynthesis in bitter melon and found that red light was the most effective wavelength.

  8. Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

    Directory of Open Access Journals (Sweden)

    Yuanjun Li

    2016-08-01

    Full Text Available Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones, which include the xanthanolides. To date, the biogenesis of xanthanolides, especiallytheir downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that were highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of sesquiterpene lactones are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides.

  9. Assembly and expression analysis of oat vitamin E biosynthesis gene homeologs during seed development

    Science.gov (United States)

    Among the cereal grains, hexaploid oats (Avena sativa L.) are particularly rich in vitamin E, an essential liposoluble vitamin that maintains membrane stability and possesses antioxidant and anti-inflammatory properties. To date, no gene sequences involved in vitamin E biosynthesis have been reporte...

  10. Sequence variation in the alpha-toxin encoding plc gene of Clostridium perfringens strains isolated from diseased and healthy chickens

    DEFF Research Database (Denmark)

    Abildgaard, L; Engberg, RM; Pedersen, Karl

    2009-01-01

    The aim of the present study was to analyse the genetic diversity of the alpha-toxin encoding plc gene and the variation in a-toxin production of Clostridium perfringens type A strains isolated from presumably healthy chickens and chickens suffering from either necrotic enteritis (NE) or cholangio......-hepatitis. The a-toxin encoding plc genes from 60 different pulsed-field gel electrophoresis (PFGE) types (strains) of C perfringens were sequenced and translated in silico to amino acid sequences and the a-toxin production was investigated in batch cultures of 45 of the strains using an enzyme...

  11. Transcriptome sequencing and expression analysis of terpenoid biosynthesis genes in Litsea cubeba.

    Directory of Open Access Journals (Sweden)

    Xiao-Jiao Han

    Full Text Available BACKGROUND: Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour. Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. RESULTS: Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56% unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG, 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. CONCLUSION: RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The

  12. Map-Based Cloning of the Fungal Toxin Sensitivity Gene Tsn1 in Wheat

    Science.gov (United States)

    The wheat Tsn1 gene on wheat chromosome arm 5BL confers sensitivity to the host-selective proteinaceous toxins Ptr ToxA and SnToxA produced by the pathogenic fungi Pyrenophora tritici-repentis and Stagonospora nodorum, respectively. Compatible Tsn1-ToxA interactions lead to extensive cell death and...

  13. Rapid detection of vip1-type genes from Bacillus cereus and characterization of a novel vip binary toxin gene.

    Science.gov (United States)

    Yu, Xiumei; Liu, Tao; Liang, Xiaoxing; Tang, Changqing; Zhu, Jun; Wang, Shiquan; Li, Shuangcheng; Deng, Qiming; Wang, Linxia; Zheng, Aiping; Li, Ping

    2011-12-01

    A PCR-restriction fragment length polymorphism (PCR-RFLP) method for identifying vegetative insecticidal protein (vip) 1-type genes from Bacillus cereus was developed by designing specific primers based on the conserved regions of the genes to amplify vip1-type gene fragments. PCR products were digested with endonuclease AciI, and four known vip1-type genes were identified. Vip1Ac and vip1Aa-type genes appeared in 17 of 26 B. cereus strains. A novel vip1-type gene, vip1Ac1, was identified from B. cereus strain HL12. The vip1Ac1 and vip2Ae3 genes were co-expressed in Escherichia coli strain BL21 by vector pCOLADuet-1. The binary toxin showed activity only against Aphis gossypii (Homoptera), but not for Coleptera (Tenebrio molitor, Holotrichia oblita), Lepidoptera (Spodoptera exigua, Helicoverpa armigera, and Chilo suppressalis), Diptera (Culex quinquefasciatus). The LC(50) of this binary toxin for A. gossypii is 87.5 (34.2-145.3) ng mL(-1) . This is probably only the second report that Vip1 and Vip2 binary toxin shows toxicity against homopteran pests. The PCR-RFLP method developed could be very useful for identifying novel Vip1-Vip2-type binary toxins, and the novel binary toxins, Vip1Ac1 and Vip2Ae3, identified in this study may have applications in biological control of insects, thus avoiding potential problems of resistance. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  14. RNA-Seq analysis for indigo biosynthesis pathway genes in Indigofera tinctoria and Polygonum tinctorium

    Directory of Open Access Journals (Sweden)

    Bijaya K. Sarangi

    2015-12-01

    Full Text Available Natural indigo is the most important blue dye for textile dyeing and valuable secondary metabolite biosynthesized in Indigofera tinctoria and Polygonum tinctorium plants. Present investigation is made to generation of gene resource for pathway enrichment and to understand possible gene expression involved in indigo biosynthesis. The data about raw reads and the transcriptome assembly project has been deposited at GenBank under the accessions SRA180766 and SRX692542 for I. tinctoria and P. tinctorium, respectively.

  15. Genes associated with 2-methylisoborneol biosynthesis in cyanobacteria: isolation, characterization, and expression in response to light.

    Directory of Open Access Journals (Sweden)

    Zhongjie Wang

    Full Text Available The volatile microbial metabolite 2-methylisoborneol (2-MIB is a root cause of taste and odor issues in freshwater. Although current evidence suggests that 2-MIB is not toxic, this compound degrades water quality and presents problems for water treatment. To address these issues, cyanobacteria and actinomycetes, the major producers of 2-MIB, have been investigated extensively. In this study, two 2-MIB producing strains, coded as Pseudanabaena sp. and Planktothricoids raciborskii, were used in order to elucidate the genetic background, light regulation, and biochemical mechanisms of 2-MIB biosynthesis in cyanobacteria. Genome walking and PCR methods revealed that two adjacent genes, SAM-dependent methyltransferanse gene and monoterpene cyclase gene, are responsible for GPP methylation and subsequent cyclization to 2-MIB in cyanobacteria. These two genes are located in between two homologous cyclic nucleotide-binding protein genes that may be members of the Crp-Fnr regulator family. Together, this sequence of genes forms a putative operon. The synthesis of 2-MIB is similar in cyanobacteria and actinomycetes. Comparison of the gene arrangement and functional sites between cyanobacteria and other organisms revealed that gene recombination and gene transfer probably occurred during the evolution of 2-MIB-associated genes. All the microorganisms examined have a common origin of 2-MIB biosynthesis capacity, but cyanobacteria represent a unique evolutionary lineage. Gene expression analysis suggested that light is a crucial, but not the only, active regulatory factor for the transcription of 2-MIB synthesis genes. This light-regulated process is immediate and transient. This study is the first to identify the genetic background and evolution of 2-MIB biosynthesis in cyanobacteria, thus enhancing current knowledge on 2-MIB contamination of freshwater.

  16. Transcriptomic Analysis Reveals Key Genes Related to Betalain Biosynthesis in Pulp Coloration of Hylocereus polyrhizus.

    Science.gov (United States)

    Qingzhu, Hua; Chengjie, Chen; Zhe, Chen; Pengkun, Chen; Yuewen, Ma; Jingyu, Wu; Jian, Zheng; Guibing, Hu; Jietang, Zhao; Yonghua, Qin

    2015-01-01

    Betalains have high nutritional value and bioactivities. Red pulp pitaya (Hylocereus polyrhizus) is the only fruit containing abundant betalains for consumer. However, no information is available about genes involved in betalain biosynthesis in H. polyrhizus. Herein, two cDNA libraries of pitaya pulps with two different coloration stages (white and red pulp stages) of Guanhuahong (H. polyrhizus) were constructed. A total of about 12 Gb raw RNA-Seq data was generated and was de novo assembled into 122,677 transcripts with an average length of 1183 bp and an N50 value of 2008. Approximately 99.99% of all transcripts were annotated based on seven public databases. A total of 8871 transcripts were significantly regulated. Thirty-three candidate transcripts related to betalain biosynthesis were obtained from the transcriptome data. Transcripts encoding enzymes involved in betalain biosynthesis were analyzed using RT-qPCR at the whole pulp coloration stages of H. polyrhizus (7-1) and H. undatus (132-4). Nine key transcripts of betalain biosynthesis were identified. They were assigned to four kinds of genes in betalain biosynthetic pathway, including tyrosinase, 4, 5-DOPA dioxygenase extradiol, cytochrome P450 and glucosyltransferase. Ultimately, a preliminary betalain biosynthetic pathway for pitaya was proposed based on betalain analyses, gene expression profiles and published documents.

  17. Transcriptomic analysis reveals key genes related to betalain biosynthesis in pulp coloration of Hylocereus polyrhizus

    Directory of Open Access Journals (Sweden)

    Hua eQingzhu

    2016-01-01

    Full Text Available Betalains have high nutritional value and bioactivities. Red pulp pitaya (Hylocereus polyrhizus is the only fruit containing abundant betalains for consumer. However, no information is available about genes involved in betalain biosynthesis in H. polyrhizus. Herein, two cDNA libraries of pitaya pulps with two different coloration stages (white and red pulp stages of Guanhuahong (H. polyrhizus were constructed. A total of about 12 Gb raw RNA-Seq data was generated and was de novo assembled into 122,677 transcripts with an average length of 1,183 bp and an N50 value of 2008. Approximately 99.99% of all transcripts were annotated based on seven public databases. A total of 8,871 transcripts were significantly regulated. Thirty-three candidate transcripts related to betalain biosynthesis were obtained from the transcriptome data. Transcripts encoding enzymes involved in betalain biosynthesis were analyzed using RT-qPCR at the whole pulp coloration stages of H. Polyrhizus (7-1 and H. Undatus (132-4. Nine key transcripts of betalain biosynthesis were identified. They were assigned to four kinds of genes in betalain biosynthetic pathway, including tyrosinase, 4, 5-DOPA dioxygenase extradiol, cytochrome P450 and glucosyltransferase. Ultimately, a preliminary betalain biosynthetic pathway for pitaya was proposed based on betalain analyses and gene expression profiles.

  18. Genome-wide survey of flavonoid biosynthesis genes and gene expression analysis between black- and yellow-seeded Brassica napus

    Directory of Open Access Journals (Sweden)

    Cunmin Qu

    2016-12-01

    Full Text Available Flavonoids, the compounds that impart color to fruits, flowers, and seeds, are the most widespread secondary metabolites in plants. However, a systematic analysis of these loci has not been performed in Brassicaceae. In this study, we isolated 649 nucleotide sequences related to flavonoid biosynthesis, i.e., the Transparent Testa (TT genes, and their associated amino acid sequences in 17 Brassicaceae species, grouped into Arabidopsis or Brassicaceae subgroups. Moreover, 36 copies of 21 genes of the flavonoid biosynthesis pathway were identified in A. thaliana, 53 were identified in B. rapa, 50 in B. oleracea, and 95 in B. napus, followed the genomic distribution, collinearity analysis and genes triplication of them among Brassicaceae species. The results showed that the extensive gene loss, whole genome triplication, and diploidization that occurred after divergence from the common ancestor. Using qRT-PCR methods, we analyzed the expression of eighteen flavonoid biosynthesis genes in 6 yellow- and black-seeded B. napus inbred lines with different genetic background, found that 12 of which were preferentially expressed during seed development, whereas the remaining genes were expressed in all B. napus tissues examined. Moreover, fourteen of these genes showed significant differences in expression level during seed development, and all but four of these (i.e., BnTT5, BnTT7, BnTT10, and BnTTG1 had similar expression patterns among the yellow- and black-seeded B. napus. Results showed that the structural genes (BnTT3, BnTT18 and BnBAN, regulatory genes (BnTTG2 and BnTT16 and three encoding transfer proteins (BnTT12, BnTT19, and BnAHA10 might play an crucial roles in the formation of different seed coat colors in B. napus. These data will be helpful for illustrating the molecular mechanisms of flavonoid biosynthesis in Brassicaceae species.

  19. Silver nanoparticles mediated altered gene expression of melanin biosynthesis genes in Bipolaris sorokiniana.

    Science.gov (United States)

    Mishra, Sandhya; Singh, H B

    2015-03-01

    Melanin production in many fungal phytopathogens has been investigated to play direct or indirect role in pathogenesis. However, in Bipolaris sorokiniana, the spot blotch pathogen of wheat, much less is known about the role melanin play in pathogenesis. As an extension of our previous report, the present study aims to investigate the plausible association between melanin production and virulence factor in B. sorokiniana. In the previous study, we carried out analysis on the antifungal efficacy of biosynthesized silver nanoparticles (AgNPs) against B. sorokiniana. The present investigation revealed the gene expression analysis of melanin biosynthesis genes viz. polyketide synthase (PKS1) and scytalone dehydratase (SCD1) under the influence of AgNPs. The 0.05mg/ml concentration of AgNPs yielded noticeable inhibition of B. sorokiniana growth, while 0.1mg/ml concentration of AgNPs accounted for complete inhibition of pathogen growth. In addition, the semiquantitative RT-PCR analysis exhibited reduced expression of PKS1 and SCD1 under the influence of AgNPs treatment. Furthermore, the qRT-PCR demonstrated 6.47 and 1.808 fold significant decrease in the expression pattern of PKS1 and SCD1, respectively, in B. sorokiniana treated with AgNPs. The present study provides probable understanding of molecular events underlying the antifungal role of AgNPs against B. sorokiniana. Copyright © 2015 Elsevier GmbH. All rights reserved.

  20. Detection of seven virulence and toxin genes of Campylobacter jejuni isolates from Danish turkeys by PCR and cytolethal distending toxin production of the isolates.

    Science.gov (United States)

    Bang, Dang Duong; Borck, Birgitte; Nielsen, Eva Møller; Scheutz, Flemming; Pedersen, Karl; Madsen, Mogens

    2004-10-01

    A total of 117 Campylobacter jejuni isolates from Danish turkeys were tested for the presence of seven virulence and toxin genes by PCR. One hundred seventeen (100%) isolates were positive for flaA, cadF, and ceuE gene primers. One hundred three (88%) isolates were positive for cdt gene cluster PCR detection (cdt gene cluster-PCR), whereas 101 (86.3%), 102 (87.2%), and 110 (94%) isolates were positive for cdtA-, cdtB-, and cdtC-PCR, respectively. Only 39 (33.3%) isolates were positive for virB11. Of 117 isolates, 114 (97.4%) produced cytolethal distending toxin (CDT) in Vero cell assays, 105 (89.7%) in Colon 205 assays, and 109 (93.2%) in chicken embryo cell assays. The CDT titers were determined in Vero cell assays. Of 117 isolates, 50 (42.7%) produced a CDT titer of 1:100, 29 (24.8%) of 1:50, and 27 (23%) of 1:5 to 1:10; 8 (6.8%) produced a CDT titer at undiluted supernatants and 3 (2.6%) produced no toxin. Twenty-nine C. jejuni isolates that were PCR negative for one or more individual cdt toxin genes also produced low or no CDT toxin. The high prevalence of the seven virulence and toxin genes demonstrates that these putative pathogenic determinants are widespread among Campylobacter isolates from turkeys and calls for further investigation for the elimination of Campylobacter infection in industrial turkey production and in industrial food chains.

  1. Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis

    International Nuclear Information System (INIS)

    Skory, C.D.; Horng, J.S.; Pestka, J.J.; Linz, J.E.

    1990-01-01

    The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per μg of DNA was developed for A. parasiticus by using homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [ 14 C]OMP to [ 14 C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [ 14 C]OMP to [ 14 C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field

  2. Toxin Gene Analysis of a Variant Strain of Clostridium difficile That Causes Human Clinical Disease

    Science.gov (United States)

    Sambol, Susan P.; Merrigan, Michelle M.; Lyerly, David; Gerding, Dale N.; Johnson, Stuart

    2000-01-01

    A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3′ end of the gene, and a point mutation in the 5′ end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5′ end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3′ end of tcdA demonstrated identical 1.8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A. PMID:10992443

  3. Identification of toxin genes encoding Cyt proteins from standard ...

    African Journals Online (AJOL)

    Polymerase chain reaction-restriction fragment length polymorphism methods for identification of cyt subclasses from Bacillus thuringiensis were established. Eight of 68 standard and ten of 107 Argentine B. thuringiensis strains harbor at least one cyt gene. The combination of cyt1Aa/cyt2Ba genes was identified in four ...

  4. Identification of a gene cluster for biosynthesis of mannosylerythritol lipids in the basidiomycetous fungus Ustilago maydis.

    Science.gov (United States)

    Hewald, Sandra; Linne, Uwe; Scherer, Mario; Marahiel, Mohamed A; Kämper, Jörg; Bölker, Michael

    2006-08-01

    Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose expression is strongly induced by nitrogen limitation. We used DNA microarray analysis to identify additional genes involved in MEL biosynthesis. Here we show that emt1 is part of a gene cluster which comprises five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and Mat1, contain short sequence motifs characteristic for acyl- and acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential for MEL production, which suggests that they are involved in the acylation of mannosylerythritol. Deletion of mat1 resulted in the secretion of completely deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in Escherichia coli and demonstrated that this enzyme acts as an acetyl coenzyme A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl groups. Based on these results, we propose a biosynthesis pathway for the generation of mannosylerythritol lipids in U. maydis.

  5. Bacteriophage-encoded shiga toxin gene in atypical bacterial host

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    Casas Veronica

    2011-07-01

    Full Text Available Abstract Background Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB. A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli. Results Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli. Conclusions The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.

  6. PREVALENCE OF TOXIN ENCODING GENES IN ESCHERICHIACOLI ISOLATES FROM URINARY TRACT INFECTIONS INSLOVENIA

    Directory of Open Access Journals (Sweden)

    Marjanca Starčič-Erjavec

    2008-06-01

    Full Text Available Methods 110 uropathogenic Escherichia coli strains (UPEC obtained from the Institute of Microbiology and Immunology of the Medical Faculty in Ljubljana were screened by PCR withprimers specific for the following toxin encoding genes: hlyA (haemolysin, cnf1 (cytotoxicnecrotising factor 1, usp (uropathogenic specific protein USP and ibeA (invasin. Dotblot hybridisation experiments were performed to validate the PCR assays.Results In 44% of the strains usp gene sequences were detected. The prevalence of hlyA and cnf1was 25% and 23%, respectively. Only 9% of the strains harbored ibeA. The majority of thetested toxin encoding genes was found in strains belonging to the B2 phylogenetic group.Conclusions The toxin encoding genes hlyA, cnf1 and usp were strongly co-associated. Further, we founda statistically significant co-association of ibeA and usp. The prevalence of the testedtoxin encoding genes in E. coli strains from urinary tract infections isolated in Slovenia iscomparable to those from studies in other geographic regions.

  7. Identification and expression profile of Halloween genes involved in ecdysteroid biosynthesis in Spodoptera littoralis.

    Science.gov (United States)

    Iga, Masatoshi; Smagghe, Guy

    2010-03-01

    20-Hydroxyecdyone (20E), an active form of ecdysteroid, is the key hormone in insect growth and development. The biosynthesis of ecdysteroid is triggered and under the control of the neuropeptide, prothoracicotropic hormone (PTTH). To date, five cytochrome P450 enzymes, namely Spook (Spo), Phantom (Phm), Disembodied (Dib), Shadow (Sad) and Shade (Shd) related to ecdysteroid biosynthesis, are identified and the character of last four enzymes is well studied in Drosophila melanogaster, Bombyx mori and Manduca sexta. These genes are called Halloween genes and mediate the biosynthesis of 20E from cholesterol. In this study, we extended these works to a major pest insect in agriculture, the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae). We identified the sequence of five Halloween genes, and the converted amino acid sequences were compared with those of other insects. The phylogenetic analysis clearly showed separated clusters of each gene and the evolutional conservation in insects with a high similarity in Lepidoptera. Spo, phm, dib and sad were predominantly expressed in prothoracic glands, and shd was expressed in fat body and Malpighian tubules at the last instar larvae. Spo expression was kept high level between day 2 and day 4 after ecdysis. The expression of phm and dib peaked at day 2, and sad and shd expressions peaked at day 2 and day 4 after ecdysis. In addition, the hemolymph ecdysteroid titer showed a small peak at day 2 and a large peak at day 4 after ecdysis. These results suggest the importance of Halloween genes in ecdysone biosynthesis by prothoracic glands and conversion of ecdysone into 20E by fat body in larval-pupal metamorphosis. (c) 2009 Elsevier Inc. All rights reserved.

  8. Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

    Science.gov (United States)

    Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

    2017-01-01

    A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

  9. Expression profiles of genes involved in tanshinone biosynthesis of ...

    Indian Academy of Sciences (India)

    tanshinone biosynthetic gene; quantitative real-time PCR; Salvia miltiorrhiza. Abstract. Author Affiliations. ZHENQIAO SONG1 2 JIANHUA WANG1 2 XINGFENG LI1 2. State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, a; Agronomy College, Shandong Agricultural University, Taian 271018, ...

  10. Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

    Energy Technology Data Exchange (ETDEWEB)

    Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

    2010-01-07

    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during

  11. Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation.

    Science.gov (United States)

    Li, Wang; Wang, Bing; Wang, Man; Chen, Min; Yin, Jing-Ming; Kaleri, Ghullam Murtaza; Zhang, Rui-Jie; Zuo, Tie-Niu; You, Xiong; Yang, Qing

    2014-04-01

    Anthocyanins are a class of products of plant secondary metabolism and are responsible for tubers color in potato. The biosynthesis of anthocyanins is a complex biological process, in which multiple genes are involved including structural genes and regulatory genes. In this study, StAN11, a WD40-repeat gene, was cloned from potato cultivar Chieftain (Solanum tuberosum L.). StAN11 (HQ599506) contained no intron and its open reading frame (ORF) was 1,029 bp long, encoding a putative protein of 342 amino acids. In order to verify its role in anthocyanin biosynthesis, StAN11 was inserted behind the CaMV-35S promoter of pCMBIA1304 and the recombination vector was introduced into the potato cultivar Désirée plants by Agrobacterium-mediated transformation. The color of transgenic tuber skin was significantly deepened, compared to the wild-type control, which was highly consistent with the accumulation of anthocyanin and expression of StAN11 in transgenic lines tuber skin. Further analysis on the expression of Flavonone-3-hydroxylase (F3H), Dihydroflavonol reductase (DFR), Anthocyanidin synthase (ANS), and Flavonoid 3-O-glucosyl transferase (3GT) in transgenic plants revealed that only DFR was upregulated. This result suggested that StAN11 regulated anthocyanin biosynthesis in potato by controlling DFR expression and accumulation of anthocyanin could be increased through overexpression of StAN11 in the tubers with the genetic background of anthocyanin biosynthesis. © 2013 Institute of Botany, Chinese Academy of Sciences.

  12. Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae)

    Science.gov (United States)

    Keeling, Christopher I.; Blomquist, Gary J.; Tittiger, Claus

    In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

  13. Arabidopsis acetyl-amido synthetase GH3.5 involvement in camalexin biosynthesis through conjugation of indole-3-carboxylic acid and cysteine and upregulation of camalexin biosynthesis genes.

    Science.gov (United States)

    Wang, Mu-Yang; Liu, Xue-Ting; Chen, Ying; Xu, Xiao-Jing; Yu, Biao; Zhang, Shu-Qun; Li, Qun; He, Zu-Hua

    2012-07-01

    Camalexin (3-thiazol-2'-yl-indole) is the major phytoalexin found in Arabidopsis thaliana. Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments. Camalexin is formed when indole-3-acetonitrile (IAN) is catalyzed by the cytochrome P450 monooxygenase CYP71A13. Here, we demonstrate that the Arabidopsis GH3.5 protein, a multifunctional acetyl-amido synthetase, is involved in camalexin biosynthesis via conjugating indole-3-carboxylic acid (ICA) and cysteine (Cys) and regulating camalexin biosynthesis genes. Camalexin levels were increased in the activation-tagged mutant gh3.5-1D in both Col-0 and cyp71A13-2 mutant backgrounds after pathogen infection. The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole-3-acyl-cysteinate (ICA(Cys)) in vitro. In support of the in vitro reaction, feeding with ICA and Cys increased camalexin levels in Col-0 and gh3.5-1D. Dihydrocamalexic acid (DHCA), the precursor of camalexin and the substrate for PAD3, was accumulated in gh3.5-1D/pad3-1, suggesting that ICA(Cys) could be an additional precursor of DHCA for camalexin biosynthesis. Furthermore, expression of the major camalexin biosynthesis genes CYP79B2, CYP71A12, CYP71A13 and PAD3 was strongly induced in gh3.5-1D. Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA(Cys), and upregulation of the major biosynthetic pathway genes. © 2012 Institute of Botany, Chinese Academy of Sciences.

  14. Differential roles of two SARP-encoding regulatory genes during tylosin biosynthesis.

    Science.gov (United States)

    Bate, Neil; Stratigopoulos, George; Cundliffe, Eric

    2002-01-01

    The tylosin biosynthetic gene cluster of Streptomyces fradiae is remarkable in harbouring at least five regulatory genes, two of which (tylS and tylT) encode proteins of the Streptomyces antibiotic regulatory protein (SARP) family. The aim of the present work was to assess the respective contributions of TylS and TylT to tylosin production. A combination of targeted gene disruption, fermentation studies and gene expression analysis via reverse transcriptase-polymerase chain reaction (RT-PCR) suggests that tylS is essential for tylosin production and controls the expression of tylR (previously shown to be a global activator of the biosynthetic pathway) plus at least one other gene involved in polyketide metabolism or regulation thereof. This is the first demonstration of a SARP acting to control another regulatory gene during antibiotic biosynthesis. In contrast, tylT is not essential for tylosin production.

  15. A potato skin SSH library yields new candidate genes for suberin biosynthesis and periderm formation.

    Science.gov (United States)

    Soler, Marçal; Serra, Olga; Fluch, Silvia; Molinas, Marisa; Figueras, Mercè

    2011-05-01

    Potato (Solanum tuberosum) tubers are underground storage organs covered by the skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Understanding the molecular processes associated with periderm formation is of great importance for a better knowledge of this protective tissue and for improving the storage life of tubers. Here, to isolate new candidate genes for potato periderm, a suppression subtractive hybridization library from potato skin was performed. This library yielded a comprehensive list of 108 candidate genes that were manually sorted in functional categories according to the main cellular and metabolic processes in periderm. As expected, the list contains Suberin and wax genes, including some genes with a demonstrated role in the biosynthesis of these cell wall aliphatic compounds. Moreover, Regulation and Stress and defence genes are highly abundant in the library in general agreement with previous potato skin proteomic studies. The putative function of the genes in periderm is discussed.

  16. Digital Gene Expression Profiling to Explore Differentially Expressed Genes Associated with Terpenoid Biosynthesis during Fruit Development in Litsea cubeba

    Directory of Open Access Journals (Sweden)

    Ming Gao

    2016-09-01

    Full Text Available Mountain pepper (Litsea cubeba (Lour. Pers. (Lauraceae is an important industrial crop as an ingredient in cosmetics, pesticides, food additives and potential biofuels. These properties are attributed to monoterpenes and sesquiterpenes. However, there is still no integrated model describing differentially expressed genes (DEGs involved in terpenoid biosynthesis during the fruit development of L. cubeba. Here, we performed digital gene expression (DGE using the Illumina NGS platform to evaluated changes in gene expression during fruit development in L. cubeba. DGE generated expression data for approximately 19354 genes. Fruit at 60 days after flowering (DAF served as the control, and a total of 415, 1255, 449 and 811 up-regulated genes and 505, 1351, 1823 and 1850 down-regulated genes were identified at 75, 90, 105 and 135 DAF, respectively. Pathway analysis revealed 26 genes involved in terpenoid biosynthesis pathways. Three DEGs had continued increasing or declining trends during the fruit development. The quantitative real-time PCR (qRT-PCR results of five differentially expressed genes were consistent with those obtained from Illumina sequencing. These results provide a comprehensive molecular biology background for research on fruit development, and information that should aid in metabolic engineering to increase the yields of L. cubeba essential oil.

  17. Identification and expression analysis of candidate genes involved in carotenoid biosynthesis in chickpea seeds

    Directory of Open Access Journals (Sweden)

    Mohammad Kazem Rezaei

    2016-12-01

    Full Text Available Plant carotenoids have a key role in preventing various diseases in human because of their antioxidant and provitamin A properties. Chickpea is a good source of carotenoid among legumes and its diverse germplasm and genome accessibility makes it a good model for carotenogenesis studies. The structure, location and copy numbers of genes involved in carotenoid biosynthesis were retrieved from the chickpea genome. The majority of the single nucleotide polymorphism (SNPs within these genes across five diverse chickpea cultivars was synonymous mutation. We examined the expression of the carotenogenesis genes and their association with carotenoid concentration at different seed development stages of five chickpea cultivars. Total carotenoid concentration ranged from 22 μg g-1 in yellow cotyledon kabuli to 44 μg g-1 in green cotyledon desi at 32 days post anthesis (DPA. The majority of carotenoids in chickpea seeds consists of lutein and zeaxanthin. The expression of the selected 19 genes involved in carotenoid biosynthesis pathway showed common pattern across five cultivars with higher expression at 8 and/or 16 DPA then dropped considerably at 24 and 32 DPA. Almost all genes were up-regulated in CDC Jade cultivar. Correlation analysis between gene expression and carotenoid concentration showed that the genes involved in the primary step of carotenoid biosynthesis pathway including carotenoid desaturase and isomerase positively correlated with various carotenoid components in chickpea seeds. A negative correlation was found between hydroxylation activity and provitamin A concentration in the seeds. The highest provitamin A concentration including β-carotene and β-cryptoxanthin were found in green cotyledon chickpea cultivars.

  18. Detection of Shiga toxin variants, virulence genes and the relationship to cytotoxicity in Shiga toxin-producing Escherichia coli (STEC) from domestic farm animals

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to life-threating diseases such as hemorrhagic colitis, and hemolytic uremic syndrome in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in STEC stra...

  19. Detection of toxic shock toxin (tst gene in Staphylococcus aureus isolated from bovine milk samples

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    S. Baniardalan

    2017-09-01

    Full Text Available Staphylococcus aureus is a major causative pathogen of clinical and subclinical mastitis in dairy cattle all over the world. This agent produces a variety of extracellular toxins and virulence factors in-cluding toxic shock syndrome toxin-1 (TSST-1 which is the major cause of toxic shock syndrome (TSS. In the present study, 76 S. aureus isolates have been obtained from milk samples collected from 7 dairy herds in Hamedan province of Iran. The isolates were identified based on the biochemical and molecular methods using PCR amplification of the femA gene. The staphylococcal isolates were also examined for the presence of TSST-1 (tst encoding gene. This gene was detected in only one S. aureus isolate (1.3%. The results revealed that S. aureus strains causing bovine mastitis may potentially produce staphylococcal toxic shock syndrome toxin-1, indicating that it is very important to follow the presence of TSST-1 producing S. aureus isolates in foodstuffs to protect consumers against the risk of toxic shock syndrome

  20. Molecular cloning of Clostridium septicum vaccine strain alpha toxin gene in E. coli

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    Bozorgkhoo

    2014-05-01

    Full Text Available Clostridium septicum a Gram positive anaerobic bacterium produces several toxins including alpha, beta, gamma and delta. C. septicum alpha toxin is lethal and is responsible for a serious disease known as gas gangrene. The aim of the present study was to molecular cloning and sequencing of C. septicum vaccine strain alpha toxin gene. Genomic DNA was extracted using standard phenol and chloroform extraction method, and the target gene was amplified through PCR by specific primers. Quality and quantity of PCR product was evaluated using agarose gel electrophoresis and confirmed with spectrophotometry. The PCR product was purified and was ligated in pJET1.2blunt cloning vector and was used for E. coli/TOP10 competent cells transformation. pJETαsep recombinant plasmid was purified and sequenced using universal primers. Sequencing and BLAST analysis of csa showed over 99% identity to other previously deposited csa in the GenBank. The csa sequence was deposited in the GenBank under accession number JN793989. E. coli/TOP10/pJETαsep as a recombinant bacterium could be used for purifying of recombinant csa gene and its expression in the suitable prokaryotic hosts.

  1. Coregulated expression of loline alkaloid-biosynthesis genes in Neotyphodium uncinatum cultures.

    Science.gov (United States)

    Zhang, Dong-Xiu; Stromberg, Arnold J; Spiering, Martin J; Schardl, Christopher L

    2009-08-01

    Epichloë endophytes (holomorphic Epichloë spp. and anamorphic Neotyphodium spp.) are systemic, often heritable symbionts of cool-season grasses (subfamily Pooideae). Many epichloae provide protection to their hosts by producing anti-insect compounds. Among these are the loline alkaloids (LA), which are toxic and deterrent to a broad range of herbivorous insects but not to mammalian herbivores. LOL, a gene cluster containing nine genes, is associated with LA biosynthesis. We investigated coordinate regulation between LOL-gene expression and LA production in minimal medium (MM) cultures of Neotyphodium uncinatum. Expression of all LOL genes significantly fit temporal quadratic patterns during LA production. LOL-gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached at close to the time of most rapid LA accumulation, and gene expression declined to a very low level as amounts of LA plateaued. Temporal expression profiles of the nine LOL genes were tightly correlated with each other, but not as tightly correlated with proC and metE (genes for biosynthesis of precursor amino acids). Furthermore, the start days and peak days of expression significantly correlated with the order of the LOL-cluster genes in the genome. Hierarchical cluster analysis indicated three pairs of genes-lolA and lolC, lolO and lolD, and lolT and lolE-expression of which was especially tightly correlated. Of these, lolA and lolC tended to be expressed early, and lolT and lolE tended to be expressed late, in keeping with the putative roles of the respective gene products in the LA-biosynthesis pathway. Several common transcriptional binding sites were discovered in the LOL upstream regions. However, low expression of P(lolC2)uidA and P(lolA2)uidA in N. uncinatum transformants suggested induced expression of LOL genes might be subject to position effect at the LOL locus.

  2. [Molecular cloning of threonine biosynthesis genes from Propionibacterium shermanii in Escherichia coli cells].

    Science.gov (United States)

    Pankova, S V; Abilev, S K

    1993-03-01

    Cloning the genes for threonine biosynthesis from Propionibacterium shermanii was performed in Escherichia coli cells using the plasmid vector pVZ361. The cloned genes were identified via complementation of thrB, thrC, thrA1 and thrA2 mutations of E. coli. The gene complementing thrB of E. coli was located within a 5.1 kb fragment of P. shermanii chromosomal DNA. The cloned DNA fragment hybridized with a chromosomal fragment of P. shermanii of the same size. The plasmid pSPt4 (with the thrB gene) was digested with Sau3A and ligated with the BamHI-restricted pUC19 vector. The 1.8 kb DNA fragment of P. shermanii was shown to complement the thrB gene function in E. coli cells.

  3. Carotenoids biosynthesis and cleavage related genes from bacteria to plants.

    Science.gov (United States)

    Liang, Ming-Hua; Zhu, Jianhua; Jiang, Jian-Guo

    2017-06-13

    Carotenoids are essential for photosynthesis and photoprotection in photosynthetic organisms and beneficial for human health. Apocarotenoids derived from carotenoid degradation can serve critical functions including hormones, volatiles, and signals. They have been used commercially as food colorants, animal feed supplements, and nutraceuticals for cosmetic and pharmaceutical purposes. This review focuses on the molecular evolution of carotenogenic enzymes and carotenoid cleavage oxygenases (CCOs) from bacteria, fungi, cyanobacteria, algae, and plants. The diversity of carotenoids and apocarotenoids as well as their complicated biosynthetic pathway in different species can shed light on the history of early molecular evolution. Some carotenogenic genes (such as phytoene synthases) have high protein sequence similarity from bacteria to land plants, but some (such as phytoene desaturases, lycopene cyclases, carotenoid hydroxylases, and CCOs) have low similarity. The broad diversity of apocarotenoid volatile compounds can be attributed to large numbers of carotenoid precursors and the various cleavage sites catalyzed by CCOs enzymes. A variety of carotenogenic enzymes and CCOs indicate the functional diversification of carotenoids and apocrotenoids in different species. New carotenoids, new apocarotenoids, new carotenogenic enzymes, new CCOs, and new pathways still need to be explored.

  4. Restriction and Recruitment—Gene Duplication and the Origin and Evolution of Snake Venom Toxins

    Science.gov (United States)

    Hargreaves, Adam D.; Swain, Martin T.; Hegarty, Matthew J.; Logan, Darren W.; Mulley, John F.

    2014-01-01

    Snake venom has been hypothesized to have originated and diversified through a process that involves duplication of genes encoding body proteins with subsequent recruitment of the copy to the venom gland, where natural selection acts to develop or increase toxicity. However, gene duplication is known to be a rare event in vertebrate genomes, and the recruitment of duplicated genes to a novel expression domain (neofunctionalization) is an even rarer process that requires the evolution of novel combinations of transcription factor binding sites in upstream regulatory regions. Therefore, although this hypothesis concerning the evolution of snake venom is very unlikely and should be regarded with caution, it is nonetheless often assumed to be established fact, hindering research into the true origins of snake venom toxins. To critically evaluate this hypothesis, we have generated transcriptomic data for body tissues and salivary and venom glands from five species of venomous and nonvenomous reptiles. Our comparative transcriptomic analysis of these data reveals that snake venom does not evolve through the hypothesized process of duplication and recruitment of genes encoding body proteins. Indeed, our results show that many proposed venom toxins are in fact expressed in a wide variety of body tissues, including the salivary gland of nonvenomous reptiles and that these genes have therefore been restricted to the venom gland following duplication, not recruited. Thus, snake venom evolves through the duplication and subfunctionalization of genes encoding existing salivary proteins. These results highlight the danger of the elegant and intuitive “just-so story” in evolutionary biology. PMID:25079342

  5. RNA-seq analysis of overexpressing ovine AANAT gene of melatonin biosynthesis in switchgrass

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    Shan Yuan

    2016-08-01

    Full Text Available Melatonin serves important functions in the promotion of growth and anti-stress regulation by efficient radical scavenging and regulation of antioxidant enzyme activity in various plants. To investigate its regulatory roles and metabolism pathways, the transcriptomic profile of overexpressing the ovine arylalkylamine N-acetyltransferase (oAANAT gene, encoding the penultimate enzyme in melatonin biosynthesis, was compared with empty vector (EV control using RNA-seq in switchgrass, a model plant of cellulosic ethanol conversion. The 85.22 million high quality reads that were assembled into 135,684 unigenes were generated by Illumina sequencing for transgenic oAANAT switchgrass with an average sequence length of 716 bp. A total of 946 differential expression genes (DEGs in transgenic line comparing to control switchgrass, including 737 up-regulated and 209 down-regulated genes, were mainly enriched with two main functional patterns of melatonin identifying by gene ontology analysis: the growth regulator and stress tolerance. Furthermore, KEGG maps indicated that the biosynthetic pathways of secondary metabolite (phenylpropanoids, flavonoids, steroids, stilbenoid, diarylheptanoid and gingerol and signaling pathways (MAPK signaling pathway, estrogen signaling pathway were involved in melatonin metabolism. This study substantially expands the transcriptome information for switchgrass and provides valuable clues for identifying candidate genes involved in melatonin biosynthesis and elucidating the mechanism of melatonin metabolism.

  6. Metazoan Remaining Genes for Essential Amino Acid Biosynthesis: Sequence Conservation and Evolutionary Analyses

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    Igor R. Costa

    2014-12-01

    Full Text Available Essential amino acids (EAA consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS and betaine-homocysteine S-methyltransferase (BHMT diverged from the expected Tree of Life (ToL relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  7. Differential expression of jasmonate biosynthesis genes in cacao genotypes contrasting for resistance against Moniliophthora perniciosa.

    Science.gov (United States)

    Litholdo, Celso G; Leal, Gildemberg A; Albuquerque, Paulo S B; Figueira, Antonio

    2015-10-01

    The resistance mechanism of cacao against M. perniciosa is likely to be mediated by JA/ET-signaling pathways due to the preferential TcAOS and TcSAM induction in a resistant genotype. The basidiomycete Moniliophthora perniciosa causes a serious disease in cacao (Theobroma cacao L.), and the use of resistant varieties is the only sustainable long-term solution. Cacao resistance against M. perniciosa is characterized by pathogen growth inhibition with reduced colonization and an attenuation of disease symptoms, suggesting a regulation by jasmonate (JA)/ethylene (ET) signaling pathways. The hypothesis that genes involved in JA biosynthesis would be active in the interaction of T. cacao and M. perniciosa was tested here. The cacao JA-related genes were evaluated for their relative quantitative expression in susceptible and resistant genotypes upon the exogenous application of ET, methyl-jasmonate (MJ), and salicylic acid (SA), or after M. perniciosa inoculation. MJ treatment triggered changes in the expression of genes involved in JA biosynthesis, indicating that the mechanism of positive regulation by exogenous MJ application occurs in cacao. However, a higher induction of these genes was observed in the susceptible genotype. Further, a contrast in JA-related transcriptional expression was detected between susceptible and resistant plants under M. perniciosa infection, with the induction of the allene oxide synthase gene (TcAOS), which encodes a key enzyme in the JA biosynthesis pathway in the resistant genotype. Altogether, this work provides additional evidences that the JA-dependent signaling pathway is modulating the defense response against M. perniciosa in a cacao-resistant genotype.

  8. Functional conservation and divergence of Miscanthus lutarioriparius GT43 gene family in xylan biosynthesis.

    Science.gov (United States)

    Wang, Xiaoyu; Tang, Qi; Zhao, Xun; Jia, Chunlin; Yang, Xuanwen; He, Guo; Wu, Aimin; Kong, Yingzhen; Hu, Ruibo; Zhou, Gongke

    2016-04-26

    Xylan is the most abundant un-cellulosic polysaccharides of plant cell walls. Much progress in xylan biosynthesis has been gained in the model plant species Arabidopsis. Two homologous pairs Irregular Xylem 9 (IRX9)/9L and IRX14/14L from glycosyltransferase (GT) family 43 have been proved to play crucial roles in xylan backbone biosynthesis. However, xylan biosynthesis in grass such as Miscanthus remains poorly understood. We characterized seven GT43 members in M. lutarioriparius, a promising bioenergy crop. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of MlGT43 genes was ubiquitously detected in the tissues examined. In-situ hybridization demonstrated that MlGT43A-B and MlGT43F-G were specifically expressed in sclerenchyma, while MlGT43C-E were expressed in both sclerenchyma and parenchyma. All seven MlGT43 proteins were localized to Golgi apparatus. Overexpression of MlGT43A-E but not MlGT43F and MlGT43G in Arabidopsis irx9 fully or partially rescued the mutant defects, including morphological changes, collapsed xylem and increased xylan contents, whereas overexpression of MlGT43F and MlGT43G but not MlGT43A-E complemented the defects of irx14, indicating that MlGT43A-E are functional orthologues of IRX9, while MlGT43F and MlGT43G are functional orthologues of IRX14. However, overexpression of all seven MlGT43 genes could not rescue the mucilage defects of irx14 seeds. Furthermore, transient transactivation analyses of MlGT43A-E reporters demonstrated that MlGT43A and MlGT43B but not MlGT43C-E were differentially activated by MlSND1, MlMYB46 or MlVND7. The results demonstrated that all seven MlGT43s are functionally conserved in xylan biosynthesis during secondary cell wall formation but diversify in seed coat mucilage xylan biosynthesis. The results obtained provide deeper insight into xylan biosynthesis in grass, which lay the foundation for genetic modification of grass cell wall components and structure to better suit for next

  9. Secondary metabolite toxins and nutrition of plant pathogenic fungi.

    Science.gov (United States)

    Howlett, Barbara J

    2006-08-01

    Fungal pathogens derive nutrition from the plants they invade. Some fungi can subvert plant defence responses such as programmed cell death to provide nutrition for their growth and colonisation. Secondary metabolite toxins produced by fungi often play a role in triggering these responses. Knowledge of the biosynthesis of these toxins, and the availability of fungal genome sequences and gene disruption techniques, allows the development of tools for experiments aimed at discovering the role of such toxins in triggering plant cell death and plant disease.

  10. Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    Reischl, Udo; Youssef, Mohammad T.; Kilwinski, Jochen; Lehn, Norbert; Zhang, Wen Lan; Karch, Helge; Strockbine, Nancy A.

    2002-01-01

    PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx1 and stx2) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx1, eae, and E-hly genes and 96 and 100%, respectively, for the stx2 gene. No stx2 genes were detected from 10 stx2f-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains. PMID:12089277

  11. The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans.

    Science.gov (United States)

    Keller, Sebastian; Treder, Aaron; von Reuss, Stephan H; Escalante-Semerena, Jorge C; Schubert, Torsten

    2016-08-15

    The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which, in comparison to other cobamides, e.g., cobalamin and pseudo-B12, lacks the methyl group in the linker moiety of the nucleotide loop. In this study, the protein SMUL_1544 was shown to be responsible for the formation of the unusual linker moiety, which is most probably derived from ethanolamine-phosphate (EA-P) as the precursor. The product of the SMUL_1544 gene successfully complemented a Salmonella enterica ΔcobD mutant. The cobD gene encodes an l-threonine-O-3-phosphate (l-Thr-P) decarboxylase responsible for the synthesis of (R)-1-aminopropan-2-ol O-2-phosphate (AP-P), required specifically for cobamide biosynthesis. When SMUL_1544 was produced in the heterologous host lacking CobD, norpseudo-B12 was formed, which pointed toward the formation of EA-P rather than AP-P. Guided cobamide biosynthesis experiments with minimal medium supplemented with l-Thr-P supported cobamide biosynthesis in S. enterica producing SMUL_1544 or S. multivorans Under these conditions, both microorganisms synthesized pseudo-B12 This observation indicated a flexibility in the SMUL_1544 substrate spectrum. From the formation of catalytically active PCE reductive dehalogenase (PceA) in S. multivorans cells producing pseudo-B12, a compatibility of the respiratory enzyme with the cofactor was deduced. This result might indicate a structural flexibility of PceA in cobamide binding. Feeding of l-[3-(13)C]serine to cultures of S. multivorans resulted in isotope labeling of the norpseudo-B12 linker moiety, which strongly supports the hypothesis of EA-P formation from l-serine-O-phosphate (l-Ser-P) in this organism. The identification of the gene product SMUL_1544 as a putative l-Ser-P decarboxylase involved in norcobamide biosynthesis in S. multivorans adds a novel module to the assembly line of cobamides (complete corrinoids) in prokaryotes. Selected cobamide

  12. [Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense strains from serogroups I and II].

    Science.gov (United States)

    Petrova, L P; Prilipov, A G; Katsy, E I

    2017-01-01

    It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

  13. Staphylococci isolated from ready-to-eat meat - Identification, antibiotic resistance and toxin gene profile.

    Science.gov (United States)

    Fijałkowski, Karol; Peitler, Dorota; Karakulska, Jolanta

    2016-12-05

    The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumer's specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of

  14. De novo Assembly of the Camellia nitidissima Transcriptome Reveals Key Genes of Flower Pigment Biosynthesis

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    Xingwen Zhou

    2017-09-01

    Full Text Available The golden camellia, Camellia nitidissima Chi., is a well-known ornamental plant that is known as “the queen of camellias” because of its golden yellow flowers. The principal pigments in the flowers are carotenoids and flavonol glycosides. Understanding the biosynthesis of the golden color and its regulation is important in camellia breeding. To obtain a comprehensive understanding of flower development in C. nitidissima, a number of cDNA libraries were independently constructed during flower development. Using the Illumina Hiseq2500 platform, approximately 71.8 million raw reads (about 10.8 gigabase pairs were obtained and assembled into 583,194 transcripts and 466, 594 unigenes. A differentially expressed genes (DEGs and co-expression network was constructed to identify unigenes correlated with flower color. The analysis of DEGs and co-expressed network involved in the carotenoid pathway indicated that the biosynthesis of carotenoids is regulated mainly at the transcript level and that phytoene synthase (PSY, β -carotene 3-hydroxylase (CrtZ, and capsanthin synthase (CCS1 exert synergistic effects in carotenoid biosynthesis. The analysis of DEGs and co-expressed network involved in the flavonoid pathway indicated that chalcone synthase (CHS, naringenin 3-dioxygenase (F3H, leucoanthocyanidin dioxygenase(ANS, and flavonol synthase (FLS play critical roles in regulating the formation of flavonols and anthocyanidin. Based on the gene expression analysis of the carotenoid and flavonoid pathways, and determinations of the pigments, we speculate that the high expression of PSY and CrtZ ensures the production of adequate levels of carotenoids, while the expression of CHS, FLS ensures the production of flavonols. The golden yellow color is then the result of the accumulation of carotenoids and flavonol glucosides in the petals. This study of the mechanism of color formation in golden camellia points the way to breeding strategies that exploit gene

  15. Profiling of the Major Phenolic Compounds and Their Biosynthesis Genes in Sophora flavescens Aiton

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    Jeongyeo Lee

    2018-01-01

    Full Text Available Sophorae Radix (Sophora flavescens Aiton has long been used in traditional medicine in East Asia due to the various biological activities of its secondary metabolites. Endogenous contents of phenolic compounds (phenolic acid, flavonol, and isoflavone and the main bioactive compounds of Sophorae Radix were analyzed based on the qualitative HPLC analysis and evaluated in different organs and at different developmental stages. In total, 11 compounds were detected, and the composition of the roots and aerial parts (leaves, stems, and flowers was significantly different. trans-Cinnamic acid and p-coumaric acid were observed only in the aerial parts. Large amounts of rutin and maackiain were detected in the roots. Four phenolic acid compounds (benzoic acid, caffeic acid, ferulic acid, and chlorogenic acid and four flavonol compounds (kaempferol, catechin hydrate, epicatechin, and rutin were higher in aerial parts than in roots. To identify putative genes involved in phenolic compounds biosynthesis, a total of 41 transcripts were investigated. Expression patterns of these selected genes, as well as the multiple isoforms for the genes, varied by organ and developmental stage, implying that they are involved in the biosynthesis of various phenolic compounds both spatially and temporally.

  16. Isolation and characterization of Carotenoid biosynthesis genes from Pantoea agglomerans pv. millettiae Wist 801

    International Nuclear Information System (INIS)

    Hirata, R.; Tominaga, H.; Kamiunten, H.

    2007-01-01

    Pantoea agglomerans pv. millettiae produces a yellow pigment and is the causal agent of wisteria stem galls. We cloned and sequenced a 6.8-kb DNA fragment containing the yellow pigment gene. Analysis of the nucleotide sequence revealed that this clone contains five open reading frames (ORFs) transcribed in the same direction, and one terminal ORF transcribed in the opposite orientation. Comparison of the nucleotide and predicted amino acid sequences of these ORFs showed that they correspond to the carotenoid biosynthesis (crt) genes crtE, -X, -Y, -I, -B and -Z, respectively, in P. ananatis pv. uredovora 20D3, and P. agglomerans strains Eho 10 and Eho 13. It was suggested that the carotenoid biosynthesis pathway of P. agglomerans pv. millettiae is basically identical to that of the three other Pantoea strains. When the crt genes from P. agglomerans pv. millettiae were expressed in E. coli WP2, this bacterium also displayed a yellow phenotype. The carotenoid pigments are known for their ability to protect various organisms against UV-induced damage, however, there were no significant differences in mutagenesis and survival after UV irradiation between yellow-pigmented and non-pigmented E. coli WP2 strains

  17. [Opine biosynthesis and catabolism genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes].

    Science.gov (United States)

    Vladimirov, I A; Matveeva, T V; Lutova, L A

    2015-02-01

    Agrobacterium is a genus of soil bacteria with the ability to transform plant cells by a T-DNA-sequence located on the pTi/pRi- plasmid containing a set of genes expressed in plant cells. Expression of these genes leads to a proliferation of transformed cells, with the subsequent formation of tumors or growths of roots and the synthesis of opines--products of the condensation of amino acids with ketoacids or sugars used by Agrobacteria as a source of carbon and nitrogen. In this review, we systematized the information about most common opines in plant--Agrobacterium systems and their biosynthesis and catabolism genes, as well as the role of opines in the interaction of pathogenic Agrobacterium with plants and with other Agrobacterium strains, including the genetic consequences of such interactions.

  18. Effects of auxins on sorgoleone accumulation and genes for sorgoleone biosynthesis in sorghum roots.

    Science.gov (United States)

    Uddin, Md Romij; Park, Woo Tae; Kim, Yong Kyoung; Pyon, Jong Yeong; Park, Sang-Un

    2011-12-28

    Sorgoleone is a major component of the hydrophobic root exudate of Sorghum bicolor and is of particular interest to plant chemical ecology as well as agriculture. Sorgoleone was evaluated in this study to observe the expression levels of genes involved in its biosynthesis in response to auxins. Sorgoleone content varied widely according to the duration of application and the concentrations of the auxins. When the application time was increased, the sorgoleone content increased accordingly for all concentrations of IBA (1, 3, and 5 mg/L) and at 1 mg/L for both IAA and NAA. In this study, five different sorgoleone biosynthetic genes were observed, namely DES2, DES3, ARS1, ARS2, and OMT3, which are upregulated in response to IAA, IBA, and NAA. Transcript accumulation was apparent for all genes, but particularly for DES2, which increased up to 475-fold and 180-fold following 72 h exposure to NAA and IBA, respectively, compared to no treatment.

  19. Relationships among superantigen toxin gene profiles, genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis.

    Science.gov (United States)

    Wang, Dong; Zhang, Limei; Yong, Changfu; Shen, Mingliang; Ali, Tariq; Shahid, Muhammad; Han, Kun; Zhou, Xuezhang; Han, Bo

    2017-06-01

    Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes

  20. Functional Characterization of 4´OMT and 7OMT Genes in BIA Biosynthesis

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    Tugba eGurkok

    2016-02-01

    Full Text Available Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L., the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4´-O-methyltransferase (4´OMT and reticuline 7-O-methyltransferase (7OMT genes were subjected tomanipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem and capsule tissues accordingly. Suppression of 4´OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4´OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4´OMT and 7OMT were observed upon manipulation of 4´OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4´OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.

  1. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway and their regulation during reproduction

    Science.gov (United States)

    Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

  2. Genetic Profiling of the Isoprenoid and Sterol Biosynthesis Pathway Genes of Trypanosoma cruzi

    Science.gov (United States)

    Cosentino, Raúl O.; Agüero, Fernán

    2014-01-01

    In Trypanosoma cruzi the isoprenoid and sterol biosynthesis pathways are validated targets for chemotherapeutic intervention. In this work we present a study of the genetic diversity observed in genes from these pathways. Using a number of bioinformatic strategies, we first identified genes that were missing and/or were truncated in the T. cruzi genome. Based on this analysis we obtained the complete sequence of the ortholog of the yeast ERG26 gene and identified a non-orthologous homolog of the yeast ERG25 gene (sterol methyl oxidase, SMO), and we propose that the orthologs of ERG25 have been lost in trypanosomes (but not in Leishmanias). Next, starting from a set of 16 T. cruzi strains representative of all extant evolutionary lineages, we amplified and sequenced ∼24 Kbp from 22 genes, identifying a total of 975 SNPs or fixed differences, of which 28% represent non-synonymous changes. We observed genes with a density of substitutions ranging from those close to the average (∼2.5/100 bp) to some showing a high number of changes (11.4/100 bp, for the putative lathosterol oxidase gene). All the genes of the pathway are under apparent purifying selection, but genes coding for the sterol C14-demethylase, the HMG-CoA synthase, and the HMG-CoA reductase have the lowest density of missense SNPs in the panel. Other genes (TcPMK, TcSMO-like) have a relatively high density of non-synonymous SNPs (2.5 and 1.9 every 100 bp, respectively). However, none of the non-synonymous changes identified affect a catalytic or ligand binding site residue. A comparative analysis of the corresponding genes from African trypanosomes and Leishmania shows similar levels of apparent selection for each gene. This information will be essential for future drug development studies focused on this pathway. PMID:24828104

  3. The Lethal Toxin from Australian Funnel-Web Spiders Is Encoded by an Intronless Gene

    Science.gov (United States)

    Pineda, Sandy Steffany; Wilson, David; Mattick, John S.; King, Glenn F.

    2012-01-01

    Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome. PMID:22928020

  4. The lethal toxin from Australian funnel-web spiders is encoded by an intronless gene.

    Directory of Open Access Journals (Sweden)

    Sandy Steffany Pineda

    Full Text Available Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome.

  5. Detection of methicillin resistant and toxin-associated genes in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Cajethan Ezeamagu

    2018-03-01

    Full Text Available Methicillin-resistant Staphylococcus aureus is a problem in both healthcare institutions and community settings. This is due to its multi-drug resistant challenges. Hence, this study assessed the prevalence of methicillin resistant gene (mecA, exfoliative toxin (eta and etb and toxic shock syndrome (tsst-1 genes in S. aureus isolated from clinical samples. A total of 120 clinical samples of patients (urine, high vagina swab (HVS, semen, wound swab, sputum and urethral swab from a hospital laboratory were obtained. S. aureus was isolated and then identified with API-staph kit. Antibiotic susceptibility of the isolates was determined by agar diffusion while PCR was used to detect the presence of mecA and toxin-associated genes. Fifty S. aureus isolates were obtained at frequencies of 26(52%, 12(24%, 4(8%, 3(6%, 3(6% and 2(4% from the HVS, urine, semen, wound, sputum and urethral swab samples respectively. All the isolates of S. aureus were resistant to the antibiotics used in this study. MecA, tsst-1, eta and etb were detected in 19(38%, 7(14%, 3(6% and 2(4% of the isolates respectively. The prevalence of MRSA and its resistance pattern observed in this study was a signal that the health-care workers and the general public are at risk.

  6. Homologues of insecticidal toxin complex genes within a genomic island in the marine bacterium Vibrio parahaemolyticus.

    Science.gov (United States)

    Tang, Kathy F J; Lightner, Donald V

    2014-12-01

    Three insecticidal toxin complex (tc)-like genes were identified in Vibrio parahaemolyticus 13-028/A3, which can cause acute hepatopancreatic necrosis disease in penaeid shrimp. The three genes are a tcdA-like gene (7710 bp), predicted to code for a 284-kDa protein; a tcdB-like gene (4272 bp), predicted to code for a 158-kDa protein; and a tccC3-like gene (2916 bp), predicted to encode a 107-kDa protein. All three predicted proteins contain conserved domains that are characteristic of their respective Tc proteins. By RT-PCR, all three tc-like genes were found to be expressed in this bacterium. Through genome walking and the use of PCR to join contigs surrounding these three genes, a genomic island (87 712 bp, named tc-GIvp) was found on chromosome II localized next to the tRNA Gly. The GC content of this island, which is not found in other Vibrio species, is 40%. The tc-GIvp is characterized to have 60 ORFs encoding regulatory or virulence factors. These include a type 6 secretion protein VgrG, EAL domain-containing proteins, fimbriae subunits and assembly proteins, invasin-like proteins, peptidoglycan-binding proteins, and Tc proteins. The tc-GIvp also contains 21 transposase genes, suggesting that it was acquired through horizontal transfer from other organisms. © 2014 Federation of European Microbiological Societies.

  7. Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461.

    Directory of Open Access Journals (Sweden)

    Liang Zhu

    Full Text Available Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB and a β-carotene hydroxylase gene (crtZ located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.

  8. Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461.

    Science.gov (United States)

    Zhu, Liang; Wu, Xuechang; Li, Ou; Qian, Chaodong; Gao, Haichun

    2012-01-01

    Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.

  9. Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

    Science.gov (United States)

    Xu, Zhong; Wang, Yemin; Chater, Keith F.; Ou, Hong-Yu; Xu, H. Howard; Deng, Zixin

    2017-01-01

    ABSTRACT Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement. IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its

  10. Lantibiotics biosynthesis genes and bacteriocinogenic activity of Lactobacillus spp. isolated from raw milk and cheese.

    Science.gov (United States)

    Perin, Luana Martins; Moraes, Paula Mendonça; Silva, Abelardo; Nero, Luís Augusto

    2012-05-01

    Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by 16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or 48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods.

  11. Genome-wide identification of GLABRA3 downstream genes for anthocyanin biosynthesis and trichome formation in Arabidopsis.

    Science.gov (United States)

    Gao, Chenhao; Li, Dong; Jin, Changyu; Duan, Shaowei; Qi, Shuanghui; Liu, Kaige; Wang, Hanchen; Ma, Haoli; Hai, Jiangbo; Chen, Mingxun

    2017-04-01

    GLABRA3 (GL3), a bHLH transcription factor, has previously proved to be involved in anthocyanin biosynthesis and trichome formation in Arabidopsis, however, its downstream targeted genes are still largely unknown. Here, we found that GL3 was widely present in Arabidopsis vegetative and reproductive organs. New downstream targeted genes of GL3 for anthocyanin biosynthesis and trichome formation were identified in young shoots and expanding true leaves by RNA sequencing. GL3-mediated gene expression was tissue specific in the two biological processes. This study provides new clues to further understand the GL3-mediated regulatory network of anthocyanin biosynthesis and trichome formation in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout

    International Nuclear Information System (INIS)

    Sandhu, Navdeep; Vijayan, Mathilakath M.

    2011-01-01

    Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000 nM) for 4 h either in the presence or absence of ACTH (0.5 IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

  13. Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout

    Energy Technology Data Exchange (ETDEWEB)

    Sandhu, Navdeep [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada); Vijayan, Mathilakath M., E-mail: mvijayan@uwaterloo.ca [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada)

    2011-05-15

    Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000 nM) for 4 h either in the presence or absence of ACTH (0.5 IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

  14. Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra; Peck, Matthew L.; Vega-Sánchez, Miguel E.; Williams, Brian; Chiniquy, Dawn M.; Saha, Prasenjit; Pattathil, Sivakumar; Conlin, Brian; Zhu, Lan; Hahn, Michael G.; Willats, William G. T.; Scheller, Henrik V.; Ronald, Pamela C.; Bartley, Laura E.

    2016-08-01

    Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.

  15. A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana

    KAUST Repository

    Meier, Stuart

    2011-05-19

    Background: The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana.Results: A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of

  16. Manipulation of regulatory genes reveals complexity and fidelity in hormaomycin biosynthesis.

    Science.gov (United States)

    Cai, Xiaofeng; Teta, Roberta; Kohlhaas, Christoph; Crüsemann, Max; Ueoka, Reiko; Mangoni, Alfonso; Freeman, Michael F; Piel, Jörn

    2013-06-20

    Hormaomycin (HRM) is a structurally remarkable peptide produced by Streptomyces griseoflavus W-384 that acts as a Streptomyces signaling metabolite and exhibits potent antibiotic activity against coryneform actinomycetes. HRM biosynthetic studies have been hampered by inconsistent and low production. To enhance fermentation titers, the role of its cluster-encoded regulatory genes was investigated. Extra copies of the putative regulators hrmA and hrmB were introduced into the wild-type strain, resulting in an increase of HRM production and its analogs up to 135-fold. For the HrmB overproducer, six bioactive analogs were isolated and characterized. This study demonstrates that HrmA and HrmB are positive regulators in HRM biosynthesis. A third gene, hrmH, was identified as encoding a protein capable of shifting the metabolic profile of HRM and its derivatives. Its manipulation resulted in the generation of an additional HRM analog. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Identification of Candidate Genes and Biosynthesis Pathways Related to Fertility Conversion by Wheat KTM3315A Transcriptome Profiling

    Directory of Open Access Journals (Sweden)

    Lingli Zhang

    2017-04-01

    Full Text Available The Aegilops kotschyi thermo-sensitive cytoplasmic male sterility (K-TCMS system may facilitate hybrid wheat (Triticum aestivum L. seed multiplication and production. The K-TCMS line is completely male sterile during the normal wheat-growing season, whereas its fertility can be restored in a high-temperature environment. To elucidate the molecular mechanisms responsible for male sterility/fertility conversion and candidate genes involved with pollen development in K-TCMS, we employed RNA-seq to sequence the transcriptomes of anthers from K-TCMS line KTM3315A during development under sterile and fertile conditions. We identified 16840 differentially expressed genes (DEGs in different stages including15157 known genes (15135 nuclear genes and 22 plasmagenes and 1683 novel genes. Bioinformatics analysis identified possible metabolic pathways involved with fertility based on KEGG pathway enrichment of the DEGs expressed in fertile and sterile plants. We found that most of the genes encoding key enzyme in the phenylpropanoid biosynthesis and jasmonate biosynthesis pathways were significant upregulated in uninucleate, binuclate or trinucleate stage, which both interact with MYB transcription factors, and that link between all play essential roles in fertility conversion. The relevant DEGs were verified by quantitative RT-PCR. Thus, we suggested that phenylpropanoid biosynthesis and jasmonate biosynthesis pathways were involved in fertility conversion of K-TCMS wheat. This will provide a new perspective and an effective foundation for the research of molecular mechanisms of fertility conversion of CMS wheat. Fertility conversion mechanism in thermo-sensitive cytoplasmic male sterile/fertile wheat involves the phenylpropanoid biosynthesis pathway, jasmonate biosynthesis pathway, and MYB transcription factors.

  18. The Ethylene Biosynthesis Gene CitACS4 Regulates Monoecy/Andromonoecy in Watermelon (Citrullus lanatus).

    Science.gov (United States)

    Manzano, Susana; Aguado, Encarnación; Martínez, Cecilia; Megías, Zoraida; García, Alicia; Jamilena, Manuel

    2016-01-01

    Monoecious and andromonoecious cultivars of watermelon are characterised by the production of male and female flower or male and hermaphrodite flowers, respectively. The segregation analysis in the offspring of crosses between monoecious and andromonoecious lines has demonstrated that this trait is controlled by a single gene pair, being the monoecious allele M semi-dominant to the andromonoecious allele A. The two studied F1 hybrids (MA) had a predominantly monoecious phenotype since both produced not only female flowers, but also bisexual flowers with incomplete stamens, and hermaphrodite flowers with pollen. Given that in other cucurbit species andromonoecy is conferred by mutations in the ethylene biosynthesis genes CmACS7, CsACS2 and CpACS27A we have cloned and characterised CitACS4, the watermelon gene showing the highest similarity with the formers. CitACS4 encoded for a type ACS type III enzyme that is predominantly expressed in pistillate flowers of watermelon. In the andromonoecious line we have detected a missense mutation in a very conserved residue of CitACS4 (C364W) that cosegregates with the andromonoecious phenotype in two independent F2 populations, concomitantly with a reduction in ethylene production in the floral buds that will develop as hermaphrodite flowers. The gene does not however co-segregates with other sex expression traits regulated by ethylene in this species, including pistillate flowering transition and the number of pistillate flowers per plant. These data indicate that CitAC4 is likely to be involved in the biosynthesis of the ethylene required for stamen arrest during the development of female flowers. The C364W mutation would reduce the production of ethylene in pistillate floral buds, promoting the conversion of female into hermaphrodite flowers, and therefore of monoecy into andromonoecy.

  19. High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea.

    Science.gov (United States)

    Gao, Yan; Yu, Ren-Cheng; Murray, Shauna A; Chen, Jian-Hua; Kang, Zhen-Jun; Zhang, Qing-Chun; Kong, Fan-Zhou; Zhou, Ming-Jiang

    2015-10-01

    The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r=0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r=0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. The essential function of B. subtilis RNase III is to silence foreign toxin genes.

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    Sylvain Durand

    Full Text Available RNase III-related enzymes play key roles in cleaving double-stranded RNA in many biological systems. Among the best-known are RNase III itself, involved in ribosomal RNA maturation and mRNA turnover in bacteria, and Drosha and Dicer, which play critical roles in the production of micro (mi-RNAs and small interfering (si-RNAs in eukaryotes. Although RNase III has important cellular functions in bacteria, its gene is generally not essential, with the remarkable exception of that of Bacillus subtilis. Here we show that the essential role of RNase III in this organism is to protect it from the expression of toxin genes borne by two prophages, Skin and SPβ, through antisense RNA. Thus, while a growing number of organisms that use RNase III or its homologs as part of a viral defense mechanism, B. subtilis requires RNase III for viral accommodation to the point where the presence of the enzyme is essential for cell survival. We identify txpA and yonT as the two toxin-encoding mRNAs of Skin and SPβ that are sensitive to RNase III. We further explore the mechanism of RNase III-mediated decay of the txpA mRNA when paired to its antisense RNA RatA, both in vivo and in vitro.

  1. PCR detection of seven virulence and toxin genes of Campylobacter jejuni and Campylobacter coli isolates from Danish pigs and cattle and cytolethal distending toxin production of the isolates

    DEFF Research Database (Denmark)

    Bang, Dang Duong; Nielsen, E.M.; Scheutz, F.

    2003-01-01

    Aims: To study the prevalence of seven virulence and toxin genes, and cytolethal distending toxin (CDT) production of Campylobacter jejuni and C. coli isolates from Danish pigs and cattle. Methods and Results: The presence of the cadF, ceuE, virB11, flaA, cdtA, cdtB, cdtC and the cdt gene cluster...... among 40 C. jejuni and C. coli isolates was detected by polymerase chain reaction. The CDT production of the isolates was determined on Vero, colon 205 and chicken embryo cells. The cadF, flaA, ceuE and cdtB genes were detected from 100% of the isolates. The cdtA and cdtC genes were found in 95.0 and 90.......0% of the isolates, respectively. The cdt gene cluster was detected in 82.5% isolates. Only 7.5% of the isolates were positive for virB11. Ninety-five per cent of the isolates produced CDT in Vero and colon 205 cell assays, and 90% of the isolates produced CDT in chicken embryo cell assays. Conclusions: High...

  2. Transfer of toxin genes to alternate bacterial hosts for mosquito control

    Directory of Open Access Journals (Sweden)

    Sergio Orduz

    1995-02-01

    Full Text Available Mosquitoes are vector of serious human and animal diseases, such as malaria, dengue, yellow fever, among others. The use of biological control agents has provide an environmentally safe and highly specific alternative to the use of chemical insecticides in the control of vector borne diseases. Bacillus thuringiensis and B. sphaericus produce toxic proteins to mosquito larvae. Great progress has been made on the biochemical and molecular characterization of such proteins and the genes encoding them. Nevertheless, the low residuality of these biological insecticides is one of the major drawbacks. This article present some interesting aspects of the mosquito larvae feeding habits and review the attempts that have been made to genetically engineer microorganisms that while are used by mosquito larvae as a food source should express the Bacillus toxin genes in order to improve the residuality and stability in the mosquito breeding ponds.

  3. Transcriptomic landscape of Dendrobium huoshanense and its genes related to polysaccharide biosynthesis

    Directory of Open Access Journals (Sweden)

    Rongchun Han

    2018-03-01

    Full Text Available Dendrobium huoshanense has long been used to treat various diseases in oriental medicine. In order to study its gene expression profile, transcripts involved in the biosynthesis of precursors of polysaccharides, as well as mechanisms underlining morphological differences between wild and cultivated plants, three organs of both wild type and cultivated D. huoshanense were collected and sequenced by Illumina HiSeq4000 platform, yielding 919,409,540 raw reads in FASTQ format. After Trinity de novo assembly and quality control, 241,242 nonredundant contigs with the average length of 967.5 bp were generated. qRT-PCR experiment on the selected transcripts showed the transcriptomic data were reliable. BLASTx was conducted against NR, SwissProt, String, Pfam, and KEGG. Gene ontology annotation revealed more than 40,000 contigs assigned to catalytic activity and metabolic process, suggesting its dynamic physiological activities. By searching KEGG pathway, six genes potentially involved in mannose biosynthetic pathway were retrieved. Gene expression analysis for stems between wild and cultivated D. huoshanense resulted in 956 genes differentially expressed. Simple sequence repeats (SSRs analysis revealed 143 SSRs with the unit size of 4 and 3,437 SSRs the size of 3. The obtained SSRs are the potential molecular markers for discriminating distinct cultivars of D. huoshanense.

  4. Identification of substituent groups and related genes involved in salecan biosynthesis in Agrobacterium sp. ZX09.

    Science.gov (United States)

    Xu, Linxiang; Cheng, Rui; Li, Jing; Wang, Yang; Zhu, Bin; Ma, Shihong; Zhang, Weiming; Dong, Wei; Wang, Shiming; Zhang, Jianfa

    2017-01-01

    Salecan, a soluble β-1,3-D-glucan produced by a salt-tolerant strain Agrobacterium sp. ZX09, has been the subject of considerable interest in recent years because of its multiple bioactivities and unusual rheological properties in solution. In this study, both succinyl and pyruvyl substituent groups on salecan were identified by an enzymatic hydrolysis following nuclear magnetic resonance (NMR), HPLC, and MS analysis. The putative succinyltransferase gene (sleA) and pyruvyltransferase gene (sleV) were determined and cloned. Disruption of the sleA gene resulted in the absence of succinyl substituent groups on salecan. This defect could be complemented by expressing the sleA cloned in a plasmid. Thus, the sleA and sleV genes located in a 19.6-kb gene cluster may be involved in salecan biosynthesis. Despite the lack of succinyl substituents, the molecular mass of salecan generated by the sleA mutant did not substantially differ from that generated by the wild-type strain. Loss of succinyl substituents on salecan changed its rheological characteristics, especially a decrease in intrinsic viscosity.

  5. Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes.

    Science.gov (United States)

    de Crécy-Lagard, V; Blanc, V; Gil, P; Naudin, L; Lorenzon, S; Famechon, A; Bamas-Jacques, N; Crouzet, J; Thibaut, D

    1997-01-01

    Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed. PMID:9006024

  6. Evolutionary acquisition and loss of saxitoxin biosynthesis in dinoflagellates: the second "core" gene, sxtG.

    Science.gov (United States)

    Orr, Russell J S; Stüken, Anke; Murray, Shauna A; Jakobsen, Kjetill S

    2013-04-01

    Saxitoxin and its derivatives are potent neurotoxins produced by several cyanobacteria and dinoflagellate species. SxtA is the initial enzyme in the biosynthesis of saxitoxin. The dinoflagellate full mRNA and partial genomic sequences have previously been characterized, and it appears that sxtA originated in dinoflagellates through a horizontal gene transfer from a bacterium. So far, little is known about the remaining genes involved in this pathway in dinoflagellates. Here we characterize sxtG, an amidinotransferase enzyme gene that putatively encodes the second step in saxitoxin biosynthesis. In this study, the entire sxtG transcripts from Alexandrium fundyense CCMP1719 and Alexandrium minutum CCMP113 were amplified and sequenced. The transcripts contained typical dinoflagellate spliced leader sequences and eukaryotic poly(A) tails. In addition, partial sxtG transcript fragments were amplified from four additional Alexandrium species and Gymnodinium catenatum. The phylogenetic inference of dinoflagellate sxtG, congruent with sxtA, revealed a bacterial origin. However, it is not known if sxtG was acquired independently of sxtA. Amplification and sequencing of the corresponding genomic sxtG region revealed noncanonical introns. These introns show a high interspecies and low intraspecies variance, suggesting multiple independent acquisitions and losses. Unlike sxtA, sxtG was also amplified from Alexandrium species not known to synthesize saxitoxin. However, amplification was not observed for 22 non-saxitoxin-producing dinoflagellate species other than those of the genus Alexandrium or G. catenatum. This result strengthens our hypothesis that saxitoxin synthesis has been secondarily lost in conjunction with sxtA for some descendant species.

  7. Expression of Genes Related to Phenylpropanoid Biosynthesis in Different Organs of Ixeris dentata var. albiflora.

    Science.gov (United States)

    Lee, Sang-Hoon; Park, Yun-Ji; Park, Sang Un; Lee, Sang-Won; Kim, Seong-Cheol; Jung, Chan-Sik; Jang, Jae-Ki; Hur, Yoonkang; Kim, Yeon Bok

    2017-05-30

    Members of the genus Ixeris have long been used in traditional medicines as stomachics, sedatives, and diuretics. Phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate: coenzyme-A (CoA) ligase (4CL), chalcone synthase (CHS), and dihydroflavonol 4-reductase (DFR) are important enzymes in the phenylpropanoid pathway. In this study, we analyzed seven genes from Ixeris dentata var. albiflora that are involved in phenylpropanoid biosynthesis, using an Illumina/Solexa HiSeq 2000 platform. The amino acid sequence alignments for IdPAL s, IdC4H, Id4CL s, IdCHS , and IdDFR showed high identity to sequences from other plants. We also investigated transcript levels using quantitative real-time PCR, and analyzed the accumulation of phenylpropanoids in different organs of I. dentata var. albiflora using high-performance liquid chromatography. The transcript levels of IdC4H, Id4CL1 , IdCHS , and IdDFR were highest in the leaf. The catechin, chlorogenic acid, ferulic acid, and quercetin contents were also highest in the leaf. We suggest that expression of IdC4H, Id4CL1 , IdCHS , and IdDFR is associated with the accumulation of phenylpropanoids. Our results may provide baseline information for elucidating the mechanism of phenylpropanoid biosynthesis in different organs of I. dentata var. albiflora .

  8. The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis.

    Science.gov (United States)

    van Zeijl, Arjan; Liu, Wei; Xiao, Ting Ting; Kohlen, Wouter; Yang, Wei-Cai; Bisseling, Ton; Geurts, René

    2015-10-26

    Strigolactones are a class of plant hormones whose biosynthesis is activated in response to phosphate starvation. This involves several enzymes, including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both proteins are essential for rhizobium induced root nodule formation in legumes. In line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is co-opted in rhizobium symbiosis. We provide evidence that MtD27 is involved in strigolactone biosynthesis in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies revealed that this gene is also highly expressed in nodule primordia and subsequently becomes restricted to the meristem and distal infection zone of a mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8. Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner. Symbiotic expression of MtD27 requires components of the symbiosis signaling pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in contrast to MtD27 expression upon phosphate starvation, which only requires MtNSP1 and MtNSP2. Our data show that the phosphate-starvation responsive strigolactone biosynthesis gene MtD27 is also rapidly induced by rhizobium LCO signals in an MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone of mature nodules.

  9. Expression of toxic shock syndrome toxin-1 gene of Staphylococcus aureus in milk: Proof of concept

    Directory of Open Access Journals (Sweden)

    Milijana Babić

    2018-01-01

    Full Text Available The aim of this study was to assess the expression of a toxic shock syndrome toxin-1 (TSST-1 gene of Staphylococcus aureus in different types of milk, depending on inadequate temperature and storage conditions. Pasteurized and UHT milk were inoculated with monotoxic TSST-1 strain of S. aureus and growth kinetics was determined by the drop plate method using Baird-Parker agar medium in accordance with EN ISO 6888-1. The patterns of gene regulation were detected by quantitative reverse transcriptase PCR. Relative quantification method for statistical significance testing was used to detect significant gene expression responses. The results of experiments showed the dependence of the growth rate and consequent up-regulation of TSST-1 encoding gene on storage time-temperature and type of milk. In contaminated pasteurized and UHT milk kept at both 15 °C and 22 °C S. aureus significantly increased its virulent potential over time. This effect was a bit more emphasized in UHT milk serving as a “proof of concept”. Possible explanation could be a presence of lactic acid bacteria in pasteurized milk which is known to have down-regulatory effect on TSST-1 gene. Maintenance of the milk storage temperature below 8 °C and employment of microbiological control measures in hygienic practices, from milk producer through retailer and on to the consumer is of utmost importance to decrease risk of non-emetic staphylococcal poisoning.

  10. Differential selection on carotenoid biosynthesis genes as a function of gene position in the metabolic pathway: a study on the carrot and dicots.

    Directory of Open Access Journals (Sweden)

    Jérémy Clotault

    Full Text Available Selection of genes involved in metabolic pathways could target them differently depending on the position of genes in the pathway and on their role in controlling metabolic fluxes. This hypothesis was tested in the carotenoid biosynthesis pathway using population genetics and phylogenetics.Evolutionary rates of seven genes distributed along the carotenoid biosynthesis pathway, IPI, PDS, CRTISO, LCYB, LCYE, CHXE and ZEP, were compared in seven dicot taxa. A survey of deviations from neutrality expectations at these genes was also undertaken in cultivated carrot (Daucus carota subsp. sativus, a species that has been intensely bred for carotenoid pattern diversification in its root during its cultivation history. Parts of sequences of these genes were obtained from 46 individuals representing a wide diversity of cultivated carrots. Downstream genes exhibited higher deviations from neutral expectations than upstream genes. Comparisons of synonymous and nonsynonymous substitution rates between genes among dicots revealed greater constraints on upstream genes than on downstream genes. An excess of intermediate frequency polymorphisms, high nucleotide diversity and/or high differentiation of CRTISO, LCYB1 and LCYE in cultivated carrot suggest that balancing selection may have targeted genes acting centrally in the pathway.Our results are consistent with relaxed constraints on downstream genes and selection targeting the central enzymes of the carotenoid biosynthesis pathway during carrot breeding history.

  11. The effect of nutrition pattern alteration on Chlorella pyrenoidosa growth, lipid biosynthesis-related gene transcription.

    Science.gov (United States)

    Fan, Jianhua; Cui, Yanbin; Zhou, Yang; Wan, Minxi; Wang, Weiliang; Xie, Jingli; Li, Yuanguang

    2014-07-01

    Heterotrophy to photoautotrophy transition leads to the accumulation of lipids in Chlorella, which has potential to produce both healthy food and biofuels. Therefore, it is of key interest to study the metabolism shift and gene expression changes that influenced by the transition. Both total and neutral lipids contents were increased rapidly within 48 h after the switch to light environment, from 24.5% and 18.0% to 35.3% and 27.4%, respectively, along with the sharp decline of starch from 42.3% to 10.4% during 24h photoinduction phase. By analyzing the correlation between lipid content and gene expression, results revealed several genes viz. me g3137, me g6562, pepc g6833, dgat g3280 and dgat g7566, which encode corresponding enzymes in the de novo lipid biosynthesis pathway, are highly related to lipid accumulation and might be exploited as target genes for genetic modification. These results represented the feasibility of lipid production through trophic converting cultivation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. The Phosducin-Like Protein PhnA Is Required for Gβγ-Mediated Signaling for Vegetative Growth, Developmental Control, and Toxin Biosynthesis in Aspergillus nidulans

    Science.gov (United States)

    Seo, Jeong-Ah; Yu, Jae-Hyuk

    2006-01-01

    Phosducin or phosducin-like protein (PhLP) is a positive regulator of Gβγ activity. The Gβ (SfaD) and Gγ (GpgA) subunits function in vegetative growth and developmental control in the model filamentous fungus Aspergillus nidulans. To better understand the nature of Gβγ-mediated signaling, phnA, encoding an A. nidulans PhLP, has been studied. Deletion of phnA resulted in phenotypes almost identical to those caused by deletion of sfaD, i.e., reduced biomass, asexual sporulation in liquid submerged culture, and defective fruiting body formation, suggesting that PhnA is necessary for Gβ function. The requirement for the RGS protein FlbA in asexual sporulation could be bypassed by the ΔphnA mutation, indicating that PhnA functions in FlbA-controlled vegetative growth signaling, primarily mediated by the heterotrimeric G protein composed of FadA (Gα), SfaD, and GpgA. However, whereas deletion of fadA restored both asexual sporulation and the production of sterigmatocystin (ST), deletion of sfaD, gpgA, or phnA failed to restore ST production in the ΔflbA mutant. Further studies revealed that SfaD, GpgA, and PhnA are necessary for the expression of aflR, encoding the transcriptional activator for the ST biosynthetic genes, and subsequent ST biosynthesis. Overexpression of aflR bypassed the need for SfaD in ST production, indicating that the results of SfaD-mediated signaling may include transcriptional activation of aflR. Potential differential roles of FadA, Gβγ, and FlbA in controlling ST biosynthesis are further discussed. PMID:16467480

  13. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  14. [Expression of the genes for lysine biosynthesis of Bacillus subtilis in Escherichia coli cells].

    Science.gov (United States)

    Shevchenko, T N; Okunev, O V; Aleksieva, Z M; Maliuta, S S

    1984-01-01

    Hybrid plasmids pLRS33 and pLRB4 containing Bac. subtilis genes coding lysin biosynthesis were subjected to genetical analysis. It is shown that after pLRS33- and pLRB4- transformation of E. coli strains, auxotrophic relative to lysin and diaminopimelic acid, there occurs complementation of dapA, dapB, dapC, dapD, dapE, lysA mutations by plasmid pLRS33 and of dapC, dapB, lysA mutations by plasmid pLRB4. The plasmids are studied for their influence on the level of lysin and its precurror synthesis in E. coli strains.

  15. Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes.

    Science.gov (United States)

    Itkin, M; Heinig, U; Tzfadia, O; Bhide, A J; Shinde, B; Cardenas, P D; Bocobza, S E; Unger, T; Malitsky, S; Finkers, R; Tikunov, Y; Bovy, A; Chikate, Y; Singh, P; Rogachev, I; Beekwilder, J; Giri, A P; Aharoni, A

    2013-07-12

    Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants--as, for example, potato--are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

  16. Dissection of tomato lycopene biosynthesis through virus-induced gene silencing.

    Science.gov (United States)

    Fantini, Elio; Falcone, Giulia; Frusciante, Sarah; Giliberto, Leonardo; Giuliano, Giovanni

    2013-10-01

    Lycopene biosynthesis in tomato (Solanum lycopersicum) fruits has been proposed to proceed through a poly-cis pathway catalyzed by phytoene synthase (PSY), two desaturases (phytoene desaturase [PDS] and ζ-carotene desaturase [ZDS]), and two cis-trans isomerases (ζ-carotene isomerase [ZISO] and prolycopene isomerase [CrtISO]). The mechanism of action of these enzymes has been studied in Escherichia coli, but a systematic study of their in vivo function is lacking. We studied the function of nine candidate genes (PSY1, PSY2, PSY3, PDS, ZDS, ZISO, CrtISO, CrtISO-Like1, and CrtISO-Like2) using virus-induced gene silencing (VIGS) coupled to high-resolution liquid chromatography coupled with diode array detector and mass spectrometry, which allowed the identification and quantitation of 45 different carotenoid isomers, including linear xanthophylls. The data confirm the confinement of the VIGS signal to the silenced fruits and the similarity of the phenotypes of PSY1- and CrtISO-silenced fruits with those of the yellow flesh and tangerine mutants. Light was able to restore lycopene biosynthesis in ZISO-silenced fruits. Isomeric composition of fruits silenced at different metabolic steps suggested the existence of three functional units, comprising PSY1, PDS/ZISO, and ZDS/CrtISO, and responsible for the synthesis of 15-cis-phytoene, 9,9'-di-cis-ζ-carotene, and all-trans-lycopene, respectively. Silencing of a desaturase (PDS or ZDS) resulted in the induction of the isomerase in the same functional unit (ZISO or CrtISO, respectively). All-trans-ζ-carotene was detectable in nonsilenced fruits, greatly increased in ZDS-silenced ones, and disappeared in CrtISO-Like1-/CrtISO-Like2-silenced ones, suggesting the existence of a metabolic side branch, comprising this compound and initiated by the latter enzymes.

  17. A Malus crabapple chalcone synthase gene, McCHS, regulates red petal color and flavonoid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Deqiang Tai

    Full Text Available Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, 'Royalty' and 'Flame', have dark red and white petals respectively, while the intermediate cultivar 'Radiant' has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in 'Radiant'. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

  18. A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

    National Research Council Canada - National Science Library

    Curiel, David T; Siegal, Gene; Wang, Minghui

    2005-01-01

    ... embodies the requisite properties of efficacy and specificity required for ovarian cancer gene therapy. This approach is based on targeting the delivered anti-cancer gene to tumor via two complimentary approaches...

  19. A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

    National Research Council Canada - National Science Library

    Curiel, David T; Siegal, Gene; Wang, Minghui

    2006-01-01

    ... embodies the requisite properties of efficacy and specificity required for ovarian cancer gene therapy. This approach is based on targeting the delivered anti-cancer gene to tumor via two complimentary approaches...

  20. Transcriptional Profiles of Hybrid Eucalyptus Genotypes with Contrasting Lignin Content Reveal That Monolignol Biosynthesis-related Genes Regulate Wood Composition.

    Science.gov (United States)

    Shinya, Tomotaka; Iwata, Eiji; Nakahama, Katsuhiko; Fukuda, Yujiroh; Hayashi, Kazunori; Nanto, Kazuya; Rosa, Antonio C; Kawaoka, Akiyoshi

    2016-01-01

    Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants.

  1. Transcriptional profiles of hybrid Eucalyptus genotypes with contrasting lignin content reveal that monolignol biosynthesis-related genes regulate wood composition

    Directory of Open Access Journals (Sweden)

    Tomotaka eShinya

    2016-04-01

    Full Text Available Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected three-year old hybrid Eucalyptus (Eucalyptus urophylla x E. grandis genotypes (AM063 and AM380 that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0% and 48.2%, -cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA and sucrose synthase (SUSY were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase (UGP and xyloglucan endotransglucoxylase (XTH than those in AM380. Most monolignol biosynthesis- related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase (PAL, cinnamate-4-hydroxylase (C4H and 4-coumarate-CoA ligase (4CL. Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents

  2. Detection of seven virulence and toxin genes of Campylobacter jejuni isolates from Danish turkeys by PCR and cytolethal distending toxin production of the isolates

    DEFF Research Database (Denmark)

    Bang, Dang Duong; Borck, Birgitte; Nielsen, Eva Møller

    2004-01-01

    A total of 117 Campylobacter jejuni isolates from Danish turkeys were tested for the presence of seven virulence and toxin genes by PCR. One hundred seventeen (100%) isolates were positive for flaA, cadF, and ceuE gene primers. One hundred three (88%) isolates were positive for cdt gene cluster PCR.......7%) in Colon 205 assays, and 109 (93.2%) in chicken embryo cell assays. The CDT titers were determined in Vero cell assays. Of 117 isolates, 50 (42.7%) produced a CDT titer of 1:100, 29 (24.8%) of 1:50, and 27 (23%) of 1:5 to 1:10; 8 (6.8%) produced a CDT titer at undiluted supernatants and 3 (2.6%) produced...

  3. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains.

    Science.gov (United States)

    Tominaga, Mihoko; Lee, Yun-Hae; Hayashi, Risa; Suzuki, Yuji; Yamada, Osamu; Sakamoto, Kazutoshi; Gotoh, Kuniyasu; Akita, Osamu

    2006-01-01

    To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40. Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.

  4. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

    DEFF Research Database (Denmark)

    Jakociune, Dziuginta; Herrero-Fresno, Ana; Jelsbak, Lotte

    2016-01-01

    , di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino......-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis...

  5. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    Directory of Open Access Journals (Sweden)

    Jianrong Wang

    2014-12-01

    Full Text Available Poria cocos (P. cocos has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%. The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP from geranyl diphosphate (GPP and isopentenyl diphosphate (IPP. Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  6. Anthocyanin accumulation and molecular analysis of anthocyanin biosynthesis-associated genes in eggplant (Solanum melongena L.).

    Science.gov (United States)

    Zhang, Yanjie; Hu, Zongli; Chu, Guihua; Huang, Cheng; Tian, Shibing; Zhao, Zhiping; Chen, Guoping

    2014-04-02

    Eggplant (Solanum melongena L.) is an edible fruit vegetable cultivated and consumed worldwide. The purple eggplant is more eye-catching and popular for the health-promoting anthocyanins contained in the fruit skin. Two kinds of anthocyanin were separated and identified from purple cultivar (Zi Chang) by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. To investigate the molecular mechanisms of anthocyanin accumulation in eggplant, the transcripts of anthocyanin biosynthetic and regulatory genes were analyzed in the fruit skin and the flesh of the purple cultivar and the white cultivar (Bai Xue). Compared with the other tissues, SmMYB1 and all anthocyanin biosynthetic genes except PAL were dramatically upregulated in the fruit skin of the purple cultivar. Overexpression of SmMYB1 activated abundant anthocyanin accumulation in the regenerating shoots of eggplant. These results prove that transcriptional activation of SmMYB1 accounts for constitutive upregulation of most anthocyanin biosynthetic genes and the onset of anthocyanin biosynthesis in the purple cultivar.

  7. The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation

    DEFF Research Database (Denmark)

    Harris, Abigail K P; Williamson, Neil R; Slater, Holly

    2004-01-01

    The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respective pig clusters revealed 14 ORFs in S. marcescens ATCC 274...... from Str. coelicolor A3(2) revealed some important differences. A modified scheme for the biosynthesis of prodigiosin, based on the pathway recently suggested for the synthesis of undecylprodigiosin, is proposed. The distribution of the pig cluster within several Serratia sp. isolates is demonstrated...

  8. RAV transcription factors are essential for disease resistance against cassava bacterial blight via activation of melatonin biosynthesis genes.

    Science.gov (United States)

    Wei, Yunxie; Chang, Yanli; Zeng, Hongqiu; Liu, Guoyin; He, Chaozu; Shi, Haitao

    2018-01-01

    With 1 AP2 domain and 1 B3 domain, 7 MeRAVs in apetala2/ethylene response factor (AP2/ERF) gene family have been identified in cassava. However, the in vivo roles of these remain unknown. Gene expression assays showed that the transcripts of MeRAVs were commonly regulated after Xanthomonas axonopodis pv manihotis (Xam) and MeRAVs were specifically located in plant cell nuclei. Through virus-induced gene silencing (VIGS) in cassava, we found that MeRAV1 and MeRAV2 are essential for plant disease resistance against cassava bacterial blight, as shown by the bacterial propagation of Xam in plant leaves. Through VIGS in cassava leaves and overexpression in cassava leave protoplasts, we found that MeRAV1 and MeRAV2 positively regulated melatonin biosynthesis genes and the endogenous melatonin level. Further investigation showed that MeRAV1 and MeRAV2 are direct transcriptional activators of 3 melatonin biosynthesis genes in cassava, as evidenced by chromatin immunoprecipitation-PCR in cassava leaf protoplasts and electrophoretic mobility shift assay. Moreover, cassava melatonin biosynthesis genes also positively regulated plant disease resistance. Taken together, this study identified MeRAV1 and MeRAV2 as common and upstream transcription factors of melatonin synthesis genes in cassava and revealed a model of MeRAV1 and MeRAV2-melatonin biosynthesis genes-melatonin level in plant disease resistance against cassava bacterial blight. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Molecular Link between Leaf Coloration and Gene Expression of Flavonoid and Carotenoid Biosynthesis in Camellia sinensis Cultivar ‘Huangjinya’

    Directory of Open Access Journals (Sweden)

    Lubin Song

    2017-05-01

    Full Text Available ‘Huangjinya’ is an excellent albino tea germplasm cultivated in China because of its bright color and high amino acid content. It is light sensitive, with yellow leaves under intense light while green leaves under weak light. As well, the flavonoid and carotenoid levels increased after moderate shading treatment. However, the mechanism underlying this interesting phenomenon remains unclear. In this study, the transcriptome of ‘Huangjinya’ plants exposed to sunlight and shade were analyzed by high-throughput sequencing followed by de novo assembly. Shading ‘Huangjinya’ made its leaf color turn green. De novo assembly showed that the transcriptome of ‘Huangjinya’ leaves comprises of 127,253 unigenes, with an average length of 914 nt. Among the 81,128 functionally annotated unigenes, 207 differentially expressed genes were identified, including 110 up-regulated and 97 down-regulated genes under moderate shading compared to full light. Gene ontology (GO indicated that the differentially expressed genes are mainly involved in protein and ion binding and oxidoreductase activity. Antioxidation-related pathways, including flavonoid and carotenoid biosynthesis, were highly enriched in these functions. Shading inhibited the expression of flavonoid biosynthesis-associated genes and induced carotenoid biosynthesis-related genes. This would suggest that decreased flavonoid biosynthetic gene expression coincides with increased flavonoids (e.g., catechin content upon moderate shading, while carotenoid levels and biosynthetic gene expression are positively correlated in ‘Huangjinya.’ In conclusion, the leaf color changes in ‘Huangjinya’ are largely determined by the combined effects of flavonoid and carotenoid biosynthesis.

  10. Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

    2015-12-08

    This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

  11. Molecular evolution accompanying functional divergence of duplicated genes along the plant starch biosynthesis pathway.

    Science.gov (United States)

    Nougué, Odrade; Corbi, Jonathan; Ball, Steven G; Manicacci, Domenica; Tenaillon, Maud I

    2014-05-15

    Starch is the main source of carbon storage in the Archaeplastida. The starch biosynthesis pathway (sbp) emerged from cytosolic glycogen metabolism shortly after plastid endosymbiosis and was redirected to the plastid stroma during the green lineage divergence. The SBP is a complex network of genes, most of which are members of large multigene families. While some gene duplications occurred in the Archaeplastida ancestor, most were generated during the sbp redirection process, and the remaining few paralogs were generated through compartmentalization or tissue specialization during the evolution of the land plants. In the present study, we tested models of duplicated gene evolution in order to understand the evolutionary forces that have led to the development of SBP in angiosperms. We combined phylogenetic analyses and tests on the rates of evolution along branches emerging from major duplication events in six gene families encoding sbp enzymes. We found evidence of positive selection along branches following cytosolic or plastidial specialization in two starch phosphorylases and identified numerous residues that exhibited changes in volume, polarity or charge. Starch synthases, branching and debranching enzymes functional specializations were also accompanied by accelerated evolution. However, none of the sites targeted by selection corresponded to known functional domains, catalytic or regulatory. Interestingly, among the 13 duplications tested, 7 exhibited evidence of positive selection in both branches emerging from the duplication, 2 in only one branch, and 4 in none of the branches. The majority of duplications were followed by accelerated evolution targeting specific residues along both branches. This pattern was consistent with the optimization of the two sub-functions originally fulfilled by the ancestral gene before duplication. Our results thereby provide strong support to the so-called "Escape from Adaptive Conflict" (EAC) model. Because none of the

  12. Transcriptome Analysis of Syringa oblata Lindl. Inflorescence Identifies Genes Associated with Pigment Biosynthesis and Scent Metabolism.

    Directory of Open Access Journals (Sweden)

    Jian Zheng

    Full Text Available Syringa oblata Lindl. is a woody ornamental plant with high economic value and characteristics that include early flowering, multiple flower colors, and strong fragrance. Despite a long history of cultivation, the genetics and molecular biology of S. oblata are poorly understood. Transcriptome and expression profiling data are needed to identify genes and to better understand the biological mechanisms of floral pigments and scents in this species. Nine cDNA libraries were obtained from three replicates of three developmental stages: inflorescence with enlarged flower buds not protruded, inflorescence with corolla lobes not displayed, and inflorescence with flowers fully opened and emitting strong fragrance. Using the Illumina RNA-Seq technique, 319,425,972 clean reads were obtained and were assembled into 104,691 final unigenes (average length of 853 bp, 41.75% of which were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 36,967 were assigned to gene ontology categories and 19,956 were assigned to eukaryoticorthologous groups. Using the Kyoto Encyclopedia of Genes and Genomes pathway database, 12,388 unigenes were sorted into 286 pathways. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at different flower stages and that were related to floral pigment biosynthesis and fragrance metabolism. This comprehensive transcriptomic analysis provides fundamental information on the genes and pathways involved in flower secondary metabolism and development in S. oblata, providing a useful database for further research on S. oblata and other plants of genus Syringa.

  13. Identification and characterization of ANAC042, a transcription factor family gene involved in the regulation of camalexin biosynthesis in Arabidopsis.

    Science.gov (United States)

    Saga, Hirohisa; Ogawa, Takumi; Kai, Kosuke; Suzuki, Hideyuki; Ogata, Yoshiyuki; Sakurai, Nozomu; Shibata, Daisuke; Ohta, Daisaku

    2012-05-01

    Camalexin is the major phytoalexin in Arabidopsis. An almost complete set of camalexin biosynthetic enzymes have been elucidated but only limited information is available regarding molecular mechanisms regulating camalexin biosynthesis. Here, we demonstrate that ANAC042, a member of the NAM, ATAF1/2, and CUC2 (NAC) transcription factor family genes, is involved in camalexin biosynthesis induction. T-DNA insertion mutants of ANAC042 failed to accumulate camalexin at the levels achieved in the wild type, and were highly susceptible to Alternaria brassicicola infection. The camalexin biosynthetic genes CYP71A12, CYP71A13, and CYP71B15/PAD3 were not fully induced in the mutants, indicating that the camalexin defects were at least partly a result of reduced expression levels of these P450 genes. β-Glucuronidase (GUS)-reporter assays demonstrated tissue-specific induction of ANAC042 in response to differential pathogen infections. Bacterial flagellin (Flg22) induced ANAC042 expression in the root-elongation zone, the camalexin biosynthetic site, and the induction was abolished in the presence of either a general kinase inhibitor (K252a), a Ca(2+)-chelator (BAPTA), or methyl jasmonate. The GUS-reporter assay revealed repression of the Flg22-dependent ANAC042 expression in the ethylene-insensitive ein2-1 background but not in sid2-2 plants defective for salicylic acid biosynthesis. We discuss ANAC042 as a key transcription factor involved in previously unknown regulatory mechanisms to induce phytoalexin biosynthesis in Arabidopsis.

  14. Regulation of anthrax toxin activator gene (atxA) expression in Bacillus anthracis: temperature, not CO2/bicarbonate, affects AtxA synthesis.

    OpenAIRE

    Dai, Z; Koehler, T M

    1997-01-01

    Anthrax toxin gene expression in Bacillus anthracis is dependent on the presence of atxA, a trans-acting regulatory gene located on the resident 185-kb plasmid pXO1. In atxA+ strains, expression of the toxin genes (pag, lef, and cya) is enhanced by two physiologically significant signals: elevated CO2/bicarbonate and temperature. To determine whether increased toxin gene expression in response to these signals is associated with increased atxA expression, we monitored steady-state levels of a...

  15. Role of Halloween genes in ecdysteroids biosynthesis of the swimming crab (Portunus trituberculatus): Implications from RNA interference and eyestalk ablation.

    Science.gov (United States)

    Xie, Xi; Liu, Zhiye; Liu, Mingxin; Tao, Tian; Shen, Xiquan; Zhu, Dongfa

    2016-09-01

    Molting, including metamorphosis molting in arthropods are controlled by the ecdysteroids that are synthesized and secreted by the crustacean Y-organ (YO) or the insect prothoracic gland (PG). The Halloween genes encoding the enzymes mainly involved in the biosynthesis of ecdysteroids are well studied in insects but not in crustaceans. Given the importance of Halloween genes in ecdysteroids biosynthesis, we have previously reported the cDNA cloning of disembodied (Dib) in P. trituberculatus. Here, cDNA sequences of another two Halloween genes, Spook (Spo) and Shadow (Sad), were further identified and characterized. The predicted amino acid sequences for these two Halloween genes of Portunus trituberculatus were compared to those of several other arthropods, and several typical domains of the cytochrome P450 mono-oxygenase (CYP) were identified. Similar to the tissue distribution of Dib, the Spo and Sad also showed high specificity to the YO. RNA interference (RNAi) of these 3 genes indicated they all play essential role in ecdysteroids biosynthesis. To investigate the relationships of the Halloween genes to the eyestalk neuropeptides such as molt-inhibiting hormone (MIH), effects of eyestalk ablation (ESA) on the expression of Dib, Spo and Sad were detected. Expression of Dib and Sad, but not Spo, was significantly induced by ESA. The result indicated that the inhibition of MIH in ecdysteroids biosynthesis may be partly through the transcriptional regulation of certain Halloween genes, such as Dib and Sad, while the Spo might not be the target for MIH signal. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Profiling and Quantifying Differential Gene Transcription Provide Insights into Ganoderic Acid Biosynthesis in Ganoderma lucidum in Response to Methyl Jasmonate

    Science.gov (United States)

    Shi, Liang; Mu, Da-Shuai; Jiang, Ai-Liang; Han, Qin; Zhao, Ming-Wen

    2013-01-01

    Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum. PMID:23762280

  17. Staphylococcal food poisoning case and molecular analysis of toxin genes in Staphylococcus aureus strains isolated from food in Sicily, Italy.

    Science.gov (United States)

    Vitale, Maria; Scatassa, Maria Luisa; Cardamone, Cinzia; Oliveri, Giuseppa; Piraino, Chiara; Alduina, Rosa; Napoli, Concetta

    2015-01-01

    A case of staphylococcal food poisoning was observed in two individuals of the same family after consumption of primosale, a semiripened sheep cheese produced in Sicily. Staphylococcus aureus isolated from the cheese produced enterotoxin C (SEC) and carried both the enterotoxin C (sec) and the toxic shock syndrome toxin (tsst-1) gene. Following this case, an extensive survey was conducted on 971 food samples (raw milk, cheese, meat, and food preparations). S. aureus was detected in 102 of 971 food samples, from all types of food with the exception of ricotta cheese. The tsst-1 gene was present in 42% of the strains, either alone or in combination with other toxin genes. The enterotoxin C gene was the most represented enterotoxin, but it was only found in dairy products. Six S. aureus isolates carried the sea gene alone, two isolates carried both sea and seb, and one isolate carried both sea and sec. A significant percentage (46%) of all isolates carried a toxin gene, creating significant concern that virulent S. aureus can be transmitted through food in Sicily.

  18. Gene coexpression network analysis of oil biosynthesis in an interspecific backcross of oil palm.

    Science.gov (United States)

    Guerin, Chloé; Joët, Thierry; Serret, Julien; Lashermes, Philippe; Vaissayre, Virginie; Agbessi, Mawussé D T; Beulé, Thierry; Severac, Dany; Amblard, Philippe; Tregear, James; Durand-Gasselin, Tristan; Morcillo, Fabienne; Dussert, Stéphane

    2016-09-01

    Global demand for vegetable oils is increasing at a dramatic rate, while our understanding of the regulation of oil biosynthesis in plants remains limited. To gain insights into the mechanisms that govern oil synthesis and fatty acid (FA) composition in the oil palm fruit, we used a multilevel approach combining gene coexpression analysis, quantification of allele-specific expression and joint multivariate analysis of transcriptomic and lipid data, in an interspecific backcross population between the African oil palm, Elaeis guineensis, and the American oil palm, Elaeis oleifera, which display contrasting oil contents and FA compositions. The gene coexpression network produced revealed tight transcriptional coordination of fatty acid synthesis (FAS) in the plastid with sugar sensing, plastidial glycolysis, transient starch storage and carbon recapture pathways. It also revealed a concerted regulation, along with FAS, of both the transfer of nascent FA to the endoplasmic reticulum, where triacylglycerol assembly occurs, and of the production of glycerol-3-phosphate, which provides the backbone of triacylglycerols. Plastid biogenesis and auxin transport were the two other biological processes most tightly connected to FAS in the network. In addition to WRINKLED1, a transcription factor (TF) known to activate FAS genes, two novel TFs, termed NF-YB-1 and ZFP-1, were found at the core of the FAS module. The saturated FA content of palm oil appeared to vary above all in relation to the level of transcripts of the gene coding for β-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  19. Halloween genes and nuclear receptors in ecdysteroid biosynthesis and signalling in the pea aphid.

    Science.gov (United States)

    Christiaens, O; Iga, M; Velarde, R A; Rougé, P; Smagghe, G

    2010-03-01

    The pea aphid (Acyrthosiphon pisum) is the first whole genome sequenced insect with a hemimetabolic development and an emerging model organism for studies in ecology, evolution and development. The insect steroid moulting hormone 20-hydroxyecdysone (20E) controls and coordinates development in insects, especially the moulting/metamorphosis process. We, therefore present here a comprehensive characterization of the Halloween genes phantom, disembodied, shadow, shade, spook and spookiest, coding for the P450 enzymes that control the biosynthesis of 20E. Regarding the presence of nuclear receptors in the pea aphid genome, we found 19 genes, representing all of the seven known subfamilies. The annotation and phylogenetic analysis revealed a strong conservation in the class of Insecta. But compared with other sequenced insect genomes, three orthologues are missing in the Acyrthosiphon genome, namely HR96, PNR-like and Knirps. We also cloned the EcR, Usp, E75 and HR3. Finally, 3D-modelling of the ligand-binding domain of Ap-EcR exhibited the typical canonical structural scaffold with 12 alpha-helices associated with a short hairpin of two antiparallel beta-strands. Upon docking, 20E was located in the hormone-binding groove, supporting the hypothesis that EcR has a role in 20E signalling.

  20. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds

    Directory of Open Access Journals (Sweden)

    Yusuke Tagashira

    2015-04-01

    Full Text Available The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP6 biosynthesis-related genes, as InsP6 is a major storage form of P in seeds. The rice (Oryza sativa L. low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP6 biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP6 and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP6 biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP6 biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  1. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium Botulinum Strain PS-5.

    Science.gov (United States)

    Singh, Ajay K; Sachdeva, Amita; Degrasse, Jeffrey A; Croley, Timothy R; Stanker, Larry H; Hodge, David; Sharma, Shashi K

    2013-04-01

    Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography-Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.

  2. Quantitative Detection of Clostridium perfringens in Broiler Chickens by Real-Time PCR Targeting the Alpha-Toxin Gene

    DEFF Research Database (Denmark)

    Abildgaard, Lone; Engberg, Ricarda M.; Schramm, Andreas

    2006-01-01

    by antibiotics (ionophores) presently used to prevent parasitic coccidiosis. From 2012 the European Union has banned these anticoccidials as feed additives, wherefore alternatives are needed to suppress C. perfringens and/or α-toxin production. A real-time PCR primer-probe set targeting the α-toxin gene...

  3. Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynthesis in Ganoderma lingzhi.

    Science.gov (United States)

    Zhang, De-Huai; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2017-02-01

    Ganoderic acids (GAs) in Ganoderma lingzhi exhibit anticancer and antimetastatic activities. GA yields can be potentially improved by manipulating G. lingzhi through genetic engineering. In this study, a putative lanosterol synthase (LS) gene was cloned and overexpressed in G. lingzhi. Results showed that its overexpression (OE) increased the ganoderic acid (GA) content and the accumulation of lanosterol and ergosterol in a submerged G. lingzhi culture. The maximum contents of GA-O, GA-Mk, GA-T, GA-S, GA-Mf, and GA-Me in transgenic strains were 46.6 ± 4.8, 24.3 ± 3.5, 69.8 ± 8.2, 28.9 ± 1.4, 15.4 ± 1.2, and 26.7 ± 3.1 μg/100 mg dry weight, respectively, these values being 6.1-, 2.2-, 3.2-, 4.8-, 2.0-, and 1.9-times higher than those in wild-type strains. In addition, accumulated amounts of lanosterol and ergosterol in transgenic strains were 2.3 and 1.4-fold higher than those in the control strains, respectively. The transcription level of LS was also increased by more than five times in the presence of the G. lingzhi glyceraldehyde-3-phosphate dehydrogenase gene promoter, whereas transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A enzyme and squalene synthase did not change significantly in transgenic strains. This study demonstrated that OE of the homologous LS gene can enhance lanosterol accumulation. A large precursor supply promotes GA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis cinerea Is Positively Controlled by VELVET

    Science.gov (United States)

    Schumacher, Julia; Gautier, Angélique; Morgant, Guillaume; Studt, Lena; Ducrot, Paul-Henri; Le Pêcheur, Pascal; Azeddine, Saad; Fillinger, Sabine; Leroux, Pierre; Tudzynski, Bettina; Viaud, Muriel

    2013-01-01

    The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus. PMID:23308280

  5. Flavonoid Biosynthesis Genes Putatively Identified in the Aromatic Plant Polygonum minus via Expressed Sequences Tag (EST Analysis

    Directory of Open Access Journals (Sweden)

    Zamri Zainal

    2012-02-01

    Full Text Available P. minus is an aromatic plant, the leaf of which is widely used as a food additive and in the perfume industry. The leaf also accumulates secondary metabolites that act as active ingredients such as flavonoid. Due to limited genomic and transcriptomic data, the biosynthetic pathway of flavonoids is currently unclear. Identification of candidate genes involved in the flavonoid biosynthetic pathway will significantly contribute to understanding the biosynthesis of active compounds. We have constructed a standard cDNA library from P. minus leaves, and two normalized full-length enriched cDNA libraries were constructed from stem and root organs in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis. Thus, large‑scale sequencing of P. minus cDNA libraries identified 4196 expressed sequences tags (ESTs which were deposited in dbEST in the National Center of Biotechnology Information (NCBI. From the three constructed cDNA libraries, 11 ESTs encoding seven genes were mapped to the flavonoid biosynthetic pathway. Finally, three flavonoid biosynthetic pathway-related ESTs chalcone synthase, CHS (JG745304, flavonol synthase, FLS (JG705819 and leucoanthocyanidin dioxygenase, LDOX (JG745247 were selected for further examination by quantitative RT-PCR (qRT-PCR in different P. minus organs. Expression was detected in leaf, stem and root. Gene expression studies have been initiated in order to better understand the underlying physiological processes.

  6. De novo transcriptome of safflower and the identification of putative genes for oleosin and the biosynthesis of flavonoids.

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    Haiyan Li

    Full Text Available Safflower (Carthamus tinctorius L. is one of the most extensively used oil crops in the world. However, little is known about how its compounds are synthesized at the genetic level. In this study, Solexa-based deep sequencing on seed, leaf and petal of safflower produced a de novo transcriptome consisting of 153,769 unigenes. We annotated 82,916 of the unigenes with gene annotation and assigned functional terms and specific pathways to a subset of them. Metabolic pathway analysis revealed that 23 unigenes were predicted to be responsible for the biosynthesis of flavonoids and 8 were characterized as seed-specific oleosins. In addition, a large number of differentially expressed unigenes, for example, those annotated as participating in anthocyanin and chalcone synthesis, were predicted to be involved in flavonoid biosynthesis pathways. In conclusion, the de novo transcriptome investigation of the unique transcripts provided candidate gene resources for studying oleosin-coding genes and for investigating genes related to flavonoid biosynthesis and metabolism in safflower.

  7. Burkholderia thailandensis harbors two identical rhl gene clusters responsible for the biosynthesis of rhamnolipids

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    Woods Donald E

    2009-12-01

    Full Text Available Abstract Background Rhamnolipids are surface active molecules composed of rhamnose and β-hydroxydecanoic acid. These biosurfactants are produced mainly by Pseudomonas aeruginosa and have been thoroughly investigated since their early discovery. Recently, they have attracted renewed attention because of their involvement in various multicellular behaviors. Despite this high interest, only very few studies have focused on the production of rhamnolipids by Burkholderia species. Results Orthologs of rhlA, rhlB and rhlC, which are responsible for the biosynthesis of rhamnolipids in P. aeruginosa, have been found in the non-infectious Burkholderia thailandensis, as well as in the genetically similar important pathogen B. pseudomallei. In contrast to P. aeruginosa, both Burkholderia species contain these three genes necessary for rhamnolipid production within a single gene cluster. Furthermore, two identical, paralogous copies of this gene cluster are found on the second chromosome of these bacteria. Both Burkholderia spp. produce rhamnolipids containing 3-hydroxy fatty acid moieties with longer side chains than those described for P. aeruginosa. Additionally, the rhamnolipids produced by B. thailandensis contain a much larger proportion of dirhamnolipids versus monorhamnolipids when compared to P. aeruginosa. The rhamnolipids produced by B. thailandensis reduce the surface tension of water to 42 mN/m while displaying a critical micelle concentration value of 225 mg/L. Separate mutations in both rhlA alleles, which are responsible for the synthesis of the rhamnolipid precursor 3-(3-hydroxyalkanoyloxyalkanoic acid, prove that both copies of the rhl gene cluster are functional, but one contributes more to the total production than the other. Finally, a double ΔrhlA mutant that is completely devoid of rhamnolipid production is incapable of swarming motility, showing that both gene clusters contribute to this phenotype. Conclusions Collectively, these

  8. Coordinated Regulation of Anthocyanin Biosynthesis Genes Confers Varied Phenotypic and Spatial-Temporal Anthocyanin Accumulation in Radish (Raphanus sativus L.).

    Science.gov (United States)

    Muleke, Everlyne M'mbone; Fan, Lianxue; Wang, Yan; Xu, Liang; Zhu, Xianwen; Zhang, Wei; Cao, Yang; Karanja, Benard K; Liu, Liwang

    2017-01-01

    Anthocyanins are natural pigments that have important functions in plant growth and development. Radish taproots are rich in anthocyanins which confer different taproot colors and are potentially beneficial to human health. The crop differentially accumulates anthocyanin during various stages of growth, yet molecular mechanisms underlying this differential anthocyanin accumulation remains unknown. In the present study, transcriptome analysis was used to concisely identify putative genes involved in anthocyanin biosynthesis in radish. Spatial-temporal transcript expressions were then profiled in four color variant radish cultivars. From the total transcript sequences obtained through illumina sequencing, 102 assembled unigenes, and 20 candidate genes were identified to be involved in anthocyanin biosynthesis. Fifteen genomic sequences were isolated and sequenced from radish taproot. The length of these sequences was between 900 and 1,579 bp, and the unigene coverage to all of the corresponding cloned sequences was more than 93%. Gene structure analysis revealed that RsF3 ' H is intronless and anthocyanin biosynthesis genes (ABGs) bear asymmetrical exons, except RsSAM . Anthocyanin accumulation showed a gradual increase in the leaf of the red radish and the taproot of colored cultivars during development, with a rapid increase at 30 days after sowing (DAS), and the highest content at maturity. Spatial-temporal transcriptional analysis of 14 genes revealed detectable expressions of 12 ABGs in various tissues at different growth levels. The investigation of anthocyanin accumulation and gene expression in four color variant radish cultivars, at different stages of development, indicated that total anthocyanin correlated with transcript levels of ABGs, particularly RsUFGT, RsF3H, RsANS, RsCHS3 and RsF3 ' H1 . Our results suggest that these candidate genes play key roles in phenotypic and spatial-temporal anthocyanin accumulation in radish through coordinated regulation

  9. Coordinated Regulation of Anthocyanin Biosynthesis Genes Confers Varied Phenotypic and Spatial-Temporal Anthocyanin Accumulation in Radish (Raphanus sativus L.

    Directory of Open Access Journals (Sweden)

    Everlyne M'mbone Muleke

    2017-07-01

    Full Text Available Anthocyanins are natural pigments that have important functions in plant growth and development. Radish taproots are rich in anthocyanins which confer different taproot colors and are potentially beneficial to human health. The crop differentially accumulates anthocyanin during various stages of growth, yet molecular mechanisms underlying this differential anthocyanin accumulation remains unknown. In the present study, transcriptome analysis was used to concisely identify putative genes involved in anthocyanin biosynthesis in radish. Spatial-temporal transcript expressions were then profiled in four color variant radish cultivars. From the total transcript sequences obtained through illumina sequencing, 102 assembled unigenes, and 20 candidate genes were identified to be involved in anthocyanin biosynthesis. Fifteen genomic sequences were isolated and sequenced from radish taproot. The length of these sequences was between 900 and 1,579 bp, and the unigene coverage to all of the corresponding cloned sequences was more than 93%. Gene structure analysis revealed that RsF3′H is intronless and anthocyanin biosynthesis genes (ABGs bear asymmetrical exons, except RsSAM. Anthocyanin accumulation showed a gradual increase in the leaf of the red radish and the taproot of colored cultivars during development, with a rapid increase at 30 days after sowing (DAS, and the highest content at maturity. Spatial-temporal transcriptional analysis of 14 genes revealed detectable expressions of 12 ABGs in various tissues at different growth levels. The investigation of anthocyanin accumulation and gene expression in four color variant radish cultivars, at different stages of development, indicated that total anthocyanin correlated with transcript levels of ABGs, particularly RsUFGT, RsF3H, RsANS, RsCHS3 and RsF3′H1. Our results suggest that these candidate genes play key roles in phenotypic and spatial-temporal anthocyanin accumulation in radish through

  10. Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis.

    OpenAIRE

    Marahiel, M A; Zuber, P; Czekay, G; Losick, R

    1987-01-01

    Tyrocidine is a cyclic decapeptide antibiotic which is produced and secreted by stationary-phase cells of the sporeforming bacterium Bacillus brevis. We identified the promoter for the B. brevis structural gene (tycA) for tyrocidine synthetase I, the enzyme catalyzing the first step in tyrocidine biosynthesis, and studied its regulation in cells of B. brevis and Bacillus subtilis. Transcription from the tycA promoter was induced at the end of the exponential phase of the growth cycle in B. br...

  11. Comprehensive assessment of the genes involved in withanolide biosynthesis from Withania somnifera: chemotype-specific and elicitor-responsive expression.

    Science.gov (United States)

    Agarwal, Aditya Vikram; Gupta, Parul; Singh, Deeksha; Dhar, Yogeshwar Vikram; Chandra, Deepak; Trivedi, Prabodh Kumar

    2017-07-01

    Withania somnifera (L.) Dunal (Family, Solanaceae), is among the most valuable medicinal plants used in Ayurveda owing to its rich reservoir of pharmaceutically active secondary metabolites known as withanolides. Withanolides are C 28 -steroidal lactones having a triterpenoidal metabolic origin synthesised via mevalonate (MVA) pathway and methyl-D-erythritol-4-phosphate (MEP) pathway involving metabolic intermediacy of 24-methylene (C 30 -terpenoid) cholesterol. Phytochemical studies suggest differences in the content and/or nature of withanolides in different tissues of different chemotypes. Though development of genomic resources has provided information about putative genes encoding enzymes for biosynthesis of intermediate steps of terpenoid backbone, not much is known about their regulation and response to elicitation. In this study, we generated detailed molecular information about genes catalysing key regulatory steps of withanolide biosynthetic pathway. The full-length sequences of genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis and their paralogs have been characterized for their functional and structural properties as well as phylogeny using bioinformatics approach. The expression analysis suggests that these genes are differentially expressed in different tissues (with maximal expression in young leaf), chemotypes and in response to salicylic acid (SA) and methyl jasmonate (MJ) treatments. Sub-cellular localization studies suggest that both paralogs of sterol ∆-7 reductase (WsDWF5-1 and WsDWF5-2) are localized in the endoplasmic reticulum (ER) thus supporting their indispensible role in withanolide biosynthesis. Comprehensive information developed, in this study, will lead to elucidation of chemotype- as well as tissue-specific withanolide biosynthesis and development of new tools for functional genomics in this important medicinal plant.

  12. Identification and functional analysis of gene cluster involvement in biosynthesis of the cyclic lipopeptide antibiotic pelgipeptin produced by Paenibacillus elgii

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    Qian Chao-Dong

    2012-09-01

    Full Text Available Abstract Background Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a β-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway. Results A potential pelgipeptin synthetase gene cluster (plp was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPSs, with one, seven, and one module(s, respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1 provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis. Conclusions In this study, a gene cluster (plp responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.

  13. Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers.

    Science.gov (United States)

    Zhang, Guang-Hui; Ma, Chun-Hua; Zhang, Jia-Jin; Chen, Jun-Wen; Tang, Qing-Yan; He, Mu-Han; Xu, Xiang-Zeng; Jiang, Ni-Hao; Yang, Sheng-Chao

    2015-03-08

    P. vietnamensis var. fuscidiscus, called "Yesanqi" in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking. To investigate the P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance

  14. Sequencing and transcriptional analysis of the Streptococcus thermophilus histamine biosynthesis gene cluster: factors that affect differential hdcA expression

    DEFF Research Database (Denmark)

    Calles-Enríquez, Marina; Hjort, Benjamin Benn; Andersen, Pia Skov

    2010-01-01

    to produce histamine. The hdc clusters of S. thermophilus CHCC1524 and CHCC6483 were sequenced, and the factors that affect histamine biosynthesis and histidine-decarboxylating gene (hdcA) expression were studied. The hdc cluster began with the hdcA gene, was followed by a transporter (hdcP), and ended...... with the hdcB gene, which is of unknown function. The three genes were orientated in the same direction. The genetic organization of the hdc cluster showed a unique organization among the lactic acid bacterial group and resembled those of Staphylococcus and Clostridium species, thus indicating possible...... acquisition through a horizontal transfer mechanism. Transcriptional analysis of the hdc cluster revealed the existence of a polycistronic mRNA covering the three genes. The histidine-decarboxylating gene (hdcA) of S. thermophilus demonstrated maximum expression during the stationary growth phase, with high...

  15. Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

    Directory of Open Access Journals (Sweden)

    Kudrna David

    2011-03-01

    Full Text Available Abstract Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1 digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb to 157 Kb (Eg_Ba, very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×, contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae

  16. ATAF2, a NAC Transcription Factor, Binds to the Promoter and Regulates NIT2 Gene Expression Involved in Auxin Biosynthesis

    Science.gov (United States)

    Huh, Sung Un; Lee, Suk-Bae; Kim, Hwang Hyun; Paek, Kyung-Hee

    2012-01-01

    The transcription factor ATAF2, one of the plant specific NAC family genes, is known as repressor of pathogenesis-related genes and responsive to the diverse defense-related hormones, pathogen infection, and wounding stress. Furthermore, it is important to consider that tryptophan-dependant IAA biosynthesis pathway can be activated by wounding and pathogen. We found that ATAF2pro::GUS reporter was induced upon indole-3-acetonitrile (IAN) treatments. And ataf2 mutant showed reduced sensitivity to IAN whereas 35S::ATAF2 plants showed hyper-sensitivity to IAN. IAN biosynthesis required nitrilase involved in the conversion of IAN to an auxin, indole-3-acetic acid (IAA). We found that the NIT2 gene was repressed in ataf2 knockout plants. Expression of both ATAF2 and NIT2 genes was induced by IAN treatment. Transgenic plants overexpressing ATAF2 showed up-regulated NIT2 expression. ATAF2 activated promoter of the NIT2 gene in Arabidopsis protoplasts. Electrophoretic mobility shift assay revealed that NIT2 promoter region from position −117 to −82 contains an ATAF2 binding site where an imperfect palindrome sequence was critical to the protein-DNA interaction. These findings indicate that ATAF2 regulates NIT2 gene expression via NIT2 promoter binding. PMID:22965747

  17. A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus.

    Science.gov (United States)

    Horbal, Liliya; Ostash, Bohdan; Luzhetskyy, Andriy; Walker, Suzanne; Kalinowski, Jorn; Fedorenko, Victor

    2016-09-01

    Moenomycins are phosphoglycolipid antibiotics notable for their extreme potency, unique mode of action, and proven record of use in animal nutrition without selection for resistant microflora. There is a keen interest in manipulation of structures of moenomycins in order to better understand their structure-activity relationships and to generate improved analogs. Only two almost identical moenomycin biosynthetic gene clusters are known, limiting our knowledge of the evolution of moenomycin pathways and our ability to genetically diversify them. Here, we report a novel gene cluster (tchm) that directs production of the phosphoglycolipid teichomycin in Actinoplanes teichomyceticus. Its overall genetic architecture is significantly different from that of the moenomycin biosynthesis (moe) gene clusters of Streptomyces ghanaensis and Streptomyces clavuligerus, featuring multiple gene rearrangements and two novel structural genes. Involvement of the tchm cluster in teichomycin biosynthesis was confirmed via heterologous co-expression of amidotransferase tchmH5 and moe genes. Our work sets the background for further engineering of moenomycins and for deeper inquiries into the evolution of this fascinating biosynthetic pathway.

  18. Three novel rice genes closely related to the Arabidopsis IRX9, IRX9L, and IRX14 genes and their roles in xylan biosynthesis

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    Dawn eChiniquy

    2013-04-01

    Full Text Available Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9, Os01g48440 (OsIRX9L, and Os06g47340 (OsIRX14, from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the overexpression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in xylosyltransferase activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength.

  19. Study of three interesting Amanita species from Thailand: Morphology, multiple-gene phylogeny and toxin analysis.

    Directory of Open Access Journals (Sweden)

    Benjarong Thongbai

    Full Text Available Amanita ballerina and A. brunneitoxicaria spp. nov. are introduced from Thailand. Amanita fuligineoides is also reported for the first time from Thailand, increasing the known distribution of this taxon. Together, those findings support our view that many taxa are yet to be discovered in the region. While both morphological characters and a multiple-gene phylogeny clearly place A. brunneitoxicaria and A. fuligineoides in sect. Phalloideae (Fr. Quél., the placement of A. ballerina is problematic. On the one hand, the morphology of A. ballerina shows clear affinities with stirps Limbatula of sect. Lepidella. On the other hand, in a multiple-gene phylogeny including taxa of all sections in subg. Lepidella, A. ballerina and two other species, including A. zangii, form a well-supported clade sister to the Phalloideae sensu Bas 1969, which include the lethal "death caps" and "destroying angels". Together, the A. ballerina-A. zangii clade and the Phalloideae sensu Bas 1969 also form a well-supported clade. We therefore screened for two of the most notorious toxins by HPLC-MS analysis of methanolic extracts from the basidiomata. Interestingly, neither α-amanitin nor phalloidin was found in A. ballerina, whereas Amanita fuligineoides was confirmed to contain both α-amanitin and phalloidin, and A. brunneitoxicaria contained only α-amanitin. Together with unique morphological characteristics, the position in the phylogeny indicates that A. ballerina is either an important link in the evolution of the deadly Amanita sect. Phalloideae species, or a member of a new section also including A. zangii.

  20. Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors.

    Science.gov (United States)

    Dupuy, Bruno; Raffestin, Stéphanie; Matamouros, Susana; Mani, Nagraj; Popoff, Michel R; Sonenshein, Abraham L

    2006-05-01

    The production of major extracellular toxins by pathogenic strains of Clostridium botulinum, Clostridium tetani and Clostridium difficile, and a bacteriocin by Clostridium perfringens is dependent on a related group of RNA polymerase sigma-factors. These sigma-factors (BotR, TetR, TcdR and UviA) were shown to be sufficiently similar that they could substitute for one another in in vitro DNA binding and run-off transcription experiments. In cells, however, the sigma-factors fell into two subclasses. BotR and TetR were able to direct transcription of their target genes in a fully reciprocal manner. Similarly, UviA and TcdR were fully interchangeable. Neither BotR nor TetR could substitute for UviA or TcdR, however, and neither UviA nor TcdR could direct transcription of the natural targets of BotR or TetR. The extent of functional interchangeability of the sigma-factors was attributed to the strong conservation of their subregion 4.2 sequences and the conserved -35 sequences of their target promoters, while restrictions on interchangeability were attributed to variations in their subregion 2.4 sequences and the target site -10 sequences. The four sigma-factors have been assigned to group 5 of the sigma(70) family and seem to have arisen from a common ancestral protein that may have co-evolved with the genes whose transcription they direct. A fifth Clostridiumsigma-factor, sigma(Y) of Clostridium acetobutylicum, resembles the TcdR family, but was not functionally interchangeable with members of this family.

  1. 454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides.

    Science.gov (United States)

    Zagrobelny, Mika; Scheibye-Alsing, Karsten; Jensen, Niels Bjerg; Møller, Birger Lindberg; Gorodkin, Jan; Bak, Søren

    2009-12-02

    An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases) were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being convergent between plants and insects

  2. 454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides

    Directory of Open Access Journals (Sweden)

    Jensen Niels

    2009-12-01

    Full Text Available Abstract Background An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Results Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being

  3. Comparison of Expression of Secondary Metabolite Biosynthesis Cluster Genes in Aspergillus flavus, A. parasiticus, and A. oryzae

    Directory of Open Access Journals (Sweden)

    Kenneth C. Ehrlich

    2014-06-01

    Full Text Available Fifty six secondary metabolite biosynthesis gene clusters are predicted to be in the Aspergillus flavus genome. In spite of this, the biosyntheses of only seven metabolites, including the aflatoxins, kojic acid, cyclopiazonic acid and aflatrem, have been assigned to a particular gene cluster. We used RNA-seq to compare expression of secondary metabolite genes in gene clusters for the closely related fungi A. parasiticus, A. oryzae, and A. flavus S and L sclerotial morphotypes. The data help to refine the identification of probable functional gene clusters within these species. Our results suggest that A. flavus, a prevalent contaminant of maize, cottonseed, peanuts and tree nuts, is capable of producing metabolites which, besides aflatoxin, could be an underappreciated contributor to its toxicity.

  4. Comparison of Expression of Secondary Metabolite Biosynthesis Cluster Genes in Aspergillus flavus, A. parasiticus, and A. oryzae

    Science.gov (United States)

    Ehrlich, Kenneth C.; Mack, Brian M.

    2014-01-01

    Fifty six secondary metabolite biosynthesis gene clusters are predicted to be in the Aspergillus flavus genome. In spite of this, the biosyntheses of only seven metabolites, including the aflatoxins, kojic acid, cyclopiazonic acid and aflatrem, have been assigned to a particular gene cluster. We used RNA-seq to compare expression of secondary metabolite genes in gene clusters for the closely related fungi A. parasiticus, A. oryzae, and A. flavus S and L sclerotial morphotypes. The data help to refine the identification of probable functional gene clusters within these species. Our results suggest that A. flavus, a prevalent contaminant of maize, cottonseed, peanuts and tree nuts, is capable of producing metabolites which, besides aflatoxin, could be an underappreciated contributor to its toxicity. PMID:24960201

  5. RNA Sequencing and Coexpression Analysis Reveal Key Genes Involved in α-Linolenic Acid Biosynthesis in Perilla frutescens Seed

    Directory of Open Access Journals (Sweden)

    Tianyuan Zhang

    2017-11-01

    Full Text Available Perilla frutescen is used as traditional food and medicine in East Asia. Its seeds contain high levels of α-linolenic acid (ALA, which is important for health, but is scarce in our daily meals. Previous reports on RNA-seq of perilla seed had identified fatty acid (FA and triacylglycerol (TAG synthesis genes, but the underlying mechanism of ALA biosynthesis and its regulation still need to be further explored. So we conducted Illumina RNA-sequencing in seven temporal developmental stages of perilla seeds. Sequencing generated a total of 127 million clean reads, containing 15.88 Gb of valid data. The de novo assembly of sequence reads yielded 64,156 unigenes with an average length of 777 bp. A total of 39,760 unigenes were annotated and 11,693 unigenes were found to be differentially expressed in all samples. According to Kyoto Encyclopedia of Genes and Genomes (KEGG pathway analysis, 486 unigenes were annotated in the “lipid metabolism” pathway. Of these, 150 unigenes were found to be involved in fatty acid (FA biosynthesis and triacylglycerol (TAG assembly in perilla seeds. A coexpression analysis showed that a total of 104 genes were highly coexpressed (r > 0.95. The coexpression network could be divided into two main subnetworks showing over expression in the medium or earlier and late phases, respectively. In order to identify the putative regulatory genes, a transcription factor (TF analysis was performed. This led to the identification of 45 gene families, mainly including the AP2-EREBP, bHLH, MYB, and NAC families, etc. After coexpression analysis of TFs with highly expression of FAD2 and FAD3 genes, 162 TFs were found to be significantly associated with two FAD genes (r > 0.95. Those TFs were predicted to be the key regulatory factors in ALA biosynthesis in perilla seed. The qRT-PCR analysis also verified the relevance of expression pattern between two FAD genes and partial candidate TFs. Although it has been reported that some TFs

  6. Assessment of the Essentiality of ERG Genes Late in Ergosterol Biosynthesis in Saccharomyces Cerevisiae

    National Research Council Canada - National Science Library

    Parks, Leo

    1997-01-01

    .... We are seeking to identify reactions that are essential to the survival of fungi. Those involved in sterol biosynthesis have been shown to be required for cell growth and amenable to control by antifungal compounds...

  7. Discovery of new regulatory genes of lipopeptide biosynthesis in Pseudomonas fluorescens

    NARCIS (Netherlands)

    Song, C.; Aundy, K.; Mortel, van de J.E.; Raaijmakers, J.M.

    2014-01-01

    Pseudomonas fluorescens SS101 produces the cyclic lipopeptide massetolide with diverse functions in antimicrobial activity, motility, and biofilm formation. To understand how massetolide biosynthesis is genetically regulated in SS101, c. 8000 random plasposon mutants were screened for reduced or

  8. Identification of a Gene Cluster for Biosynthesis of Mannosylerythritol Lipids in the Basidiomycetous Fungus Ustilago maydis

    OpenAIRE

    Hewald, Sandra; Linne, Uwe; Scherer, Mario; Marahiel, Mohamed A.; Kämper, Jörg; Bölker, Michael

    2006-01-01

    Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose exp...

  9. Data on the presence or absence of genes encoding essential proteins for ochratoxin and fumonisin biosynthesis in Aspergillus niger and Aspergillus welwitschiae

    Directory of Open Access Journals (Sweden)

    Fernanda Pelisson Massi

    2016-06-01

    Full Text Available We present the multiplex PCR data for the presence/absence of genes involved in OTA and FB2 biosynthesis in Aspergillus niger/Aspergillus welwitschiae strains isolated from different food substrates in Brazil. Among the 175 strains analyzed, four mPCR profiles were found: Profile 1 (17% highlights strains harboring in their genome the pks, radH and the fum8 genes. Profile 2 (3.5% highlights strains harboring genes involved in OTA biosynthesis i.e. radH and pks. Profile 3 (51.5% highlights strains harboring the fum8 gene. Profile 4 (28% highlights strains not carrying the genes studied herein. This research content is supplemental to our original research article, “Prospecting for the incidence of genes involved in ochratoxin and fumonisin biosynthesis in Brazilian strains of A. niger and A. welwitschiae” [1].

  10. RNA-Seq mediated root transcriptome analysis of Chlorophytum borivilianum for identification of genes involved in saponin biosynthesis.

    Science.gov (United States)

    Kumar, Sunil; Kalra, Shikha; Singh, Baljinder; Kumar, Avneesh; Kaur, Jagdeep; Singh, Kashmir

    2016-01-01

    Chlorophytum borivilianum is an important species of liliaceae family, owing to its vital medicinal properties. Plant roots are used for aphrodisiac, adaptogen, anti-aging, health-restorative and health-promoting purposes. Saponins, are considered to be the principal bioactive components responsible for the wide variety of pharmacological properties of this plant. In the present study, we have performed de novo root transcriptome sequencing of C. borivilianum using Illumina Hiseq 2000 platform, to gain molecular insight into saponins biosynthesis. A total of 33,963,356 high-quality reads were obtained after quality filtration. Sequences were assembled using various programs which generated 97,344 transcripts with a size range of 100-5,216 bp and N50 value of 342. Data was analyzed against non-redundant proteins, gene ontology (GO), and enzyme commission (EC) databases. All the genes involved in saponins biosynthesis along with five full-length genes namely farnesyl pyrophosphate synthase, cycloartenol synthase, β-amyrin synthase, cytochrome p450, and sterol-3-glucosyltransferase were identified. Read per exon kilobase per million (RPKM)-based comparative expression profiling was done to study the differential regulation of the genes. In silico expression analysis of seven selected genes of saponin biosynthetic pathway was validated by qRT-PCR.

  11. Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus.

    Science.gov (United States)

    Lin, W S; Cunneen, T; Lee, C Y

    1994-11-01

    We previously cloned a 19.4-kb DNA region containing a cluster of genes affecting type 1 capsule production from Staphylococcus aureus M. Subcloning experiments showed that these capsule (cap) genes are localized in a 14.6-kb region. Sequencing analysis of the 14.6-kb fragment revealed 13 open reading frames (ORFs). Using complementation tests, we have mapped a collection of Cap- mutations in 10 of the 13 ORFs, indicating that these 10 genes are involved in capsule biosynthesis. The requirement for the remaining three ORFs in the synthesis of the capsule was demonstrated by constructing site-specific mutations corresponding to each of the three ORFs. Using an Escherichia coli S30 in vitro transcription-translation system, we clearly identified 7 of the 13 proteins predicted from the ORFs. Homology search between the predicted proteins and those in the data bank showed very high homology (52.3% identity) between capL and vipA, moderate homology (29% identity) between capI and vipB, and limited homology (21.8% identity) between capM and vipC. The vipA, vipB, and vipC genes have been shown to be involved in the biosynthesis of Salmonella typhi Vi antigen, a homopolymer polysaccharide consisting of N-acetylgalactosamino uronic acid, which is also one of the components of the staphylococcal type 1 capsule. The homology between these sets of genes therefore suggests that capL, capI, and capM may be involved in the biosynthesis of amino sugar, N-acetylgalactosamino uronic acid. In addition, the search showed that CapG aligned well with the consensus sequence of a family of acetyltransferases from various prokaryotic organisms, suggesting that CapG may be an acetyltransferase. Using the isogenic Cap- and Cap+ strains constructed in this study, we have confirmed that type 1 capsule is an important virulence factor in a mouse lethality test.

  12. Bacillus subtilis HJ18-4 from traditional fermented soybean food inhibits Bacillus cereus growth and toxin-related genes.

    Science.gov (United States)

    Eom, Jeong Seon; Lee, Sun Young; Choi, Hye Sun

    2014-11-01

    Bacillus subtilis HJ18-4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad-spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18-4. Expression of B. cereus toxin-related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18-4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18-4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18-4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus. © 2014 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists®

  13. Development,Identification and Validation of Markers for Mmarker-Assisted Selection Against the Stagonospora nodorum Toxin Ssensitivity Genes Tsn1 and Snn2 in Wheat

    Science.gov (United States)

    The wheat-Stagonospora nodorum pathosystem involves a number of pathogen-produced host-selective toxins that interact with host genes in an inverse gene-for-gene manner to cause disease. The wheat intervarietal recombinant inbred population derived from BR34 and Grandin (BG population) segregates f...

  14. Transforming a NEP1 toxin gene into two Fusarium spp. to enhance mycoherbicide activity on Orobanche--failure and success.

    Science.gov (United States)

    Meir, Sagit; Amsellem, Ziva; Al-Ahmad, Hani; Safran, Einat; Gressel, Jonathan

    2009-05-01

    The NEP1 gene encoding a fungal toxin that successfully conferred hypervirulence when transformed into Colletotrichum coccodes (Wallr.) Hughes attacking Abutilon theophrasti (L.) Medic. was tested to ascertain if it would enhance pathogenicity of Fusarium species to Orobanche aegyptiaca Pers. parasitising crops. None of the Fusarium oxysporum (#CNCM I-1622) NEP1 transformants was hypervirulent. NEP1 transformants of a new but unnamed Fusarium sp. (#CNCM I-1621--previously identified as F. arthrosporioides) killed Orobanche more rapidly than the wild type. Transformed lines of both species were NEP1 PCR positive, as was the wild type of F. oxysporum #CNCM I-1622 and five other formae speciales of F. oxysporum. All six wild-type formae speciales of F. oxysporum tested excrete minute amounts of immunologically and bioassay-detectable NEP1-like protein. NEP1 expression of most F. oxysporum transformants was suppressed, suggesting that the native gene and the transgene silence each other. The sequence of the putative NEP1 gene in Fusarium oxysporum #CNCM I-1622 differs from the sequence in the toxin-overproducing strain of F. oxysporum f. sp. erythroxyli in four or five amino acids in the first exon. Wild-type Fusarium sp. #CNCM I-1621 does not contain a NEP1-like gene, explaining why it seemed amenable to transformation with high expression, and its virulence was probably enhanced by not cosuppressing the endogenous gene as occurred with Fusarium oxysporum #CNCM I-1622.

  15. Isolation and characterization of the betalain biosynthesis gene involved in hypocotyl pigmentation of the allotetraploid Chenopodium quinoa.

    Science.gov (United States)

    Imamura, Tomohiro; Takagi, Hiroki; Miyazato, Akio; Ohki, Shinya; Mizukoshi, Hiroharu; Mori, Masashi

    2018-02-05

    In quinoa seedlings, the pigment betalain accumulates in the hypocotyl. To isolate the genes involved in betalain biosynthesis in the hypocotyl, we performed ethyl methanesulfonate (EMS) mutagenesis on the CQ127 variety of quinoa seedlings. While putative amaranthin and celosianin II primarily accumulate in the hypocotyls, this process produced a green hypocotyl mutant (ghy). This MutMap+ method using the quinoa draft genome revealed that the causative gene of the mutant is CqCYP76AD1-1. Our results indicated that the expression of CqCYP76AD1-1 was light-dependent. In addition, the transient expression of CqCYP76AD1-1 in Nicotiana benthamiana leaves resulted in the accumulation of betanin but not isobetanin, and the presence of a polymorphism in CqCYP76A1-2 in the CQ127 variety was shown to have resulted in its loss of function. These findings suggested that CqCYP76AD1-1 is involved in betalain biosynthesis during the hypocotyl pigmentation process in quinoa. To our knowledge, CqCYP76AD1-1 is the first quinoa gene identified by EMS mutagenesis using a draft gene sequence. Copyright © 2018. Published by Elsevier Inc.

  16. Transcription of genes involved in sulfolipid and polyacyltrehalose biosynthesis of Mycobacterium tuberculosis in experimental latent tuberculosis infection.

    Directory of Open Access Journals (Sweden)

    Jimmy E Rodríguez

    Full Text Available The Influence of trehalose-based glycolipids in the virulence of Mycobacterium tuberculosis (Mtb is recognised; however, the actual role of these cell-wall glycolipids in latent infection is unknown. As an initial approach, we determined by two-dimensional thin-layer chromatography the sulfolipid (SL and diacyltrehalose/polyacyltrehalose (DAT/PAT profile of the cell wall of hypoxic Mtb. Then, qRT-PCR was extensively conducted to determine the transcription profile of genes involved in the biosynthesis of these glycolipids in non-replicating persistent 1 (NRP1 and anaerobiosis (NRP2 models of hypoxia (Wayne model, and murine models of chronic and progressive pulmonary tuberculosis. A diminished content of SL and increased amounts of glycolipids with chromatographic profile similar to DAT were detected in Mtb grown in the NRP2 stage. A striking decrease in the transcription of mmpL8 and mmpL10 transporter genes and increased transcription of the pks (polyketidesynthase genes involved in SL and DAT biosynthesis were detected in both the NRP2 stage and the murine model of chronic infection. All genes were found to be up-regulated in the progressive disease. These results suggest that SL production is diminished during latent infection and the DAT/PAT precursors can be accumulated inside tubercle bacilli and are possibly used in reactivation processes.

  17. [Effect of endophytic fungi on expression amount of key enzyme genes in saponins biosynthesis and Eleutherococcus senticosus saponins content].

    Science.gov (United States)

    Xing, Zhaobin; Long, Yuehong; Lao, Fengyun; He, Shan; Liang, Nengsong; Li, Baocai

    2012-07-01

    To analyze the effect of endophytic fungi on expression amount of key enzyme genes SS (squalene synthase gene), SE (squalene epoxidase gene) and bAS (beta-amyrin synthase gene) in saponin biosynthesis and saponins content in Eleutherococcus senticosus. Wound method was used for back meeting the endophytic fungi to E. senticosus. With GAPDH as internal control gene, the expression of key enzyme genes was detected by real time PCR method. E. senticosus saponins content was measured by spectrophotometry method. When wound method back meeting P116-1a and P116-1b after 30 d, the expression content of SS improved significantly (P endophytic fungi, the expression of SE were significantly higher than the control (P endophytic fungi form 60 d to 120 d, the expression of bAS was significantly higher than the control (P endophytic fungi improved E. senticosus saponins content significantly (P Endophytic fungi P116-1a, P116-1b, P1094 and P312-1 significantly effected the expression of key enzyme genes SS, SE and bAS and then affected E. senticosus saponins content. Among the genes, bAS was key target gene.

  18. High prevalence of diarrheagenic Escherichia coli carrying toxin-encoding genes isolated from children and adults in southeastern Brazil.

    Science.gov (United States)

    Spano, Liliana Cruz; da Cunha, Keyla Fonseca; Monfardini, Mariane Vedovatti; de Cássia Bergamaschi Fonseca, Rita; Scaletsky, Isabel Christina Affonso

    2017-12-18

    Diarrheagenic Escherichia coli (DEC) are important bacterial causes of childhood diarrhea in Brazil, but its impact in adults is unknown. This study aimed at investigating DEC among children and adults living in endemic areas. A total of 327 stools specimens were collected from children (n = 141) and adults (n = 186) with diarrhea attending health centers. Diarrheagenic E. coli (DEC) were identified by their virulence genes (multiplex polymerase chain reaction) and HEp-2 cell adherence patterns. DEC were detected in 56 (40%) children and 74 (39%) adults; enteroaggregative E. coli (EAEC) (23%) was the most prevalent pathotype, followed by diffusely adherent E. coli (DAEC) (13%), and occurred at similar frequencies in both diarrheal groups. Atypical enteropathogenic E. coli (aEPEC) strains were recovered more frequently from children (6%) than from adults (1%). Twenty-six percent of the EAEC were classified as typical EAEC possessing aggR gene, and carried the aap gene. EAEC strains carrying aggR-aap-aatA genes were significantly more frequent among children than adults (p < 0.05). DAEC strains possessing Afa/Dr. genes were detected from children (10%) and adults (6%). EAEC and DAEC strains harboring genes for the EAST1 (astA), Pet, Pic, and Sat toxins were common in both diarrheal groups. The astA and the porcine AE/associated adhesin (paa) genes were found in most of aEPEC strains. High levels of resistance to antimicrobial drugs were found among DAEC and aEPEC isolates. The results show a high proportion of EAEC and DAEC carrying toxin-encoding genes among adults with diarrhea.

  19. Silencing of BnTT1 family genes affects seed flavonoid biosynthesis and alters seed fatty acid composition in Brassica napus.

    Science.gov (United States)

    Lian, Jianping; Lu, Xiaochun; Yin, Nengwen; Ma, Lijuan; Lu, Jing; Liu, Xue; Li, Jiana; Lu, Jun; Lei, Bo; Wang, Rui; Chai, Yourong

    2017-01-01

    TRANSPARENT TESTA1 (TT1) is a zinc finger protein that contains a WIP domain. It plays important roles in controlling differentiation and pigmentation of the seed coat endothelium, and can affect the expression of early biosynthetic genes and late biosynthetic genes of flavonoid biosynthesis in Arabidopsis thaliana. In Brassica napus (AACC, 2n=38), the functions of BnTT1 genes remain unknown and few studies have focused on their roles in fatty acid (FA) biosynthesis. In this study, BnTT1 family genes were silenced by RNA interference, which resulted in yellow rapeseed, abnormal testa development (a much thinner testa), decreased seed weight, and altered seed FA composition in B. napus. High-throughput sequencing of genes differentially expressed between developing transgenic B. napus and wild-type seeds revealed altered expression of numerous genes involved in flavonoid and FA biosynthesis. As a consequence of this altered expression, we detected a marked decrease of oleic acid (C18:1) and notable increases of linoleic acid (C18:2) and α-linolenic acid (C18:3) in mature transgenic B. napus seeds by gas chromatography and near-infrared reflectance spectroscopy. Meanwhile, liquid chromatography-mass spectrometry showed reduced accumulation of flavonoids in transgenic seeds. Therefore, we propose that BnTT1s are involved in the regulation of flavonoid biosynthesis, and may also play a role in FA biosynthesis in B. napus. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Evidence for horizontally transferred genes involved in the biosynthesis of vitamin B(1), B(5), and B(7) in Heterodera glycines.

    Science.gov (United States)

    Craig, James P; Bekal, Sadia; Niblack, Terry; Domier, Leslie; Lambert, Kris N

    2009-12-01

    Heterodera glycines is a nematode that is highly adapted to manipulate and parasitize plant hosts. The molecular players involved in these interactions have only recently begun to be identified. Here, the sequencing of the second stage juvenile transcriptome, followed by a bioinformatic screen for novel genes, identified seven new genes involved in biosynthesis and salvage of vitamins B₁, B₅, and B₇. With no confirmed reports in the literature, each of these biosynthesis pathways is believed to have been lost in multicellular animals. However, eukaryotic-like introns in the genomic sequences of the genes confirmed eukaryotic origin and nematode-specific splice leaders found on five of the cDNAs confirmed their nematode origin. Two of the genes were found to be flanked by known nematode sequences and quantitative polymerase chain reactions on individual nematodes showed similar and consistent amplification between the vitamin B biosynthesis genes and other known H. glycines genes. This further confirmed their presence in the nematode genome. Similarity to bacterial sequences at the amino acid level suggested a prokaryotic ancestry and phylogenetic analysis of the genes supported a likely horizontal gene transfer event, suggesting H. glycines re-appropriated the genes from the prokaryotic kingdom. This finding complements the previous discovery of a vitamin B₆ biosynthesis pathway within the nematode. However, unlike the complete vitamin B₆ pathway, many of these vitamin B pathways appear to be missing the initial enzymes required for full de novo biosynthesis, suggesting that initial substrates in the pathways are obtained exogenously. These partial vitamin B biosynthesis enzymes have recently been identified in other single-celled eukaryotic parasites and on rhizobia symbiosis plasmids, indicating that they may play an important role in host-parasite interactions and survival within the plant environment.

  1. Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content

    Directory of Open Access Journals (Sweden)

    Ning Yan

    2016-11-01

    Full Text Available Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.. In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS, two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD, four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE, two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF, four 1-hydroxy-2-methyl-2-(E-butenyl 4-diphosphate synthase (IspG, two 1-hydroxy-2-methyl-2-(E-butenyl 4-diphosphate reductase (IspH, six isopentenyl diphosphate isomerase (IPI, and two solanesyl diphosphate synthase (SPS candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2, which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.

  2. Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis.

    Science.gov (United States)

    Sievert, Christian; Beuerle, Till; Hollmann, Julien; Ober, Dietrich

    2015-09-01

    Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. A molecular genetic analysis of carotenoid biosynthesis and the effects of carotenoid mutations on other photosynthetic genes in Rhodobacter capsulatus

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, G.A.

    1989-04-01

    The nine known R. capsulatus carotenoid genes are contained within the 46 kilobase (kb) photosynthesis gene cluster. An 11 kb subcluster containing eight of these genes has been cloned and its nucleotide sequence determined. A new gene, crtK, has been located in the middle of the subcluster. The carotenoid gene cluster contains sequences homologous to Escherichia coli ..omega../sup 70/ promoters, rho-independent transcription terminators, and prokaryotic transcriptional factor binding sites. The phenotypes and genotypes of ten transposon Tn5.7 insertion mutations within the carotenoid gene cluster have been analyzed, by characterization of the carotenoids accumulated and high resolution mapping of the Tn5.7 insertions. The enzymatic blockages in previously uncharacterized early carotenoid mutants have been determined using a new in vitro synthesis system, suggesting specific roles for the CrtB and CrtE gene products. The expression of six of the eight carotenoid genes in the cluster is induced upon the shift from dark chemoheterotrophic to anaerobic photosynthetic growth. The magnitude of the induction is equivalent to that of genes encoding structural photosynthesis polypeptides, although the carotenoid genes are induced earlier after the growth shift. Different means of regulating photosynthesis genes in R. capsulatus are discussed, and a rationale for the temporal pattern of expression of the carotenoid genes during photosynthetic adaptation is presented. Comparison of the deduced amino acid sequences of the two dehydrogenases of the R. capsulatus carotenoid biosynthesis pathway reveals two regions of strong similarity. The effect of carotenoid mutations on the photosynthetic phenotype has been studied by examining growth rates, pigments, pigment-protein complexes and gene expression for a complete set of carotenoid mutants. 161 refs.

  4. A molecular genetic analysis of carotenoid biosynthesis and the effects of carotenoid mutations on other photosynthetic genes in Rhodobacter capsulatus

    International Nuclear Information System (INIS)

    Armstrong, G.A.

    1989-04-01

    The nine known R. capsulatus carotenoid genes are contained within the 46 kilobase (kb) photosynthesis gene cluster. An 11 kb subcluster containing eight of these genes has been cloned and its nucleotide sequence determined. A new gene, crtK, has been located in the middle of the subcluster. The carotenoid gene cluster contains sequences homologous to Escherichia coli ω 70 promoters, rho-independent transcription terminators, and prokaryotic transcriptional factor binding sites. The phenotypes and genotypes of ten transposon Tn5.7 insertion mutations within the carotenoid gene cluster have been analyzed, by characterization of the carotenoids accumulated and high resolution mapping of the Tn5.7 insertions. The enzymatic blockages in previously uncharacterized early carotenoid mutants have been determined using a new in vitro synthesis system, suggesting specific roles for the CrtB and CrtE gene products. The expression of six of the eight carotenoid genes in the cluster is induced upon the shift from dark chemoheterotrophic to anaerobic photosynthetic growth. The magnitude of the induction is equivalent to that of genes encoding structural photosynthesis polypeptides, although the carotenoid genes are induced earlier after the growth shift. Different means of regulating photosynthesis genes in R. capsulatus are discussed, and a rationale for the temporal pattern of expression of the carotenoid genes during photosynthetic adaptation is presented. Comparison of the deduced amino acid sequences of the two dehydrogenases of the R. capsulatus carotenoid biosynthesis pathway reveals two regions of strong similarity. The effect of carotenoid mutations on the photosynthetic phenotype has been studied by examining growth rates, pigments, pigment-protein complexes and gene expression for a complete set of carotenoid mutants. 161 refs

  5. Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene.

    NARCIS (Netherlands)

    Teng, S.; Keurentjes, J.J.B.; Bentsink, L.; Koornneef, M.; Smeekens, S.

    2005-01-01

    Sugar-induced anthocyanin accumulation has been observed in many plant species. We observed that sucrose (Suc) is the most effective inducer of anthocyanin biosynthesis in Arabidopsis (Arabidopsis thaliana) seedlings. Other sugars and osmotic controls are either less effective or ineffective.

  6. Overexpression of an ABA biosynthesis gene using a stress inducible promoter enhances drought resistance in petunia

    Science.gov (United States)

    Plants respond to drought stress by closing their stomata and reducing transpirational water loss. The plant hormone abscisic acid (ABA) regulates growth and stomatal closure particularly when the plant is under environmental stresses. One of the key enzymes in the ABA biosynthesis of higher plants ...

  7. Heteroconium chaetospira induces resistance to clubroot via upregulation of host genes involved in jasmonic acid, ethylene, and auxin biosynthesis.

    Directory of Open Access Journals (Sweden)

    Rachid Lahlali

    Full Text Available An endophytic fungus, Heteroconium chaetospira isolate BC2HB1 (Hc, suppressed clubroot (Plasmodiophora brassicae -Pb on canola in growth-cabinet trials. Confocal microscopy demonstrated that Hc penetrated canola roots and colonized cortical tissues. Based on qPCR analysis, the amount of Hc DNA found in canola roots at 14 days after treatment was negatively correlated (r = 0.92, P<0.001 with the severity of clubroot at 5 weeks after treatment at a low (2×10(5 spores pot(-1 but not high (2×10(5 spores pot(-1 dose of pathogen inoculum. Transcript levels of nine B. napus (Bn genes in roots treated with Hc plus Pb, Pb alone and a nontreated control were analyzed using qPCR supplemented with biochemical analysis for the activity of phenylalanine ammonia lyases (PAL. These genes encode enzymes involved in several biosynthetic pathways related potentially to plant defence. Hc plus Pb increased the activity of PAL but not that of the other two genes (BnCCR and BnOPCL involved also in phenylpropanoid biosynthesis, relative to Pb inoculation alone. In contrast, expression of several genes involved in the jasmonic acid (BnOPR2, ethylene (BnACO, auxin (BnAAO1, and PR-2 protein (BnPR-2 biosynthesis were upregulated by 63, 48, 3, and 3 fold, respectively, by Hc plus Pb over Pb alone. This indicates that these genes may be involved in inducing resistance in canola by Hc against clubroot. The upregulation of BnAAO1 appears to be related to both pathogenesis of clubroot and induced defence mechanisms in canola roots. This is the first report on regulation of specific host genes involved in induced plant resistance by a non-mycorrhizal endophyte.

  8. Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica.

    Science.gov (United States)

    Morita, Tomotake; Ito, Emi; Kitamoto, Hiroko K; Takegawa, Kaoru; Fukuoka, Tokuma; Imura, Tomohiro; Kitamoto, Dai

    2010-11-01

    The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast. Copyright © 2010 John Wiley & Sons, Ltd.

  9. A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines β-Diketone Biosynthesis and Glaucousness.

    Science.gov (United States)

    Hen-Avivi, Shelly; Savin, Orna; Racovita, Radu C; Lee, Wing-Sham; Adamski, Nikolai M; Malitsky, Sergey; Almekias-Siegl, Efrat; Levy, Matan; Vautrin, Sonia; Bergès, Hélène; Friedlander, Gilgi; Kartvelishvily, Elena; Ben-Zvi, Gil; Alkan, Noam; Uauy, Cristobal; Kanyuka, Kostya; Jetter, Reinhard; Distelfeld, Assaf; Aharoni, Asaph

    2016-06-01

    The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular β-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of β-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating β-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a β-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in β-diketone biosynthesis, demonstrating a gene cluster also in the β-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response. © 2016 American Society of Plant Biologists. All rights reserved.

  10. Stool C difficile toxin

    Science.gov (United States)

    ... toxin; Colitis - toxin; Pseudomembranous - toxin; Necrotizing colitis - toxin; C difficile - toxin ... be analyzed. There are several ways to detect C difficile toxin in the stool sample. Enzyme immunoassay ( ...

  11. Strengthening Triterpene Saponins Biosynthesis by Over-Expression of Farnesyl Pyrophosphate Synthase Gene and RNA Interference of Cycloartenol Synthase Gene in Panax notoginseng Cells

    Directory of Open Access Journals (Sweden)

    Yan Yang

    2017-04-01

    Full Text Available To conform to the multiple regulations of triterpene biosynthesis, the gene encoding farnesyl pyrophosphate synthase (FPS was transformed into Panax notoginseng (P. notoginseng cells in which RNA interference (RNAi of the cycloartenol synthase (CAS gene had been accomplished. Transgenic cell lines showed both higher expression levels of FPS and lower expression levels of CAS compared to the wild-type (WT cells. In the triterpene and phytosterol analysis, transgenic cell lines provided a higher accumulation of total triterpene saponins, and a lower amount of phytosterols in comparison with the WT cells. Compared with the cells in which RNAi of the CAS gene was achieved, the cells with simultaneously over-expressed FPS and silenced CAS showed higher triterpene contents. These results demonstrate that over-expression of FPS can break the rate-limiting reaction catalyzed by FPS in the triterpene saponins biosynthetic pathway; and inhibition of CAS expression can decrease the synthesis metabolic flux of the phytosterol branch. Thus, more precursors flow in the direction of triterpene synthesis, and ultimately promote the accumulation of P. notoginseng saponins. Meanwhile, silencing and over-expressing key enzyme genes simultaneously is more effective than just manipulating one gene in the regulation of saponin biosynthesis.

  12. Virulence genes, Shiga toxin subtypes, major O-serogroups, and phylogenetic background of Shiga toxin-producing Escherichia coli strains isolated from cattle in Iran.

    Science.gov (United States)

    Jajarmi, Maziar; Imani Fooladi, Abbas Ali; Badouei, Mahdi Askari; Ahmadi, Ali

    2017-08-01

    The aim of this study was to investigate the virulence potential of the isolated bovine STEC for humans in Iran. In this study a collection of STEC strains (n = 50) had been provided via four stages, including sampling from feces of cattle, E. coli isolation, molecular screening of Shiga toxin (stx) genes, and saving the STEC strains from various geographical areas in Iran. The STEC isolates were subjected to stx-subtyping, O-serogrouping, and phylo-grouping by conventional polymerase chain reaction (PCR). Occurrence of stx1 (52%) and stx2 (64%) was not significantly different (p = 0.1), and 16% of isolates carried both stx1 and stx2, simultaneously. In addition, 36% and 80% of the isolates were positive for eae and ehxA, respectively. Molecular subtyping showed that stx1a (52%), stx2a (44%), stx2c (44%), and stx2d (30%) were the most prevalent subtypes; two combinations stx2a/stx2c and stx2c/stx2d coexisted in 18% and 10% of STEC strains, respectively. Three important non-O157 serogroups, including O113 (20%), O26 (12%), and O111 (10%), were predominant, and none of the isolates belonged to O157. Importantly, one O26 isolate carried stx1, stx2, eae and ehxA and revealed highly virulent stx subtypes. Moreover, all the 21 serogrouped strains belonged to the B1 phylo-type. Our study highlights the significance of non-O157 STEC strains carrying highly pathogenic virulence genes in cattle population as the source of this pathogen in Iran. Since non-O157 STEC strains are not routinely tried in most diagnostic laboratories, majority of the STEC-associated human infections appear to be overlooked in the clinical settings. Copyright © 2017. Published by Elsevier Ltd.

  13. Generation of divalent DNA vaccine based on p39 and shiga-like toxin 2 (Stx2 genes

    Directory of Open Access Journals (Sweden)

    Doosti Abbas

    2015-01-01

    Full Text Available The virulence factors such as shiga-like toxin (Stx and immunogenic P39 protein in Escherichia coli and Brucella melitensis are related to disease of digestive system in human worldwide. In the present study the stx2 and p39 genes were cloned into expression plasmid pEEF1D-FLAG (pcDNA 3.1+ as a divalent DNA vaccine candidate. The Enterohemorrhagic E. coli ATCC 3081 and smooth virulent B. melitensis strain M5 were obtained and cultured on specific media. Bacterial DNA was extracted from colonies and was used for p39 and stx2 genes amplification by PCR. The amplified products on 2% agarose gel electrophoresis were revealed 285 and 1220 bp fragments for stx2 and p39 genes, respectively. Each amplified genes were T/A cloned into pGEM-T easy vector and pGEM-T-stx2 and pGEM-T-p39 were produced. The stx2 and p39 genes were sub-cloned in linearized expression vector (pcDNA 3.1+ using HindIII, XhoI and XbaI restriction enzymes and pCDNA3-stx2-p39 was generated. This final construct was confirmed by PCR and enzymes digestion. The results were showed stx2 and p39 genes were sub-cloned, successfully into pcDNA 3.1+ to generate pcDNA 3.1+-stx2-p39 recombinant vector. According to these findings novel recombinant pcDNA 3.1+-stx2- p39 construct that was produced in this study could be useful as DNA vaccine candidate in animal models against shiga-like toxin producing E. coli and virulence B. melitensis strains in future studies.

  14. Effects of subinhibitory concentrations of antibiotics on alpha-toxin (hla) gene expression of methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates.

    Science.gov (United States)

    Ohlsen, K; Ziebuhr, W; Koller, K P; Hell, W; Wichelhaus, T A; Hacker, J

    1998-11-01

    Concentrations of antibiotics below the MIC are able to modulate the expression of virulence-associated genes. In this study, the influence of subinhibitory doses of 31 antibiotics on the expression of the gene encoding the staphylococcal alpha-toxin (hla), a major virulence factor of Staphylococcus aureus, was investigated with a novel gene fusion protocol. The most striking observation was a strong induction of hla expression by subinhibitory concentrations of beta-lactams and an almost complete inhibition of alpha-toxin expression by clindamycin. Whereas glycopeptide antibiotics had no effect, the macrolide erythromycin and several aminoglycosides reduced and fluoroquinolones slightly stimulated hla expression. Furthermore, Northern blot analysis of hla mRNA and Western blot (immunoblot) analysis of culture supernatants of both methicillin-sensitive and methicillin-resistant S. aureus strains revealed that methicillin-induced alpha-toxin expression is a common phenomenon of alpha-toxin-producing strains. Some methicillin-resistant S. aureus isolates produced up to 30-fold more alpha-toxin in the presence of 10 microg of methicillin per ml than in its absence. The results indicate that the novel gene fusion technique is a useful tool for studying the modulation of virulence gene expression by antibiotics. Moreover, the results suggest that the effects of certain antibiotics on virulence properties may be relevant for the management of S. aureus infections.

  15. Effects of a natural toxin on life history and gene expression of Eisenia andrei

    NARCIS (Netherlands)

    van Ommen Kloeke, A.E.E.; Gong, Ping; Ellers, J.; Roelofs, D.

    2014-01-01

    Earthworms perform key functions for a healthy soil ecosystem, such as bioturbation. The soil ecosystem can be challenged by natural toxins such as isothiocyanates (ITCs), produced by many commercial crops. Therefore, the effects of 2-phenylethyl ITC were investigated on the earthworm Eisenia andrei

  16. De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis.

    Science.gov (United States)

    Cárdenas-Conejo, Yair; Carballo-Uicab, Víctor; Lieberman, Meric; Aguilar-Espinosa, Margarita; Comai, Luca; Rivera-Madrid, Renata

    2015-10-28

    Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds from B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. The identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.

  17. Comprehensive Transcriptome Analysis of Phytohormone Biosynthesis and Signaling Genes in the Flowers of Chinese Chinquapin (Castanea henryi).

    Science.gov (United States)

    Fan, Xiaoming; Yuan, Deyi; Tian, Xiaoming; Zhu, Zhoujun; Liu, Meilan; Cao, Heping

    2017-11-29

    Chinese chinquapin (Castanea henryi) nut provides a rich source of starch and nutrients as food and feed, but its yield is restricted by a low ratio of female to male flowers. Little is known about the developmental programs underlying sex differentiation of the flowers. To investigate the involvement of phytohormones during sex differentiation, we described the morphology of male and female floral organs and the cytology of flower sex differentiation, analyzed endogenous levels of indole-3-acetic acid (IAA), gibberellins (GAs), cytokinins (CKs), and abscisic acid (ABA) in the flowers, investigated the effects of exogenous hormones on flower development, and evaluated the expression profiles of genes related to biosyntheses and signaling pathways of these four hormones using RNA-Seq combined with qPCR. Morphological results showed that the flowers consisted of unisexual and bisexual catkins, and could be divided into four developmental stages. HPLC results showed that CK accumulated much more in the female flowers than that in the male flowers, GA and ABA showed the opposite results, while IAA did not show a tendency. The effects of exogenous hormones on sex differentiation were consistent with those of endogenous hormones. RNA-Seq combined with qPCR analyses suggest that several genes may play key roles in hormone biosynthesis and sex differentiation. This study presents the first comprehensive report of phytohormone biosynthesis and signaling during sex differentiation of C. henryi, which should provide a foundation for further mechanistic studies of sex differentiation in Castanea Miller species and other nonmodel plants.

  18. Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

    Science.gov (United States)

    Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

    2016-08-02

    Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F₁-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata.

  19. Exogenous GA3 Application Enhances Xylem Development and Induces the Expression of Secondary Wall Biosynthesis Related Genes in Betula platyphylla

    Directory of Open Access Journals (Sweden)

    Huiyan Guo

    2015-09-01

    Full Text Available Gibberellin (GA is a key signal molecule inducing differentiation of tracheary elements, fibers, and xylogenesis. However the molecular mechanisms underlying the effect of GA on xylem elongation and secondary wall development in tree species remain to be determined. In this study, Betula platyphylla (birch seeds were treated with 300 ppm GA3 and/or 300 ppm paclobutrazol (PAC, seed germination was recorded, and transverse sections of hypocotyls were stained with toluidine blue; the two-month-old seedlings were treated with 50 μM GA3 and/or 50 μM PAC, transverse sections of seedling stems were stained using phloroglucinol–HCl, and secondary wall biosynthesis related genes expression was analyzed by real-time quantitative PCR. Results indicated that germination percentage, energy and time of seeds, hypocotyl height and seedling fresh weight were enhanced by GA3, and reduced by PAC; the xylem development was wider in GA3-treated plants than in the control; the expression of NAC and MYB transcription factors, CESA, PAL, and GA oxidase was up-regulated during GA3 treatment, suggesting their role in GA3-induced xylem development in the birch. Our results suggest that GA3 induces the expression of secondary wall biosynthesis related genes to trigger xylogenesis in the birch plants.

  20. Discovering the role of the apolipoprotein gene and the genes in the putative pullulan biosynthesis pathway on the synthesis of pullulan, heavy oil and melanin in Aureobasidium pullulans.

    Science.gov (United States)

    Guo, Jian; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

    2017-12-18

    Pullulan produced by Aureobasidium pullulans presents various applications in food manufacturing and pharmaceutical industry. However, the pullulan biosynthesis mechanism remains unclear. This work proposed a pathway suggesting that heavy oil and melanin may correlate with pullulan production. The effects of overexpression or deletion of genes encoding apolipoprotein, UDPG-pyrophosphorylase, glucosyltransferase, and α-phosphoglucose mutase on the production of pullulan, heavy oil, and melanin were examined. Pullulan production increased by 16.93 and 8.52% with the overexpression of UDPG-pyrophosphorylase and apolipoprotein genes, respectively. Nevertheless, the overexpression or deletion of other genes exerted little effect on pullulan biosynthesis. Heavy oil production increased by 146.30, 64.81, and 33.33% with the overexpression of UDPG-pyrophosphorylase, α-phosphoglucose mutase, and apolipoprotein genes, respectively. Furthermore, the syntheses of pullulan, heavy oil, and melanin can compete with one another. This work may provide new guidance to improve the production of pullulan, heavy oil, and melanin through genetic approach.

  1. Comparative Transcriptome Analysis of Genes Involved in Anthocyanin Biosynthesis in Red and Green Walnut (Juglans regia L.

    Directory of Open Access Journals (Sweden)

    Yongzhou Li

    2017-12-01

    Full Text Available Fruit color is an important economic trait. The color of red walnut cultivars is mainly attributed to anthocyanins. The aim of this study was to explore the differences in the molecular mechanism of leaf and peel color change between red and green walnut. A reference transcriptome of walnut was sequenced and annotated to identify genes related to fruit color at the ripening stage. More than 290 million high-quality reads were assembled into 39,411 genes using a combined assembly strategy. Using Illumina digital gene expression profiling, we identified 4568 differentially expressed genes (DEGs between red and green walnut leaf and 3038 DEGs between red and green walnut peel at the ripening stage. We also identified some transcription factor families (MYB, bHLH, and WD40 involved in the control of anthocyanin biosynthesis. The trends in the expression levels of several genes encoding anthocyanin biosynthetic enzymes and transcription factors in the leaf and peel of red and green walnut were verified by quantitative real-time PCR. Together, our results identified the genes involved in anthocyanin accumulation in red walnut. These data provide a valuable resource for understanding the coloration of red walnut.

  2. A Comparative Study of Phenolic Antioxidant Activity and Flavonoid Biosynthesis-Related Gene Expression Between Summer and Winter Strawberry Cultivars.

    Science.gov (United States)

    Park, Doori; Park, Yeri; Lee, Young Hun; Choi, Ik-Young; Park, Kyong Cheul; Park, Sang Un; Kim, Byung Sup; Yeoung, Young Rog; Park, Nam Il

    2017-02-01

    Strawberry (Fragaria ananassa Duch.) possesses good antioxidant properties. Phenolic compounds in strawberries, such as anthocyanins and ellagic acid, mainly act as antioxidants. This study aimed to compare the phenolic content and expression patterns of genes involved in flavonoid biosynthesis between summer and winter strawberry cultivars affected by seasonal variation, degree of ripeness, and genotype. Antioxidant activity and the total content of phenols and flavonoids decreased with fruit ripening. Most notably, summer strawberry cultivars showed higher antioxidant activity than winter cultivars. The expression patterns of flavonoid biosynthetic genes tested were cultivar-dependent and were also affected by ripening. These results help us understand the nutritional and physiological characteristics of selected cultivars and provide a range of information for strawberry consumption. © 2017 Institute of Food Technologists®.

  3. Differential expression of ethylene biosynthesis genes in drupelets and receptacle of raspberry (Rubus idaeus).

    Science.gov (United States)

    Fuentes, Lida; Monsalve, Liliam; Morales-Quintana, Luis; Valdenegro, Mónika; Martínez, Juan-Pablo; Defilippi, Bruno G; González-Agüero, Mauricio

    2015-05-01

    Red Raspberry (Rubus idaeus) is traditionally classified as non-climacteric, and the role of ethylene in fruit ripening is not clear. The available information indicates that the receptacle, a modified stem that supports the drupelets, is involved in ethylene production of ripe fruits. In this study, we report receptacle-related ethylene biosynthesis during the ripening of fruits of cv. Heritage. In addition, the expression pattern of ethylene biosynthesis transcripts was evaluated during the ripening process. The major transcript levels of 1-aminocyclopropane-1-carboxylic acid synthase (RiACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (RiACO1) were concomitant with ethylene production, increased total soluble solids (TSS) and decreased titratable acidity (TA) and fruit firmness. Moreover, ethylene biosynthesis and transcript levels of RiACS1 and RiACO1 were higher in the receptacle, sustaining the receptacle's role as a source of ethylene in regulating the ripening of raspberry. Copyright © 2015 Elsevier GmbH. All rights reserved.

  4. An Investigation of Toxic Shock Syndrome Toxin-1 Gene in Methicillin-Resistant Clinical Strains of Staphylococcus aureus using Multiplex PCR Method

    Directory of Open Access Journals (Sweden)

    mohammad bokaiean

    2017-03-01

    Full Text Available Background and Objectives: Toxins produced by the bacteria are one of the most common cases, which can, together with other bacterial pathogens, cause or aggravate the disease. One of the diseases caused by bacterial toxins, is toxic shock syndrome. The tst gene encodes this toxin that can be easily transferred between different strains of Staphylococcus aureus. In this study, toxic shock syndrome toxin-1 gene was investigated in methicillin-resistant clinical strains of Staphylococcus aureus using multiplex PCR method. Methods: This study is a cross-sectional study, during a 9 month period, 470 samples were collected from patients hospitalized in different wards of treatment centers of Zahedan University of Medical Sciences in 2015. Phenotypic method was used for isolation and initial screening. Oxacillin and Cefoxitin discs were used. After isolation of resistant strains, femA and mecA genes and tst gene were investigated using phenotypic method and multiplex PCR method, respectively. Results: Of 170 clinical isolates of Staphylococcus aureus, 93 isolates were phenotypically methicillin-resistant, among which 89 isolates had mecA gene and 14 isolates had tst gene. Conclusion: The results indicated that the prevalence of methicillin-resistant strains and the strains carrying causative gene for TSST1, is high in Zahedan. Also, circulation of these isolates can lead to much more severe effects in individuals with weak immune system.

  5. Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate

    Science.gov (United States)

    Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

    2016-01-01

    Background and Objectives: Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Materials and Methods: Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. Results: The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. Conclusion: We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development. PMID:28210460

  6. Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by ventral veins lacking and knirps.

    Directory of Open Access Journals (Sweden)

    E Thomas Danielsen

    2014-06-01

    Full Text Available Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl and the nuclear receptor Knirps (Kni, have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld, controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

  7. Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes.

    Science.gov (United States)

    Qi, F; Chen, P; Caufield, P W

    1999-09-01

    Previously, members of our group reported the isolation and characterization of mutacin II from Streptococcus mutans T8 and the genetic analyses of the mutacin II biosynthesis genes (J. Novak, P. W. Caufield, and E. J. Miller, J. Bacteriol. 176:4316-4320, 1994; F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:652-658, 1999; P. Chen, F. Qi, J. Novak, and P. W. Caufield, Appl. Environ. Microbiol. 65:1356-1360, 1999). In this study, we cloned and sequenced the mutacin III biosynthesis gene locus from a group III strain of S. mutans, UA787. DNA sequence analysis revealed eight open reading frames, which we designated mutR, -A, -A', -B, -C, -D, -P, and -T. MutR bears strong homology with MutR of mutacin II, while MutA, -B, -C, -D, -P, and -T are counterparts of proteins in the lantibiotic epidermin group. MutA' has 60% amino acid identity with MutA and therefore appears to be a duplicate of MutA. Insertional inactivation demonstrated that mutA is an essential gene for mutacin III production, while mutA' is not required. Mutacin III was purified to homogeneity by using reverse-phase high-pressure liquid chromatography. N-terminal peptide sequencing of the purified mutacin III determined mutA to be the structural gene for prepromutacin III. The molecular mass of the purified peptide was measured by laser disorption mass spectrophotometry and found to be 2,266.43 Da, consistent with our supposition that mutacin III has posttranslational modifications similar to those of the lantibiotic epidermin.

  8. Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays.

    Science.gov (United States)

    Laprade, Natacha; Cloutier, Michel; Lapen, David R; Topp, Edward; Wilkes, Graham; Villemur, Richard; Khan, Izhar U H

    2016-05-01

    Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological

  9. Combining Metabolic Profiling and Gene Expression Analysis to Reveal the Biosynthesis Site and Transport of Ginkgolides in Ginkgo biloba L.

    Science.gov (United States)

    Lu, Xu; Yang, Hua; Liu, Xinguang; Shen, Qian; Wang, Ning; Qi, Lian-wen; Li, Ping

    2017-01-01

    The most unique components of Ginkgo biloba extracts are terpene trilactones (TTLs) including ginkgolides and bilobalide. Study of TTLs biosynthesis has been stagnant in recent years. Metabolic profiling of 40 compounds, including TTLs, flavonoids, and phenolic acids, were globally analyzed in leaf, fibrous root, main root, old stem and young stem extracts of G. biloba. Most of the flavonoids were mainly distributed in the leaf and old stem. Most of phenolic acids were generally distributed among various tissues. The total content of TTLs decreased in the order of the leaf, fibrous root, main root, old stem and young stem. The TTLs were further analyzed in different parts of the main root and old stem. The content of TTLs decreases in the order of the main root periderm, the main root cortex and phloem and the main root xylem. In old stems, the content of TTLs in the cortex and phloem was much higher than both the old stem periderm and xylem. The expression patterns of five key genes in the ginkgolide biosynthetic pathway were measured by real-time quantitative polymerase chain reaction (RT-Q-PCR). Combining metabolic profiling and RT-Q-PCR, the results showed that the fibrous root and main root periderm tissues were the important biosynthesis sites of ginkgolides. Based on the above results, a model of the ginkgolide biosynthesis site and transport pathway in G. biloba was proposed. In this putative model, ginkgolides are synthesized in the fibrous root and main root periderm, and these compounds are then transported through the old stem cortex and phloem to the leaves. PMID:28603534

  10. Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves.

    Science.gov (United States)

    Ji, Xia-Jie; Mao, Xue; Hao, Qing-Ting; Liu, Bao-Ling; Xue, Jin-Ai; Li, Run-Zhi

    2018-01-05

    The plant-specific WRINKLED1 (WRI1) is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean ( Ricinus connunis L.), an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1 , designated as RcWRI1-A and RcWRI1-B . The open reading frames of RcWRI1-A (1341 bp) and RcWRI1-B (1332 bp) differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues "VYL" that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsis wri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3-4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A . Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

  11. Disruption of Transporters Affiliated with Enantio-Pyochelin Biosynthesis Gene Cluster of Pseudomonas protegens Pf-5 Has Pleiotropic Effects.

    Science.gov (United States)

    Lim, Chee Kent; Penesyan, Anahit; Hassan, Karl A; Loper, Joyce E; Paulsen, Ian T

    2016-01-01

    Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens) is a biocontrol bacterium that produces the siderophore enantio-pyochelin under conditions of iron starvation in a process that is often accompanied by the secretion of its biosynthesis intermediates, salicylic acid and dihydroaeruginoic acid. In this study, we investigated whether several transporters that are encoded by genes within or adjacent to the enantio-pyochelin biosynthetic cluster, serve as efflux systems for enantio-pyochelin and/or its intermediates. In addition, we determined whether these transporters have broad substrates range specificity using a Phenotype Microarray system. Intriguingly, knockouts of the pchH and fetF transporter genes resulted in mutant strains that secrete higher levels of enantio-pyochelin as well as its intermediates salicylic acid and dihydroaeruginoic acid. Analyses of these mutants did not indicate significant change in transcription of biosynthetic genes involved in enantio-pyochelin production. In contrast, the deletion mutant of PFL_3504 resulted in reduced transcription of the biosynthetic genes as well as decreased dihydroaeruginoic acid concentrations in the culture supernatant, which could either point to regulation of gene expression by the transporter or its role in dihydroaeruginoic acid transport. Disruption of each of the transporters resulted in altered stress and/or chemical resistance profile of Pf-5, which may reflect that these transporters could have specificity for rather a broad range of substrates.

  12. Disruption of Transporters Affiliated with Enantio-Pyochelin Biosynthesis Gene Cluster of Pseudomonas protegens Pf-5 Has Pleiotropic Effects.

    Directory of Open Access Journals (Sweden)

    Chee Kent Lim

    Full Text Available Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens is a biocontrol bacterium that produces the siderophore enantio-pyochelin under conditions of iron starvation in a process that is often accompanied by the secretion of its biosynthesis intermediates, salicylic acid and dihydroaeruginoic acid. In this study, we investigated whether several transporters that are encoded by genes within or adjacent to the enantio-pyochelin biosynthetic cluster, serve as efflux systems for enantio-pyochelin and/or its intermediates. In addition, we determined whether these transporters have broad substrates range specificity using a Phenotype Microarray system. Intriguingly, knockouts of the pchH and fetF transporter genes resulted in mutant strains that secrete higher levels of enantio-pyochelin as well as its intermediates salicylic acid and dihydroaeruginoic acid. Analyses of these mutants did not indicate significant change in transcription of biosynthetic genes involved in enantio-pyochelin production. In contrast, the deletion mutant of PFL_3504 resulted in reduced transcription of the biosynthetic genes as well as decreased dihydroaeruginoic acid concentrations in the culture supernatant, which could either point to regulation of gene expression by the transporter or its role in dihydroaeruginoic acid transport. Disruption of each of the transporters resulted in altered stress and/or chemical resistance profile of Pf-5, which may reflect that these transporters could have specificity for rather a broad range of substrates.

  13. Juvenile hormone biosynthesis gene expression in the corpora allata of honey bee (Apis mellifera L.) female castes.

    Science.gov (United States)

    Bomtorin, Ana Durvalina; Mackert, Aline; Rosa, Gustavo Conrado Couto; Moda, Livia Maria; Martins, Juliana Ramos; Bitondi, Márcia Maria Gentile; Hartfelder, Klaus; Simões, Zilá Luz Paulino

    2014-01-01

    Juvenile hormone (JH) controls key events in the honey bee life cycle, viz. caste development and age polyethism. We quantified transcript abundance of 24 genes involved in the JH biosynthetic pathway in the corpora allata-corpora cardiaca (CA-CC) complex. The expression of six of these genes showing relatively high transcript abundance was contrasted with CA size, hemolymph JH titer, as well as JH degradation rates and JH esterase (jhe) transcript levels. Gene expression did not match the contrasting JH titers in queen and worker fourth instar larvae, but jhe transcript abundance and JH degradation rates were significantly lower in queen larvae. Consequently, transcriptional control of JHE is of importance in regulating larval JH titers and caste development. In contrast, the same analyses applied to adult worker bees allowed us inferring that the high JH levels in foragers are due to increased JH synthesis. Upon RNAi-mediated silencing of the methyl farnesoate epoxidase gene (mfe) encoding the enzyme that catalyzes methyl farnesoate-to-JH conversion, the JH titer was decreased, thus corroborating that JH titer regulation in adult honey bees depends on this final JH biosynthesis step. The molecular pathway differences underlying JH titer regulation in larval caste development versus adult age polyethism lead us to propose that mfe and jhe genes be assayed when addressing questions on the role(s) of JH in social evolution.

  14. Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B

    Directory of Open Access Journals (Sweden)

    Riley Laura M

    2012-03-01

    Full Text Available Abstract Background Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT™ and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle. Results Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network. Conclusion Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.

  15. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis.

    Science.gov (United States)

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  16. Evolutionary Acquisition and Loss of Saxitoxin Biosynthesis in Dinoflagellates: the Second “Core” Gene, sxtG

    Science.gov (United States)

    Orr, Russell J. S.; Stüken, Anke; Murray, Shauna A.

    2013-01-01

    Saxitoxin and its derivatives are potent neurotoxins produced by several cyanobacteria and dinoflagellate species. SxtA is the initial enzyme in the biosynthesis of saxitoxin. The dinoflagellate full mRNA and partial genomic sequences have previously been characterized, and it appears that sxtA originated in dinoflagellates through a horizontal gene transfer from a bacterium. So far, little is known about the remaining genes involved in this pathway in dinoflagellates. Here we characterize sxtG, an amidinotransferase enzyme gene that putatively encodes the second step in saxitoxin biosynthesis. In this study, the entire sxtG transcripts from Alexandrium fundyense CCMP1719 and Alexandrium minutum CCMP113 were amplified and sequenced. The transcripts contained typical dinoflagellate spliced leader sequences and eukaryotic poly(A) tails. In addition, partial sxtG transcript fragments were amplified from four additional Alexandrium species and Gymnodinium catenatum. The phylogenetic inference of dinoflagellate sxtG, congruent with sxtA, revealed a bacterial origin. However, it is not known if sxtG was acquired independently of sxtA. Amplification and sequencing of the corresponding genomic sxtG region revealed noncanonical introns. These introns show a high interspecies and low intraspecies variance, suggesting multiple independent acquisitions and losses. Unlike sxtA, sxtG was also amplified from Alexandrium species not known to synthesize saxitoxin. However, amplification was not observed for 22 non-saxitoxin-producing dinoflagellate species other than those of the genus Alexandrium or G. catenatum. This result strengthens our hypothesis that saxitoxin synthesis has been secondarily lost in conjunction with sxtA for some descendant species. PMID:23335767

  17. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.

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    Jie Chen

    Full Text Available BACKGROUND: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1 and membrane-bound (Tre-2 trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. PRINCIPAL FINDINGS: The membrane-bound trehalase of Spodoptera exigua (SeTre-2 was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1 and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. CONCLUSIONS: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2

  18. Genomics-Based Discovery of Plant Genes for Synthetic Biology of Terpenoid Fragrances: A Case Study in Sandalwood oil Biosynthesis.

    Science.gov (United States)

    Celedon, J M; Bohlmann, J

    2016-01-01

    Terpenoid fragrances are powerful mediators of ecological interactions in nature and have a long history of traditional and modern industrial applications. Plants produce a great diversity of fragrant terpenoid metabolites, which make them a superb source of biosynthetic genes and enzymes. Advances in fragrance gene discovery have enabled new approaches in synthetic biology of high-value speciality molecules toward applications in the fragrance and flavor, food and beverage, cosmetics, and other industries. Rapid developments in transcriptome and genome sequencing of nonmodel plant species have accelerated the discovery of fragrance biosynthetic pathways. In parallel, advances in metabolic engineering of microbial and plant systems have established platforms for synthetic biology applications of some of the thousands of plant genes that underlie fragrance diversity. While many fragrance molecules (eg, simple monoterpenes) are abundant in readily renewable plant materials, some highly valuable fragrant terpenoids (eg, santalols, ambroxides) are rare in nature and interesting targets for synthetic biology. As a representative example for genomics/transcriptomics enabled gene and enzyme discovery, we describe a strategy used successfully for elucidation of a complete fragrance biosynthetic pathway in sandalwood (Santalum album) and its reconstruction in yeast (Saccharomyces cerevisiae). We address questions related to the discovery of specific genes within large gene families and recovery of rare gene transcripts that are selectively expressed in recalcitrant tissues. To substantiate the validity of the approaches, we describe the combination of methods used in the gene and enzyme discovery of a cytochrome P450 in the fragrant heartwood of tropical sandalwood, responsible for the fragrance defining, final step in the biosynthesis of (Z)-santalols. © 2016 Elsevier Inc. All rights reserved.

  19. The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean.

    Science.gov (United States)

    Barraza, Aarón; Cabrera-Ponce, José L; Gamboa-Becerra, Roberto; Luna-Martínez, Francisco; Winkler, Robert; Álvarez-Venegas, Raúl

    2015-01-01

    Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

  20. The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean

    Directory of Open Access Journals (Sweden)

    Aarón eBarraza

    2015-07-01

    Full Text Available Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

  1. TetR Is a Positive Regulator of the Tetanus Toxin Gene in Clostridium tetani and Is Homologous to BotR

    NARCIS (Netherlands)

    Marvaud, Jean-Christophe; Eisel, Ulrich; Binz, Thomas; Niemann, Heiner; Popoff, Michel R.

    1998-01-01

    The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) in Clostridium botulinum. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a

  2. Study on ecdysteroid levels and gene expression of enzymes related to ecdysteroid biosynthesis in the larval testis of Spodoptera littoralis.

    Science.gov (United States)

    Iga, Masatoshi; Blais, Catherine; Smagghe, Guy

    2013-01-01

    We investigated here the ecdysteroid titers and the expression of six genes coding for known enzymes of the ecdysteroid biosynthesis in the testes of last instar larvae of the pest cotton leafworm, Spodoptera littoralis. We showed that the timing of the ecdysteroid profile was the same in testes and in hemolymph, with a small peak at day 2 and a large one at day 4 after ecdysis. Ecdysone and 20-hydroxyecdysone (20E) were detected in both tissues. 20E was the major ecdysteroid in testes and in hemolymph from day 4. Interestingly, the gene expression of the steroidogenetic enzymes, Neverland, and the five cytochrome P450 enzymes encoded by the Halloween genes was confirmed in the testes, and varied during the instar. However, from the data obtained so far, we cannot conclude that the measured ecdysteroids in the testes result from the activity of the genes under study. Indeed, it is suggested that the ecdysone produced centrally in the prothoracic glands, could have been transformed into 20E in the testes, where Sl-shade is well expressed. © 2012 Wiley Periodicals, Inc.

  3. Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily (Lilium spp.).

    Science.gov (United States)

    Lai, Yun-Song; Shimoyamada, Yoshihiro; Nakayama, Masayoshi; Yamagishi, Masumi

    2012-09-01

    Anthocyanin biosynthesis is often regulated by MYB transcription factors that are classified into AN2 and C1 subgroups. The AN2 subgroup regulates the late genes in the anthocyanin biosynthesis pathway of eudicots, whereas the C1 subgroup controls both early and late genes in monocots. Anthocyanin is a major pigment in Asiatic hybrid lilies (Lilium spp.), with LhMYB12 being the first AN2 subgroup in monocots. In this study, the accumulation of pigments and gene transcripts during flower bud development was evaluated to determine the genes regulated by LhMYB12. LhMYB12 and anthocyanin biosynthesis genes showed the same transcription profiles, with LhMYB12 directly activating the promoters of chalcone synthase and dihydroflavonol 4-reductase. This indicates that LhMYB12 regulates both early and late genes, despite belonging to the AN2 subgroup. The cultivar Landini accumulated anthocyanin and flavonol. The contents of these pigments increased during the late stages of flower bud development; this might result from the coordinated expression of early and late genes. During the early stages of flower bud development, the tepals contained no flavonoids but accumulated cinnamic acid derivatives. These results indicate that the profiles of pigment accumulation and gene transcription in lily tepals are unique among angiosperm flowers. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  4. Genome-Wide Characterization of bHLH Genes in Grape and Analysis of their Potential Relevance to Abiotic Stress Tolerance and Secondary Metabolite Biosynthesis

    Science.gov (United States)

    Wang, Pengfei; Su, Ling; Gao, Huanhuan; Jiang, Xilong; Wu, Xinying; Li, Yi; Zhang, Qianqian; Wang, Yongmei; Ren, Fengshan

    2018-01-01

    Basic helix-loop-helix (bHLH) transcription factors are involved in many abiotic stress responses as well as flavonol and anthocyanin biosynthesis. In grapes (Vitis vinifera L.), flavonols including anthocyanins and condensed tannins are most abundant in the skins of the berries. Flavonols are important phytochemicals for viticulture and enology, but grape bHLH genes have rarely been examined. We identified 94 grape bHLH genes in a genome-wide analysis and performed Nr and GO function analyses for these genes. Phylogenetic analyses placed the genes into 15 clades, with some remaining orphans. 41 duplicate gene pairs were found in the grape bHLH gene family, and all of these duplicate gene pairs underwent purifying selection. Nine triplicate gene groups were found in the grape bHLH gene family and all of these triplicate gene groups underwent purifying selection. Twenty-two grape bHLH genes could be induced by PEG treatment and 17 grape bHLH genes could be induced by cold stress treatment including a homologous form of MYC2, VvbHLH007. Based on the GO or Nr function annotations, we found three other genes that are potentially related to anthocyanin or flavonol biosynthesis: VvbHLH003, VvbHLH007, and VvbHLH010. We also performed a cis-acting regulatory element analysis on some genes involved in flavonoid or anthocyanin biosynthesis and our results showed that most of these gene promoters contained G-box or E-box elements that could be recognized by bHLH family members. PMID:29449854

  5. Genome-Wide Characterization of bHLH Genes in Grape and Analysis of their Potential Relevance to Abiotic Stress Tolerance and Secondary Metabolite Biosynthesis

    Directory of Open Access Journals (Sweden)

    Pengfei Wang

    2018-02-01

    Full Text Available Basic helix-loop-helix (bHLH transcription factors are involved in many abiotic stress responses as well as flavonol and anthocyanin biosynthesis. In grapes (Vitis vinifera L., flavonols including anthocyanins and condensed tannins are most abundant in the skins of the berries. Flavonols are important phytochemicals for viticulture and enology, but grape bHLH genes have rarely been examined. We identified 94 grape bHLH genes in a genome-wide analysis and performed Nr and GO function analyses for these genes. Phylogenetic analyses placed the genes into 15 clades, with some remaining orphans. 41 duplicate gene pairs were found in the grape bHLH gene family, and all of these duplicate gene pairs underwent purifying selection. Nine triplicate gene groups were found in the grape bHLH gene family and all of these triplicate gene groups underwent purifying selection. Twenty-two grape bHLH genes could be induced by PEG treatment and 17 grape bHLH genes could be induced by cold stress treatment including a homologous form of MYC2, VvbHLH007. Based on the GO or Nr function annotations, we found three other genes that are potentially related to anthocyanin or flavonol biosynthesis: VvbHLH003, VvbHLH007, and VvbHLH010. We also performed a cis-acting regulatory element analysis on some genes involved in flavonoid or anthocyanin biosynthesis and our results showed that most of these gene promoters contained G-box or E-box elements that could be recognized by bHLH family members.

  6. Genome-Wide Characterization of bHLH Genes in Grape and Analysis of their Potential Relevance to Abiotic Stress Tolerance and Secondary Metabolite Biosynthesis.

    Science.gov (United States)

    Wang, Pengfei; Su, Ling; Gao, Huanhuan; Jiang, Xilong; Wu, Xinying; Li, Yi; Zhang, Qianqian; Wang, Yongmei; Ren, Fengshan

    2018-01-01

    Basic helix-loop-helix (bHLH) transcription factors are involved in many abiotic stress responses as well as flavonol and anthocyanin biosynthesis. In grapes ( Vitis vinifera L.), flavonols including anthocyanins and condensed tannins are most abundant in the skins of the berries. Flavonols are important phytochemicals for viticulture and enology, but grape bHLH genes have rarely been examined. We identified 94 grape bHLH genes in a genome-wide analysis and performed Nr and GO function analyses for these genes. Phylogenetic analyses placed the genes into 15 clades, with some remaining orphans. 41 duplicate gene pairs were found in the grape bHLH gene family, and all of these duplicate gene pairs underwent purifying selection. Nine triplicate gene groups were found in the grape bHLH gene family and all of these triplicate gene groups underwent purifying selection. Twenty-two grape bHLH genes could be induced by PEG treatment and 17 grape bHLH genes could be induced by cold stress treatment including a homologous form of MYC2, VvbHLH007. Based on the GO or Nr function annotations, we found three other genes that are potentially related to anthocyanin or flavonol biosynthesis: VvbHLH003, VvbHLH007, and VvbHLH010. We also performed a cis-acting regulatory element analysis on some genes involved in flavonoid or anthocyanin biosynthesis and our results showed that most of these gene promoters contained G-box or E-box elements that could be recognized by bHLH family members.

  7. A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine.

    Science.gov (United States)

    Guillén, P; Guis, M; Martínez-Reina, G; Colrat, S; Dalmayrac, S; Deswarte, C; Bouzayen, M; Roustan, J P; Fallot, J; Pech, J C; Latché, A

    1998-11-01

    Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA. This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii. Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3-substituted benzaldehydes. Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.

  8. Hemagglutinin gene shuffling among Clostridium botulinum serotypes C and D yields distinct sugar recognition of the botulinum toxin complex.

    Science.gov (United States)

    Miyata, Keita; Suzuki, Tomonori; Hayashi, Shintaro; Miyashita, Shin-Ichiro; Ohyama, Tohru; Niwa, Koichi; Watanabe, Toshihiro; Sagane, Yoshimasa

    2015-10-01

    Clostridium botulinum strains produce a large-sized toxin complex (TC) that is composed of botulinum neurotoxin (BoNT), non-toxic non-hemagglutinin and three different hemagglutinins (HA-70, HA-33 and HA-17). HA components enhance toxin delivery across the intestinal cell wall in a sugar chain-dependent manner. Here we characterized the sugar recognition of serotype D strain 1873 (D-1873) botulinum L-TC. Most L-TCs produced by serotype C and D strains bind to cells via interactions between HA-33 and cell surface sialo-oligosaccharides. However, like the previously reported L-TC produced by serotype C strain Yoichi (C-Yoichi), D-1873 L-TC binds only to cells that have been treated with neuraminidase, indicating that they recognize asialo-oligosaccharides. The D-1873 HA-33 amino acid sequence is similar to that of C-Yoichi, but had lower similarity to the majority of serotype C and D HA-33s. A comparison of TC component primary structures for 12 serotype C and D strains suggested that at least three types of HA-33 genes exist, and these are shuffled among the serotype C and D strains independently of BoNT serotype. This shuffling produces the distinct sugar recognition of serotype C and D botulinum TCs. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Polyamines are essential for virulence in Salmonella enterica serovar Gallinarum despite evolutionary decay of polyamine biosynthesis genes

    DEFF Research Database (Denmark)

    Schroll, Casper; Christensen, Jens P.; Christensen, Henrik

    2014-01-01

    -specificity coincides with accumulation of pseudogenes, indicating adaptation of host-restricted serovars to their narrow niches. Polyamines are small cationic amines and in Salmonella they can be synthesized through two alternative pathways directly from l-ornithine to putrescine and from l-arginine via agmatine...... to putrescine. The first pathway is not active in S. Gallinarum and S. Typhi, and this prompted us to investigate the importance of polyamines for virulence in S. Gallinarum. Bioinformatic analysis of all sequenced genomes of Salmonella revealed that pseudogene formation of the speC gene was exclusive for S....... Typhi and S. Gallinarum and happened through independent events. The remaining polyamine biosynthesis pathway was found to be essential for oral infection with S. Gallinarum since single and double mutants in speB and speE, encoding the pathways from agmatine to putrescine and from putrescine...

  10. Identification and Interrogation of the Herbicidin Biosynthetic Gene Cluster: First Insight into the Biosynthesis of a Rare Undecose Nucleoside Antibiotic.

    Science.gov (United States)

    Lin, Geng-Min; Romo, Anthony J; Liem, Priscilla H; Chen, Zhang; Liu, Hung-Wen

    2017-11-22

    Herbicidins are adenosine-based nucleoside antibiotics with an unusual tricyclic undecose core decorated with a (5-hydroxy)tiglyl moiety. Feeding studies are herein reported demonstrating that the tricyclic core is derived from d-glucose and d-ribose, whereas the tiglyl moiety is derived from an intermediate of l-isoleucine catabolism. Identification of the gene cluster for herbicidin A biosynthesis in Streptomyces sp. L-9-10 as well as its verification by heterologous expression in a nonproducing host are described, and the results of in vitro characterization of a carboxyl methyltransferase encoded in the cluster, Her8, are presented. Based on these observations, a biosynthetic pathway is proposed for herbicidins.

  11. The border sequence of the balhimycin biosynthesis gene cluster from Amycolatopsis balhimycina contains bbr, encoding a StrR-like pathway-specific regulator

    NARCIS (Netherlands)

    Shawky, Riham M.; Puk, Oliver; Wietzorrek, Andreas; Pelzer, Stefan; Takano, Eriko; Wohlleben, Wolfgang; Stegmann, Efthimia

    2007-01-01

    Balhimycin, produced by the actinomycete Amycolatopsis balhimycina DSM5908, is a glycopeptide antibiotic highly similar to vancomycin, the antibiotic of 'last resort' used for the treatment of resistant Gram-positive pathogenic bacteria. Partial sequence of the balhimycin biosynthesis gene cluster

  12. Subgenome-specific assembly of vitamin E biosynthesis genes and expression patterns during seed development provide insight into the evolution of the oat genome

    Science.gov (United States)

    Vitamin E is essential for humans and thus must be a component of a healthy diet. Among the cereal grains, hexaploid oats (Avena sativa L.) have high vitamin E content. To date, no gene sequences in the vitamin E biosynthesis pathway have been reported for oats. Using deep sequencing and orthology-g...

  13. Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal.

    Science.gov (United States)

    Pal, Shaifali; Yadav, Akhilesh Kumar; Singh, Anup Kumar; Rastogi, Shubhra; Gupta, Madan Mohan; Verma, Rajesh Kumar; Nagegowda, Dinesh A; Pal, Anirban; Shasany, Ajit Kumar

    2017-01-01

    The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

  14.  Mutations of noncollagen genes in osteogenesis imperfecta – implications of the gene products in collagen biosynthesis and pathogenesis of disease

    Directory of Open Access Journals (Sweden)

    Anna Galicka

    2012-06-01

    Full Text Available  Recent investigations revealed that the “brittle bone” phenotype in osteogenesis imperfecta (OI is caused not only by dominant mutations in collagen type I genes, but also by recessively inherited mutations in genes responsible for the post-translational processing of type I procollagen as well as for bone formation. The phenotype of patients with mutations in noncollagen genes overlaps with very severe type III and lethal type II OI caused by mutations in collagen genes. Mutations in genes that encode proteins involved in collagen prolyl 3-hydroxylation (P3H1/CRTAP/CyPB eliminated Pro986 hydroxylation and caused an increase in modification of collagen helix by prolyl 4-hydroxylase and lysyl hydroxylase. However, the importance of these disturbances in the disease pathomechanism is not known. Loss of complex proteins’ function as collagen chaperones may dominate the disease mechanism. The latest findings added to the spectrum of OI-causing and collagen-influencing factors other chaperones (HSP47 and FKBP65 and protein BMP-1, which emphasizes the complexity of collagen folding and secretion as well as their importance in bone formation. Furthermore, mutations in genes encoding transcription factor SP7/Osterix and pigment epithelium-derived factor (PEDF constitute a novel mechanism for OI, which is independent of changes in biosynthesis and processing of collagen.

  15. Carbohydrate-related genes and cell wall biosynthesis in vascular tissues of loblolly pine (Pinus taeda).

    Science.gov (United States)

    Nairn, Campbell J; Lennon, Denise M; Wood-Jones, Alicia; Nairn, Alison V; Dean, Jeffrey F D

    2008-07-01

    Loblolly pine (Pinus taeda L.), the most widely planted tree species in the United States, is an important source of wood and wood fibers for a multitude of consumer products. Wood fibers are primarily composed of secondary cell walls, and cellulose, hemicelluloses and lignin are major components of wood. Fiber morphology and cell wall composition are important determinants of wood properties. We used comparative genomics to identify putative genes for cellulose and hemicellulose synthesis in loblolly pine that are homologous to genes implicated in cell wall synthesis in angiosperms. Sequences encoding putative secondary cell wall cellulose synthase genes, cellulose synthase-like genes, a membrane-bound endoglucanase gene, a sucrose synthase gene, a UDP-glucose pyrophosphorylase gene and GDP-mannose pyrophosphorylase genes were identified in expressed sequence tag (EST) collections from loblolly pine. Full-length coding sequences were obtained from cDNA clones isolated from a library constructed from developing xylem. Phylogenetic relationships between the genes from loblolly pine and angiosperm taxa were examined and transcriptional profiling in vascular tissues was conducted by real-time quantitative, reverse transcriptase-polymerase chain reaction. The putative cell wall synthesis genes were expressed at high levels in vascular tissues and a subset was differentially regulated in xylem and phloem tissues. Inferred phylogenetic relationships and expression patterns for the genes from loblolly pine were consistent with roles in synthesis of complex carbohydrates of the cell wall. These studies suggest functional conservation of homologous wood formation genes in gymnosperm and angiosperm taxa.

  16. Purification and biochemical characterization of mutacin I from the group I strain of Streptococcus mutans, CH43, and genetic analysis of mutacin I biosynthesis genes.

    Science.gov (United States)

    Qi, F; Chen, P; Caufield, P W

    2000-08-01

    Previously, we reported isolation and characterization of mutacin III and genetic analysis of mutacin III biosynthesis genes from the group III strain of Streptococcus mutans, UA787 (F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:3880-3887, 1999). During the same process of isolating the mutacin III structural gene, we also cloned the structural gene for mutacin I. In this report, we present purification and biochemical characterization of mutacin I from the group I strain CH43 and compare mutacin I and mutacin III biosynthesis genes. The mutacin I biosynthesis gene locus consists of 14 genes in the order mutR, -A, -A', -B, -C, -D, -P, -T, -F, -E, -G, orfX, orfY, orfZ. mutA is the structural gene for mutacin I, while mutA' is not required for mutacin I activity. DNA and protein sequence analysis revealed that mutacins I and III are homologous to each other, possibly arising from a common ancestor. The mature mutacin I is 24 amino acids in size and has a molecular mass of 2, 364 Da. Ethanethiol modification and peptide sequencing of mutacin I revealed that it contains six dehydrated serines, four of which are probably involved with thioether bridge formation. Comparison of the primary sequence of mutacin I with that of mutacin III and epidermin suggests that mutacin I likely has the same bridging pattern as epidermin.

  17. Genome-Wide Identification and Functional Characterization of UDP-Glucosyltransferase Genes Involved in Flavonoid Biosynthesis in Glycine max.

    Science.gov (United States)

    Yin, Qinggang; Shen, Guoan; Di, Shaokang; Fan, Cunying; Chang, Zhenzhan; Pang, Yongzhen

    2017-09-01

    Flavonoids, natural products abundant in the model legume Glycine max, confer benefits to plants and to animal health. Flavonoids are present in soybean mainly as glycoconjugates. However, the mechanisms of biosynthesis of flavonoid glycosides are largely unknown in G. max. In the present study, 212 putative UDP-glycosyltransferase (UGT) genes were identified in G. max by genome-wide searching. The GmUGT genes were distributed differentially among the 20 chromosomes, and they were expressed in various tissues with distinct expression profiles. We further analyzed the enzymatic activities of 11 GmUGTs that are potentially involved in flavonoid glycosylation, and found that six of them (UGT72X4, UGT72Z3, UGT73C20, UGT88A13, UGT88E19 and UGT92G4) exhibited activity toward flavonol, isoflavone, flavone and flavanol aglycones with different kinetic properties. Among them, UGT72X4, UGT72Z3 and UGT92G4 are flavonol-specific UGTs, and UGT73C20 and UGT88E19 exhibited activity toward both flavonol and isoflavone aglycones. In particular, UGT88A13 exhibited activity toward epicatechin, but not for the flavonol aglycones kaempferol and quercetin. Overexpression of these six GmUGT genes significantly increased the contents of isoflavone and flavonol glucosides in soybean hairy roots. In addition, overexpression of these six GmUGT genes also affected flavonol glycoside contents differently in seedlings and seeds of transgenic Arabidopsis thaliana. We provide valuable information on the identification of all UGT genes in soybean, and candidate GmUGT genes for potential metabolic engineering of flavonoid compounds in both Escherichia coli and plants. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Point-of-care and visual detection of P. aeruginosa and its toxin genes by multiple LAMP and lateral flow nucleic acid biosensor.

    Science.gov (United States)

    Chen, Yuting; Cheng, Nan; Xu, Yuancong; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-07-15

    This study describes a simple and sensitive approach for visual and point-of-care detection of P. aeruginosa and its toxin genes based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow nucleic acid biosensor (LFNAB). Differentiation of the internal standard gene ecfX and toxin genes (ExoS and ExoU) in P. aeruginosa was determined using FITC-, hex-and digoxin-modified primers in the mLAMP process. In the presence of biotin-and FITC- (hex-, digoxin-) modified primers and Bst DNA polymerase large fragments, the mLAMP produced numerous biotin- and FITC- (hex-, digoxin-) attached duplex DNA products. The products were detected by LFNAB through dual immunoreactions (anti-biotin antibodies on the gold nanoparticle (Au-NP) and biotin on the duplex, anti-FITC (hex, digoxin) antibodies on the LFNAB test line and FITC (hex, digoxin) on the duplex). The accumulation of Au-NPs produced a characteristic red band, enabling visual detection of P. aeruginosa and its toxin genes without instrumentation. After systematic optimization of LFNAB preparation and detecting conditions, the current approach was capable of detecting concentrations as low as 20 CFU/mL P. aeruginosa or its toxin genes within 50min without complicated instrument, which is more sensitive than PCR. Therefore, this approach provides a simple, pollution free, sensitive, and low-cost point-of-care test for the detection of P. aeruginosa and its toxin genes. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Role of the precorrin 6-X reductase gene in cobamide biosynthesis in Methanococcus maripaludis.

    Science.gov (United States)

    Kim, Wonduck; Major, Tiffany A; Whitman, William B

    2005-12-01

    In Methanococcus maripaludis strain JJ, deletion of the homolog to cbiJ, which encodes the corrin biosynthetic enzyme precorrin 6-X reductase, yielded an auxotroph that required either cobamide or acetate for good growth. This phenotype closely resembled that of JJ117, a mutant in which tandem repeats were introduced into the region immediately downstream of the homolog of cbiJ. Mutant JJ117 also produced low quantities of cobamides, about 15 nmol g(-1) protein or 1-2% of the amount found in wild-type cells. These results confirm the role of the cbiJ homolog in cobamide biosynthesis in the Archaea and suggest the presence of low amounts of a bypass activity in these organisms.

  20. Role of the precorrin 6-X reductase gene in cobamide biosynthesis in Methanococcus maripaludis

    Directory of Open Access Journals (Sweden)

    Wonduck Kim

    2005-01-01

    Full Text Available In Methanococcus maripaludis strain JJ, deletion of the homolog to cbiJ, which encodes the corrin biosynthetic enzyme precorrin 6-X reductase, yielded an auxotroph that required either cobamide or acetate for good growth. This phenotype closely resembled that of JJ117, a mutant in which tandem repeats were introduced into the region immediately downstream of the homolog of cbiJ. Mutant JJ117 also produced low quantities of cobamides, about 15 nmol g–1 protein or 1–2% of the amount found in wild-type cells. These results confirm the role of the cbiJ homolog in cobamide biosynthesis in the Archaea and suggest the presence of low amounts of a bypass activity in these organisms.

  1. Expression of the Vibrio cholerae gene encoding aldehyde dehydrogenase is under control of ToxR, the cholera toxin transcriptional activator.

    OpenAIRE

    Parsot, C; Mekalanos, J J

    1991-01-01

    The toxR gene of Vibrio cholerae encodes a transcriptional activator required for the expression of the cholera toxin genes (ctxAB) and more than 15 other genes encoding secreted or membrane proteins. The latter group includes virulence genes involved in the biogenesis of the TCP pilus, the accessory colonization factor, and such ToxR-activated genes as tagA, mutations in which cause no detectable virulence defect in the suckling mouse model. To analyze the regulation of expression and the st...

  2. Relationship between virulence gene profiles of atypical enteropathogenic Escherichia coli and Shiga toxin-producing E. coli isolates from cattle and sheep in New Zealand.

    Science.gov (United States)

    Cookson, Adrian L; Cao, Mingshu; Bennett, Jenny; Nicol, Carolyn; Thomson-Carter, Fiona; Attwood, Graeme T

    2010-06-01

    Virulence gene profiles of atypical enteropathogenic Escherichia coli (aEPEC) and Shiga toxin-producing E. coli (STEC) from cattle, sheep, and humans were examined to determine the relationship between pathotypes. Shared virulence factors (intimin, EHEC hemolysin, serine protease, and a type II secretion system) were identified, suggesting a dynamic evolutionary relationship between aEPEC and STEC.

  3. Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli.

    NARCIS (Netherlands)

    Noguera, P.S.; Posthuma-Trumpie, G.A.; Tuil, Van M.; Wal, van der F.J.; Boer, De A.; Moers, A.P.H.A.; Amerongen, Van A.

    2011-01-01

    The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1,

  4. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammal...

  5. Tissue-specific gene-expression patterns of genes associated with thymol/carvacrol biosynthesis in thyme (Thymus vulgaris L.) and their differential changes upon treatment with abiotic elicitors

    DEFF Research Database (Denmark)

    Majdi, Mohammad; Malekzadeh-Mashhady, Atefe; Maroufi, Asad

    2017-01-01

    leaves and transcript levels of early (DXR) and late (TvTPS1, CYP71D178 and CYP71D180) biosynthetic genes of thymol and carvacrol were measured. The results showed that early step and late step genes in thymol/carvacrol biosynthesis are differentially regulated. DXR was not found to be exclusively...

  6. Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

    DEFF Research Database (Denmark)

    Schembri, Mark; Neilan, B.A.; Saint, C.P.

    2001-01-01

    of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena...

  7. Characterization and expression patterns of key ecdysteroid biosynthesis and signaling genes in a spider mite (Panonychus citri).

    Science.gov (United States)

    Li, Gang; Niu, Jin-Zhi; Zotti, Moises; Sun, Qin-Zhe; Zhu, Lin; Zhang, Jun; Liao, Chong-Yu; Dou, Wei; Wei, Dan-Dan; Wang, Jin-Jun; Smagghe, Guy

    2017-08-01

    Ecdysteroids play a crucial role in regulating molting in the phylum of Arthropoda and much is known with members of the subphylum of Hexapoda including the Insecta. However, this is still unclear in key pests as spider mites belonging to the subphylum of Chelicerata that originated earlier in the Cambrian period. In this study, we investigated 14 key genes of ecdysteroid biosynthesis and signaling and their expression over the different developmental stages in the citrus red mite, Panonychus citri (Acari: Stigmaeidae). P. citri is an economically important and widespread pest of citrus crops and it has five developmental stages of egg, larva, protonymph, deutonymph and adult. Typically, the expression of the ecdysteroid-synthesizing Halloween gene Spook (PcSpo) followed a positive zigzag-like pattern with a peak in the first half of each developmental stage and a drop in the second half prior to the molting to the next stage. Similar to PcSpo, PcDib, PcSad, PcRXR2, PcE75 and PcHR38 showed a positive zigzag-like expression pattern, while that of PcE78, PcHR3 and PcFTZ-F1 was opposite that we called a negative zigzag-like pattern. Silencing of the PcSpo gene by RNAi showed that molting was inhibited. Interestingly, we could rescue these RNAi effects by supplementing ponasterone A (PonA) and not by 20E, which is indicative that mites use PonA rather than 20E as ecdysteroid hormone. Modeling of the ecdysteroid receptor (PcEcR) hormone binding cavity also predicted binding of PonA, but showed a steric hindrance for 20E. We believe our data provide insight into the evolution and expression patterns of key ecdysteroid biosynthesis and signaling genes in a distant, non-insect species, and can become a foundation to develop new targets for controlling important agricultural pests such as spider mites. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Cell wall composition and candidate biosynthesis gene expression during rice development

    DEFF Research Database (Denmark)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra

    2016-01-01

    , we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had...... strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically...

  9. Botrydial and botcinins produced by Botrytis cinerea regulate the expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis.

    Science.gov (United States)

    Malmierca, Mónica G; Izquierdo-Bueno, Inmaculada; Mccormick, Susan P; Cardoza, Rosa E; Alexander, Nancy J; Moraga, Javier; Gomes, Eriston V; Proctor, Robert H; Collado, Isidro G; Monte, Enrique; Gutiérrez, Santiago

    2016-09-01

    Trichoderma arundinaceum IBT 40837 (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides and botcinins (Botcs), respectively. We analysed the role of BOT and Botcs in the Ta37-B. cinerea interaction, including the transcriptomic changes in the genes involved in HA (tri) and ergosterol biosynthesis, as well as changes in the level of HA and squalene-ergosterol. We found that, when confronted with B. cinerea, the tri biosynthetic genes were up-regulated in all dual cultures analysed, but at higher levels when Ta37 was confronted with the BOT non-producer mutant bcbot2Δ. The production of HA was also higher in the interaction area with this mutant. In Ta37-bcbot2Δ confrontation experiments, the expression of the hmgR gene, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the first enzyme of the terpene biosynthetic pathway, was also up-regulated, resulting in an increase in squalene production compared with the confrontation with B. cinerea B05.10. Botcs had an up-regulatory effect on the tri biosynthetic genes, with BotcA having a stronger effect than BotcB. The results indicate that the interaction between Ta37 and B. cinerea exerts a stimulatory effect on the expression of the tri biosynthetic genes, which, in the interaction zone, can be attenuated by BOT produced by B. cinerea B05.10. The present work provides evidence for a metabolic dialogue between T. arundinaceum and B. cinerea that is mediated by sesquiterpenes and polyketides, and that affects the outcome of the interaction of these fungi with each other and their environment. © 2015 BSPP and John Wiley & Sons Ltd.

  10. Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization.

    Science.gov (United States)

    Nasopoulou, Constantina; Pohjanen, Johanna; Koskimäki, Janne J; Zabetakis, Ioannis; Pirttilä, Anna Maria

    2014-08-15

    Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF. Copyright © 2014. Published by Elsevier GmbH.

  11. Genes involved in sex pheromone biosynthesis of Ephestia cautella, an important food storage pest, are determined by transcriptome sequencing

    KAUST Repository

    Antony, Binu

    2015-07-18

    Background Insects use pheromones, chemical signals that underlie all animal behaviors, for communication and for attracting mates. Synthetic pheromones are widely used in pest control strategies because they are environmentally safe. The production of insect pheromones in transgenic plants, which could be more economical and effective in producing isomerically pure compounds, has recently been successfully demonstrated. This research requires information regarding the pheromone biosynthetic pathways and the characterization of pheromone biosynthetic enzymes (PBEs). We used Illumina sequencing to characterize the pheromone gland (PG) transcriptome of the Pyralid moth, Ephestia cautella, a destructive storage pest, to reveal putative candidate genes involved in pheromone biosynthesis, release, transport and degradation. Results We isolated the E. cautella pheromone compound as (Z,E)-9,12-tetradecadienyl acetate, and the major pheromone precursors 16:acyl, 14:acyl, E14-16:acyl, E12-14:acyl and Z9,E12-14:acyl. Based on the abundance of precursors, two possible pheromone biosynthetic pathways are proposed. Both pathways initiate from C16:acyl-CoA, with one involving ∆14 and ∆9 desaturation to generate Z9,E12-14:acyl, and the other involving the chain shortening of C16:acyl-CoA to C14:acyl-CoA, followed by ∆12 and ∆9 desaturation to generate Z9,E12-14:acyl-CoA. Then, a final reduction and acetylation generates Z9,E12-14:OAc. Illumina sequencing yielded 83,792 transcripts, and we obtained a PG transcriptome of ~49.5 Mb. A total of 191 PBE transcripts, which included pheromone biosynthesis activating neuropeptides, fatty acid transport proteins, acetyl-CoA carboxylases, fatty acid synthases, desaturases, β-oxidation enzymes, fatty acyl-CoA reductases (FARs) and fatty acetyltransferases (FATs), were selected from the dataset. A comparison of the E. cautella transcriptome data with three other Lepidoptera PG datasets revealed that 45 % of the sequences were shared

  12. The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098

    NARCIS (Netherlands)

    Santos, F.; Vera, J.L.; van der Heijden, R.; Valdez, G.; de Vos, W.M.; Sesma, F.; Hugenholtz, J.

    2008-01-01

    The coenzyme B12 production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B12 gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29

  13. The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL 1098

    NARCIS (Netherlands)

    Santos, dos F.; Vera, J.L.; Heijden, van der R.; Valdez, G.F.; Vos, de W.M.; Sesma, F.; Hugenholtz, J.

    2008-01-01

    The coenzyme B12 production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B12 gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29

  14. Distribution and evolution of genes responsible for biosynthesis of mycotoxins in Fusarium

    Science.gov (United States)

    Fusarium secondary metabolites (SMs) include some of the mycotoxins of greatest concern to food and feed safety. In fungi, genes directly involved in synthesis of the same SM are typically located adjacent to one another in gene clusters. To better understand the distribution and evolution of mycoto...

  15. Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes.

    Science.gov (United States)

    Brückner, Kathleen; Božić, Dragana; Manzano, David; Papaefthimiou, Dimitra; Pateraki, Irini; Scheler, Ulschan; Ferrer, Albert; de Vos, Ric C H; Kanellis, Angelos K; Tissier, Alain

    2014-05-01

    Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

    Science.gov (United States)

    Douëllou, T; Delannoy, S; Ganet, S; Mariani-Kurkdjian, P; Fach, P; Loukiadis, E; Montel, Mc; Thevenot-Sergentet, D

    2016-09-02

    Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Incidence, Antimicrobial Susceptibility, and Toxin Genes Possession Screening of Staphylococcus aureus in Retail Chicken Livers and Gizzards

    Directory of Open Access Journals (Sweden)

    Lubna S. Abdalrahman

    2015-04-01

    Full Text Available Few recent outbreaks in Europe and the US involving Campylobacter and Salmonella were linked to the consumption of chicken livers. Studies investigating Staphylococcus aureus in chicken livers and gizzards are very limited. The objectives of this study were to determine the prevalence, antimicrobial resistance, and virulence of S. aureus and MRSA (Methicillin-Resistant Staphylococcus aureus in retail chicken livers and gizzards in Tulsa, Oklahoma. In this study, 156 chicken livers and 39 chicken gizzards samples of two brands were collected. While one of the brands showed very low prevalence of 1% (1/100 for S. aureus in chicken livers and gizzards, the second brand showed prevalence of 37% (31/95. No MRSA was detected since none harbored the mecA or mecC gene. Eighty seven S. aureus isolates from livers and 28 from gizzards were screened for antimicrobial resistance to 16 antimicrobials and the possession of 18 toxin genes. Resistance to most of the antimicrobials screened including cefoxitin and oxacillin was higher in the chicken gizzards isolates. While the prevalence of enterotoxin genes seg and sei was higher in the gizzards isolates, the prevalence of hemolysin genes hla, hlb, and hld was higher in the livers ones. The lucocidin genes lukE-lukD was equally prevalent in chicken livers and gizzards isolates. Using spa typing, a subset of the recovered isolates showed that they are not known to be livestock associated and, hence, may be of a human origin. In conclusion, this study stresses the importance of thorough cooking of chicken livers and gizzards since it might contain multidrug resistant enterotoxigenic S. aureus. To our knowledge this is the first study to specifically investigate the prevalence of S. aureus in chicken livers and gizzards in the US.

  18. Arabidopsis PAD3, a gene required for camalexin biosynthesis, encodes a putative cytochrome P450 monooxygenase.

    Science.gov (United States)

    Zhou, N; Tootle, T L; Glazebrook, J

    1999-12-01

    Phytoalexins are low molecular weight antimicrobial compounds that are synthesized in response to pathogen attack. The phytoalexin camalexin, an indole derivative, is produced by Arabidopsis in response to infection with the bacterial pathogen Pseudomonas syringae. The phytoalexin deficient 3 (pad3) mutation, which causes a defect in camalexin production, has no effect on resistance to P. syringae but compromises resistance to the fungal pathogen Alternaria brassicicola. We have now isolated PAD3 by map-based cloning. The predicted PAD3 protein appears to be a cytochrome P450 monooxygenase, similar to those from maize that catalyze synthesis of the indole-derived secondary metabolite 2,4-dihydroxy-1, 4-benzoxazin-3-one. The expression of PAD3 is tightly correlated with camalexin synthesis and is regulated by PAD4 and PAD1. On the basis of these findings, we conclude that PAD3 almost certainly encodes an enzyme required for camalexin biosynthesis. Moreover, these results strongly support the idea that camalexin does not play a major role in plant resistance to P. syringae infection, although it is involved in resistance to a fungal pathogen.

  19. Gene fusions for the directed modification of the carotenoid biosynthesis pathway in Mucor circinelloides.

    Science.gov (United States)

    Iturriaga, Enrique A; Papp, Tamás; Alvarez, María Isabel; Eslava, Arturo P

    2012-01-01

    Several fungal species, particularly some included in the Mucorales, have been used to develop fermentation processes for the production of β-carotene. Oxygenated derivatives of β-carotene are more valuable products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides as a model organism to develop strains able to produce new, more valuable, and with an increased content of carotenoids. In this chapter we describe part of our efforts to construct active gene fusions which could advance in the diversification of carotenoid production by this fungus. The main carotenoid accumulated by M. circinelloides is β-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. Two enzymatic activities are required for the production of astaxanthin from β-carotene: a hydroxylase and a ketolase. We used the ctrW gene of Paracoccus sp. N81106, encoding a bacterial β-carotene ketolase, to construct gene fusions with two fungal genes essential for the modification of the pathway in M. circinelloides. First we fused it to the carRP gene of M. circinelloides, which is responsible for the phytoene synthase and lycopene cyclase activities in this fungus. The expected activity of this fusion gene would be the accumulation by M. circinelloides of canthaxanthin and probably some astaxanthin. A second construction was the fusion of the crtW gene of Paracoccus sp. to the crtS gene of Xanthophyllomyces dendrorhous, responsible for the synthesis of astaxanthin from β-carotene in this fungus, but which was shown to have only hydroxylase activity in M. circinelloides. The expected result in M. circinelloides transformants was the accumulation of astaxanthin. Here we describe a detailed and empirically tested protocol for the construction of these gene fusions.

  20. In vitro polyploidy induction: changes in morphology, podophyllotoxin biosynthesis, and expression of the related genes in Linum album (Linaceae).

    Science.gov (United States)

    Javadian, Neda; Karimzadeh, Ghasem; Sharifi, Mohsen; Moieni, Ahmad; Behmanesh, Mehrdad

    2017-06-01

    Induction of tetraploidy was performed and podophyllotoxin production increased by upregulating the expression level and enzyme activity of genes related to its biosynthesis in tetraploid compared to diploid Linum album. Linum album is a valuable medicinal plant that produces antiviral and anticancer compounds including podophyllotoxin (PTOX). To achieve homogeneous materials, in vitro diploid clones were established, and their nodal segments were exposed to different concentrations and durations of colchicine. This resulted in successful tetraploidy induction, confirmed by flow cytometry, and is being reported for the first time. The highest efficiency of tetraploid induction (22%) was achieved after 72 h exposure to 2.5-mM colchicine treatment. The stable tetraploids were produced after being subcultured three times, and their ploidy stability was confirmed after each subculture. The effects of autopolyploidy were measured on the morphological and phytochemical characteristics, as well as enzyme activity and the expression levels of some key genes involved in the PTOX biosynthetic pathway, including phenylalanine ammonia-lyase (PAL), cinnamoyl-Coa reductase (CCR), cinnamyl-alcohol dehydrogenase (CAD), and pinoresinol-lariciresinol reductase (PLR). The tetraploid plants had larger leaves and stomata (length and width) and lower density stomata. Increasing the ploidy level from diploid to tetraploid resulted in 1.39- and 1.23-fold enhancement of PTOX production, respectively, in the leaves and stem. The increase in PTOX content was associated with upregulated activities of some enzymes studied related to its biosynthetic pathway and the expression of the corresponding genes. The expression of the PAL gene and PLR enzymatic activity had the most positive correlation with the ploidy level in both leaf and stem tissues. Our results verified that autotetraploid induction is a useful breeding method, remarkably increasing the PTOX content in the leaves and stem of L

  1. Transcriptional profiling of genes involved in ascorbic acid biosynthesis, recycling, and degradation during three leaf developmental stages in celery.

    Science.gov (United States)

    Huang, Wei; Wang, Guang-Long; Li, Hui; Wang, Feng; Xu, Zhi-Sheng; Xiong, Ai-Sheng

    2016-12-01

    Ascorbic acid (AsA) is an important nutrient in the human body and performs various healthy functions. With considerable medicinal properties, celery (Apium graveolens L.) could be a good source of AsA for human health. However, the biosynthetic, recycling, and degradation pathways of AsA in celery have yet to be characterized. To study the metabolic pathways involved in AsA, the genes involved in AsA biosynthesis, recycling, and degradation were isolated from celery, and their expression profiles and AsA levels were analyzed in the leaf blades and petioles of two celery varieties at three different growth stages. AsA levels were higher in 'Ventura' compared with 'Liuhehuangxinqin' in both tissues possibly because of different transcription levels of genes, such as L-galactose dehydrogenase (GalDH), L-galactono-1,4-lactone dehydrogenase (GalLDH), and glutathione reductase (GR). Results revealed that the D-mannose/L-galactose pathway may be the predominant pathway in celery, and the D-galacturonic acid pathway appeared to contribute largely to AsA accumulation in petioles than in leaf blades in 'Liuhehuangxinqin.' AsA contents are regulated by complex regulatory mechanisms and vary at different growth stages, tissues, and varieties in celery. The results provide novel insights into AsA metabolic pathways in leaf during celery growth and development.

  2. Transcriptome Analysis to Identify the Putative Biosynthesis and Transport Genes Associated with the Medicinal Components of Achyranthes bidentata Bl.

    Directory of Open Access Journals (Sweden)

    Jinting Li

    2016-12-01

    Full Text Available Achyranthes bidentata is a popular perennial medicine herb used for thousands of years in China to treat various diseases. Although this herb has multiple pharmaceutical purposes in China, no transcriptomic information has been reported for this species. In addition, the understanding of several key pathways and enzymes involved in the biosynthesis of oleanolic acid and ecdysterone, two pharmacologically active classes of metabolites and major chemical constituents of A. bidentata root extracts, is limited. The aim of the present study was to characterize the transcriptome profile of the roots and leaves of A. bidentata to uncover the biosynthetic and transport mechanisms of the active components. In this study, we identified 100,987 transcripts, with an average length of 973.64 base pairs. A total of 31,634 (31.33% unigenes were annotated, and 12,762 unigenes were mapped to 303 pathways according to the Kyoto Encyclopedia of Genes and Genomes (KEGG pathway database. Moreover, we identified a total of 260 oleanolic acid and ecdysterone genes encoding biosynthetic enzymes. Furthermore, the key enzymes involved in the oleanolic acid and ecdysterone synthesis pathways were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR, revealing that the roots expressed these enzymes to a greater extent than the leaves. In addition, we identified 85 ATP-binding cassette (ABC transporters, some of which might be involved in the translocation of secondary metabolites.

  3. Expression and Anthocyanin Biosynthesis-Modulating Potential of Sweet Cherry (Prunus avium L.) MYB10 and bHLH Genes.

    Science.gov (United States)

    Starkevič, Pavel; Paukštytė, Jurgita; Kazanavičiūtė, Vaiva; Denkovskienė, Erna; Stanys, Vidmantas; Bendokas, Vidmantas; Šikšnianas, Tadeušas; Ražanskienė, Aušra; Ražanskas, Raimundas

    2015-01-01

    Anthocyanins are essential contributors to fruit coloration, an important quality feature and a breed determining trait of a sweet cherry fruit. It is well established that the biosynthesis of anthocyanins is regulated by an interplay of specific transcription factors belonging to MYB and bHLH families accompanied by a WD40 protein. In this study, we isolated and analyzed PaWD40, PabHLH3, PabHLH33, and several closely related MYB10 gene variants from different cultivars of sweet cherry, analyzed their expression in fruits with different anthocyanin levels at several developmental stages, and determined their capabilities to modulate anthocyanin synthesis in leaves of two Nicotiana species. Our results indicate that transcription level of variant PaMYB10.1-1 correlates with fruit coloration, but anthocyanin synthesis in Nicotiana was induced by another variant, PaMYB10.1-3, which is moderately expressed in fruits. The analysis of two fruit-expressed bHLH genes revealed that PabHLH3 enhances MYB-induced anthocyanin synthesis, whereas PabHLH33 has strong inhibitory properties.

  4. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; Bassard, Jean-Étienne André; Andersen-Ranberg, Johan

    2014-01-01

    describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly...... making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps...

  5. Mapping of polyketide biosynthesis pathways in Aspergillus nidulans using a genome wide PKS gene deletion library

    DEFF Research Database (Denmark)

    Larsen, Thomas Ostenfeld; Rank, Christian; Klejnstrup, Marie Louise

    In order to map new links between PKS genes and their products in Aspergillus nidulans we have systematically deleted all thirty-two individual genes predicted to encode polyketide synthases in this model organism. This number greatly exceeds the number of currently known PKs calling for new...... approaches for triggering “cryptic” or “silent” genes to see production of compounds not previously observed under laboratory conditions. We therefore decided to challenge our deletion library on eight different complex media, spanning a large variety of alternating carbon and nitrogen sources, vitamins...

  6. Sequencing and transcriptional analysis of the biosynthesis gene cluster of abscisic acid-producing Botrytis cinerea.

    Science.gov (United States)

    Gong, Tao; Shu, Dan; Yang, Jie; Ding, Zhong-Tao; Tan, Hong

    2014-09-29

    Botrytis cinerea is a model species with great importance as a pathogen of plants and has become used for biotechnological production of ABA. The ABA cluster of B. cinerea is composed of an open reading frame without significant similarities (bcaba3), followed by the genes (bcaba1 and bcaba2) encoding P450 monooxygenases and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). In B. cinerea ATCC58025, targeted inactivation of the genes in the cluster suggested at least three genes responsible for the hydroxylation at carbon atom C-1' and C-4' or oxidation at C-4' of ABA. Our group has identified an ABA-overproducing strain, B. cinerea TB-3-H8. To differentiate TB-3-H8 from other B. cinerea strains with the functional ABA cluster, the DNA sequence of the 12.11-kb region containing the cluster of B. cinerea TB-3-H8 was determined. Full-length cDNAs were also isolated for bcaba1, bcaba2, bcaba3 and bcaba4 from B. cinerea TB-3-H8. Sequence comparison of the four genes and their flanking regions respectively derived from B. cinerea TB-3-H8, B05.10 and T4 revealed that major variations were located in intergenic sequences. In B. cinerea TB-3-H8, the expression profiles of the four function genes under ABA high-yield conditions were also analyzed by real-time PCR.

  7. Small and Smaller-sRNAs and MicroRNAs in the Regulation of Toxin Gene Expression in Prokaryotic Cells: A Mini-Review.

    Science.gov (United States)

    Bloch, Sylwia; Węgrzyn, Alicja; Węgrzyn, Grzegorz; Nejman-Faleńczyk, Bożena

    2017-05-30

    Non-coding small RNAs (sRNAs) have been identified in the wide range of bacteria (also pathogenic species) and found to play an important role in the regulation of many processes, including toxin gene expression. The best characterized prokaryotic sRNAs regulate gene expression by base pairing with mRNA targets and fall into two broad classes: cis -encoded sRNAs (also called antisense RNA) and trans -acting sRNAs. Molecules from the second class are frequently considered as the most related to eukaryotic microRNAs. Interestingly, typical microRNA-size RNA molecules have also been reported in prokaryotic cells, although they have received little attention up to now. In this work we have collected information about all three types of small prokaryotic RNAs in the context of the regulation of toxin gene expression.

  8. The Onion (Allium cepa L.) R2R3-MYB Gene MYB1 Regulates Anthocyanin Biosynthesis

    Science.gov (United States)

    Schwinn, Kathy E.; Ngo, Hanh; Kenel, Fernand; Brummell, David A.; Albert, Nick W.; McCallum, John A.; Pither-Joyce, Meeghan; Crowhurst, Ross N.; Eady, Colin; Davies, Kevin M.

    2016-01-01

    Bulb color is an important consumer trait for onion (Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow), and chalcones (bright yellow). Flavonoid regulation is poorly characterized in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressed and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic (Allium sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum majus of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species. PMID:28018399

  9. The onion (Allium cepa L. R2R3-MYB gene MYB1 regulates anthocyanin biosynthesis

    Directory of Open Access Journals (Sweden)

    Kathy Schwinn

    2016-12-01

    Full Text Available Bulb colour is an important consumer trait for onion (Allium cepa L., Allioideae, Asparagales. The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red, flavonols (pale yellow and chalcones (bright yellow. Flavonoid regulation is poorly characterised in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs that commonly activate anthocyanin (SG6, MYB1 or flavonol (SG7, MYB29 production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5. MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressd and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic (A. sativum L. plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum majus of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species.

  10. The Onion (Allium cepa L.) R2R3-MYB Gene MYB1 Regulates Anthocyanin Biosynthesis.

    Science.gov (United States)

    Schwinn, Kathy E; Ngo, Hanh; Kenel, Fernand; Brummell, David A; Albert, Nick W; McCallum, John A; Pither-Joyce, Meeghan; Crowhurst, Ross N; Eady, Colin; Davies, Kevin M

    2016-01-01

    Bulb color is an important consumer trait for onion ( Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow), and chalcones (bright yellow). Flavonoid regulation is poorly characterized in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressed and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic ( Allium sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum maju s of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species.

  11. Expression of the Native Cholera Toxin B Subunit Gene and Assembly as Functional Oligomers in Transgenic Tobacco Chloroplasts

    Science.gov (United States)

    Daniell, Henry; Lee, Seung-Bum; Panchal, Tanvi; Wiebe, Peter O.

    2012-01-01

    The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast-synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast- synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of

  12. Prevalence of shiga toxin-producing Escherichia coli stx1, stx2, eaeA, and rfbE genes and survival of E. coli O157:H7 in manure from organic and low-input conventional dairy farms

    NARCIS (Netherlands)

    Franz, E.; Klerks, M.M.; Vos, de O.J.; Termorshuizen, A.J.; Bruggen, van A.H.C.

    2007-01-01

    Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx(1) (encoding Shiga toxin 1), stx(2) (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as

  13. RAPTOR gene polymorphism is independently correlated with urothelial cancer susceptibility compared with environmental toxin exposure

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    Hao Lun Luo

    2017-12-01

    Conclusion: RAPTOR gene polymorphisms are important SNPs with significantly association with the risk of urothelial cancer in Taiwan. Further researches about raptor-mTOR complex interfering malignant transformation of urothelium is worthy of further investigation.

  14. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

    Directory of Open Access Journals (Sweden)

    Daniel Mihálik

    2015-12-01

    Full Text Available The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v and 0%–1.53% (v/v from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat.

  15. Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

    Science.gov (United States)

    Maurer, John J.; Schmidt, Denise; Petrosko, Patricia; Sanchez, Susan; Bolton, Lance; Lee, Margie D.

    1999-01-01

    The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157. PMID:10388689

  16. Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis.

    Science.gov (United States)

    Liu, L; Shaw, P D

    1997-01-01

    The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis. PMID:8990304

  17. Genetics of Dothistromin Biosynthesis of Dothistroma septosporum: An Update

    Directory of Open Access Journals (Sweden)

    Rosie E. Bradshaw

    2010-11-01

    Full Text Available Dothistroma needle blight is one of the most devastating fungal pine diseases worldwide. The disease is characterized by accumulation in pine needles of a red toxin, dothistromin, that is chemically related to aflatoxin (AF and sterigmatocystin (ST. This review updates current knowledge of the genetics of dothistromin biosynthesis by the Dothistroma septosporum pathogen and highlights differences in gene organization and regulation that have been discovered between the dothistromin and AF/ST systems. Some previously reported genes are promoted or demoted as ‘dothistromin genes’ based on recent research. A new dothistromin gene, norB, is reported, and evidence of dothistromin gene homologs in other Dothideomycete fungi is presented. A hypothesis for the biological role of dothistromin is outlined. Finally, the impact that the availability of the D. septosporum genome sequence will have on dothistromin research is discussed.

  18. Biosynthesis of Antinutritional Alkaloids in Solanaceous Crops Is Mediated by Clustered Genes

    NARCIS (Netherlands)

    Itkin, M.; Heinig, U.; Tzfadia, O.; Bhide, A.J.; Shinde, B.; Cardenas, P.D.; Bocobza, S.E.; Unger, T.; Malitsky, S.; Finkers, H.J.; Tikunov, Y.M.; Bovy, A.G.; Chikate, Y.; Singh, P.; Rogachev, I.; Beekwilder, J.; Giri, A.P.; Aharoni, A.

    2013-01-01

    Steroidal glycoalkaloids (SGAs) such as a-solanine found in solanaceous food plants—as, for example, potato—are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA

  19. Comparative Analysis and Distribution of Omega-3 lcPUFA Biosynthesis Genes in Marine Molluscs.

    Directory of Open Access Journals (Sweden)

    Joachim M Surm

    Full Text Available Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs, based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad and elongation of very long-chain fatty acid (Elovl genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess

  20. Comparative Analysis and Distribution of Omega-3 lcPUFA Biosynthesis Genes in Marine Molluscs.

    Science.gov (United States)

    Surm, Joachim M; Prentis, Peter J; Pavasovic, Ana

    2015-01-01

    Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the

  1. Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis.

    Science.gov (United States)

    Kato, Shota; Takaichi, Shinichi; Ishikawa, Takahiro; Asahina, Masashi; Takahashi, Senji; Shinomura, Tomoko

    2016-01-05

    Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 μmol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 μmol m(-2) s(-1)). When grown under continuous light at 920 μmol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined. We identified genes

  2. Biosynthesis of mono-acylated mannosylerythritol lipid in an acyltransferase gene-disrupted mutant of Pseudozyma tsukubaensis.

    Science.gov (United States)

    Saika, Azusa; Utashima, Yu; Koike, Hideaki; Yamamoto, Shuhei; Kishimoto, Takahide; Fukuoka, Tokuma; Morita, Tomotake

    2018-02-01

    The basidiomycetous yeast genus Pseudozyma produce large amounts of mannosylerythritol lipids (MELs), which are biosurfactants. A few Pseudozyma strains produce mono-acylated MEL as a minor compound using excess glucose as the sole carbon source. Mono-acylated MEL shows higher hydrophilicity than di-acylated MEL and has great potential for aqueous applications. Recently, the gene cluster involved in the MEL biosynthesis pathway was identified in yeast. Here, we generated an acyltransferase (PtMAC2) deletion strain of P. tsukubaensis 1E5 with uracil auxotrophy as a selectable marker. A PtURA5-mutant with a frameshift mutation in PtURA5 was generated as a uracil auxotroph of strain 1E5 by ultraviolet irradiation on plate medium containing 5-fluoro-orotic acid (5-FOA). In the mutant, PtMAC2 was replaced with a PtURA5 cassette containing the 5' untranslated region (UTR) (2000 bp) and 3' UTR (2000 bp) of PtMAC2 by homologous recombination, yielding strain ΔPtMAC2. Based on TLC and NMR analysis, we found that ΔPtMAC2 accumulates MEL acylated at the C-2' position of the mannose moiety. These results indicate that PtMAC2p catalyzes acylation at the C-3' position of the mannose of MEL.

  3. Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis▿

    Science.gov (United States)

    Botts, Michael R.; Giles, Steven S.; Gates, Marcellene A.; Kozel, Thomas R.; Hull, Christina M.

    2009-01-01

    Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts. PMID:19181873

  4. Isolation and characterisation of starch biosynthesis genes from cassava (Manihot esculenta Crantz)

    OpenAIRE

    Munyikwa, T.R.I.

    1997-01-01


    Cassava (Manihot esculenta Crantz) is a tropical crop grown for its starchy thickened roots, mainly by peasant farmers, in the tropics, for whom it is a staple food. There is an increasing demand for the use of cassava in processed food and feed products, and in the paper and textile industries amongst others. This thesis describes research on the cloning of the genes encoding ADP-glucose pyrophosphorylase small and large subunits (AGPase B and S, respectively)...

  5. Gene cluster analysis for the biosynthesis of elgicins, novel lantibiotics produced by paenibacillus elgii B69

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    Teng Yi

    2012-03-01

    Full Text Available Abstract Background The recent increase in bacterial resistance to antibiotics has promoted the exploration of novel antibacterial materials. As a result, many researchers are undertaking work to identify new lantibiotics because of their potent antimicrobial activities. The objective of this study was to provide details of a lantibiotic-like gene cluster in Paenibacillus elgii B69 and to produce the antibacterial substances coded by this gene cluster based on culture screening. Results Analysis of the P. elgii B69 genome sequence revealed the presence of a lantibiotic-like gene cluster composed of five open reading frames (elgT1, elgC, elgT2, elgB, and elgA. Screening of culture extracts for active substances possessing the predicted properties of the encoded product led to the isolation of four novel peptides (elgicins AI, AII, B, and C with a broad inhibitory spectrum. The molecular weights of these peptides were 4536, 4593, 4706, and 4820 Da, respectively. The N-terminal sequence of elgicin B was Leu-Gly-Asp-Tyr, which corresponded to the partial sequence of the peptide ElgA encoded by elgA. Edman degradation suggested that the product elgicin B is derived from ElgA. By correlating the results of electrospray ionization-mass spectrometry analyses of elgicins AI, AII, and C, these peptides are deduced to have originated from the same precursor, ElgA. Conclusions A novel lantibiotic-like gene cluster was shown to be present in P. elgii B69. Four new lantibiotics with a broad inhibitory spectrum were isolated, and these appear to be promising antibacterial agents.

  6. Steroids and genes related to steroid biosynthesis in the female giant freshwater prawn, Macrobrachium rosenbergii.

    Science.gov (United States)

    Thongbuakaew, Tipsuda; Siangcham, Tanapan; Suwansa-ard, Saowaros; Elizur, Abigail; Cummins, Scott F; Sobhon, Prasert; Sretarugsa, Prapee

    2016-03-01

    The giant freshwater prawn, Macrobrachium rosenbergii, is important to many Asian countries due to its high economic value as an aquaculture product. With demand increasing, there is requirement for a better understanding of the biosynthetic components that regulate its growth and reproduction, including steroids, in order to help increase production. Vertebrate-type steroids and their receptors were identified in crustaceans and implicated in reproduction. In this study, we presented the sex steroids estradiol and progesterone by LC-MS/MS in female M. rosenbergii, and reveal steroidogenic-related genes by in silico analysis of de novo assembled transcriptomes. Comparative analysis with other species was performed to confirm their putative role, as well as tissue-specific and quantitative gene expression. We reveal 29 transcripts that encode for steroidogenic-related proteins, including steroidogenic enzymes, a nuclear steroid hormone receptors, and a steroidogenic factor. Moreover, we identified for the first time the presence of steroidogenic factor 1, StAR-related lipid transfer protein, estradiol receptor- and progesterone-like protein in M. rosenbergii. Those targeted for gene expression analysis (3 beta-hydroxysteroid dehydrogenase, 17 beta-hydroxysteroid dehydrogenase, estrogen sulfotransferase and progesterone receptor-like) showed widespread expression within many tissues, and at relatively high levels in the central nervous system (CNS) during ovarian maturation. In summary, we provide further evidence for the existence of steroidogenic pathways in crustaceans, which may be useful for advancing prawn aquaculture. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries.

    Science.gov (United States)

    Castellarin, Simone D; Matthews, Mark A; Di Gaspero, Gabriele; Gambetta, Gregory A

    2007-12-01

    Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera "Cabernet Sauvignon", and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3'4'5'-hydroxylated forms through the differential regulation of F3'H and F3'5'H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.

  8. Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster.

    Science.gov (United States)

    Gilbert, Lawrence I

    2004-02-27

    Mutation of members of the Halloween gene family results in embryonic lethality. We have shown that two of these genes code for enzymes responsible for specific steps in the synthesis of ecdysone, a polyhydroxylated sterol that is the precursor of the major molting hormone of all arthropods, 20-hydroxyecdysone. These two mitochondrial P450 enzymes, coded for by disembodied (dib) (CYP302A1) and shadow (sad) (CYP315A1), are the C22 and C2 hydroxylases, respectively, as shown by transfection of the gene into S2 cells and subsequent biochemical analysis. These are the last two enzymes in the ecdysone biosynthetic pathway. A third enzyme, necessary for the critical conversion of ecdysone to 20-hydroxyecdysone, the 20-monooxygenase, is encoded by shade (shd) (CYP314A1). All three enzymes are mitochondrial although shade has motifs suggesting both mitochondrial and microsomal locations. By tagging these enzymes, their subcellular location has been confirmed by confocal microscopy. Shade is present in several tissues as expected while disembodied and shadow are restricted to the ring gland. The paradigm used should allow us to define the enzymes mediating the entire ecdysteroid biosynthetic pathway.

  9. Expression Comparison of Oil Biosynthesis Genes in Oil Palm Mesocarp Tissue Using Custom Array

    Science.gov (United States)

    Wong, Yick Ching; Kwong, Qi Bin; Lee, Heng Leng; Ong, Chuang Kee; Mayes, Sean; Chew, Fook Tim; Appleton, David R.; Kulaveerasingam, Harikrishna

    2014-01-01

    Gene expression changes that occur during mesocarp development are a major research focus in oil palm research due to the economic importance of this tissue and the relatively rapid increase in lipid content to very high levels at fruit ripeness. Here, we report the development of a transcriptome-based 105,000-probe oil palm mesocarp microarray. The expression of genes involved in fatty acid (FA) and triacylglycerol (TAG) assembly, along with the tricarboxylic acid cycle (TCA) and glycolysis pathway at 16 Weeks After Anthesis (WAA) exhibited significantly higher signals compared to those obtained from a cross-species hybridization to the Arabidopsis (p-value < 0.01), and rice (p-value < 0.01) arrays. The oil palm microarray data also showed comparable correlation of expression (r2 = 0.569, p < 0.01) throughout mesocarp development to transcriptome (RNA sequencing) data, and improved correlation over quantitative real-time PCR (qPCR) (r2 = 0.721, p < 0.01) of the same RNA samples. The results confirm the advantage of the custom microarray over commercially available arrays derived from model species. We demonstrate the utility of this custom microarray to gain a better understanding of gene expression patterns in the oil palm mesocarp that may lead to increasing future oil yield. PMID:27600348

  10. Expression Comparison of Oil Biosynthesis Genes in Oil Palm Mesocarp Tissue Using Custom Array

    Directory of Open Access Journals (Sweden)

    Yick Ching Wong

    2014-11-01

    Full Text Available Gene expression changes that occur during mesocarp development are a major research focus in oil palm research due to the economic importance of this tissue and the relatively rapid increase in lipid content to very high levels at fruit ripeness. Here, we report the development of a transcriptome-based 105,000-probe oil palm mesocarp microarray. The expression of genes involved in fatty acid (FA and triacylglycerol (TAG assembly, along with the tricarboxylic acid cycle (TCA and glycolysis pathway at 16 Weeks After Anthesis (WAA exhibited significantly higher signals compared to those obtained from a cross-species hybridization to the Arabidopsis (p-value < 0.01, and rice (p-value < 0.01 arrays. The oil palm microarray data also showed comparable correlation of expression (r2 = 0.569, p < 0.01 throughout mesocarp development to transcriptome (RNA sequencing data, and improved correlation over quantitative real-time PCR (qPCR (r2 = 0.721, p < 0.01 of the same RNA samples. The results confirm the advantage of the custom microarray over commercially available arrays derived from model species. We demonstrate the utility of this custom microarray to gain a better understanding of gene expression patterns in the oil palm mesocarp that may lead to increasing future oil yield.

  11. Osmotic, Oxidative and Salinity Stresses Up regulate the Expressions of Thiamine (Vitamin B1) Biosynthesis Genes (THIC and THI1/ THI4) in Oil Palm (Elaeis guineensis)

    International Nuclear Information System (INIS)

    Aisamuddin Ardi Zainal Abidin; Yee, W.S.; Nur Syuhadah Abdul Rahman; Zainor Hafisah Che Idris; Zetty Norhana Balia Yusof

    2016-01-01

    Thiamine or vitamin B1 comprises a pyrimidine moiety and a thiazole moiety. Thiamine pyrophosphate (TPP), the active form of thiamine, acts as a cofactor for various major enzymes for example transketolase (TK), α-ketoglutarate dehydrogenase (KGDH) and pyruvate dehydrogenase (PDH). In this study, the gene transcripts of first two enzymes in thiamine biosynthesis pathway, THIC and THI1/ THI4 were identified and amplified from oil palm tissues. Primers were designed based on sequence comparison of the genes from Arabidopsis thaliana, Zea mays, Oryza sativa and Alnus glutinosa. The oil palm's response in terms of the expression profiles of these two thiamine biosynthesis genes to osmotic stress, salinity stress and oxidative stress were analysed in this project. This is due to the suggestion that TPP plays another important role, which is protecting plants against abiotic and biotic stresses. Both THIC and THI1/ THI4 gene transcripts were successfully amplified and the expressions of the gene transcripts were detected using reverse transcriptase polymerase chain reaction (RT-PCR). The levels of expressions were measured and the results showed that the expression of THIC gene transcripts showed increase in gene expression in conjunction with increase concentration of stress inducer. (author)

  12. Identification and expression patterns of Halloween genes encoding cytochrome P450s involved in ecdysteroid biosynthesis in the cotton bollworm Helicoverpa armigera.

    Science.gov (United States)

    Zheng, J; Tian, K; Yuan, Y; Li, M; Qiu, X

    2017-02-01

    20-Hydroxyecdysone (20E) is a key hormone which regulates growth, development and reproduction in insects. Although cytochrome P450 enzymes (P450s) participating in the ecdysteroid biosynthesis of 20E have been characterized in a few model insects, no work has been published on the molecular entity of their orthologs in the cotton bollworm Helicoverpa armigera, a major pest insect in agriculture worldwide. In this study, four cytochrome P450 homologs, namely HarmCYP302A1, HarmCYP306A1, HarmCYP314A1 and HarmCYP315A1 from H. armigera, were identified and evolutional conservation of these Halloween genes were revealed among lepidopteran. Expression analyses showed that HarmCYP302A1 and HarmCYP315A1 were predominantly expressed in larval prothoracic glands, whereas this predominance was not always observed for HarmCYP306A1 and CYP314A1. The expression patterns of Halloween genes indicate that the fat bodies may play an important role in the conversion of ecdysone into 20E in larval-larval molt and in larval-pupal metamorphosis, and raise the possibility that HarmCYP315A1 plays a role in tissue-specific regulation in the steroid biosynthesis in H. armigera. These findings represent the first identification and expression characterization of four steriodogenic P450 genes and provide the groundwork for future functional and evolutionary study of steroid biosynthesis in this agriculturally important pest.

  13. A PKS/NRPS/FAS hybrid gene cluster from Serratia plymuthica RVH1 encoding the biosynthesis of three broad spectrum, zeamine-related antibiotics.

    Science.gov (United States)

    Masschelein, Joleen; Mattheus, Wesley; Gao, Ling-Jie; Moons, Pieter; Van Houdt, Rob; Uytterhoeven, Birgit; Lamberigts, Chris; Lescrinier, Eveline; Rozenski, Jef; Herdewijn, Piet; Aertsen, Abram; Michiels, Chris; Lavigne, Rob

    2013-01-01

    Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.

  14. A PKS/NRPS/FAS hybrid gene cluster from Serratia plymuthica RVH1 encoding the biosynthesis of three broad spectrum, zeamine-related antibiotics.

    Directory of Open Access Journals (Sweden)

    Joleen Masschelein

    Full Text Available Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.

  15. Detection of toxin genes in Escherichia coli isolated from normal dogs and dogs with diarrhea.

    OpenAIRE

    Hammermueller, J; Kruth, S; Prescott, J; Gyles, C

    1995-01-01

    The etiology of acute, nonviral diarrhea in dogs is poorly understood. Enterotoxigenic and verotoxigenic Escherichia coli are causal agents of diarrhea in humans, pigs, and cattle, but the association of these toxigenic E. coli with diarrhea in dogs has not been explored to a significant extent. In this study, DNA hybridization and PCR amplification were used to identify the frequency with which the genes for E. coli enterotoxins (STap, STb, and LTI) and verotoxins (VT1 and VT2) occur in asso...

  16. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

    Science.gov (United States)

    Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

    2015-03-01

    Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

  17. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

    KAUST Repository

    Li, Yongxin

    2015-03-24

    Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning plug-and-playa approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

  18. Transcript profiling of structural genes involved in cyanidin-based anthocyanin biosynthesis between purple and non-purple carrot (Daucus carota L.) cultivars reveals distinct patterns.

    Science.gov (United States)

    Xu, Zhi-Sheng; Huang, Ying; Wang, Feng; Song, Xiong; Wang, Guang-Long; Xiong, Ai-Sheng

    2014-10-01

    Carrots (Daucus carota L.) are among the 10 most economically important vegetable crops grown worldwide. Purple carrot cultivars accumulate rich cyanidin-based anthocyanins in a light-independent manner in their taproots whereas other carrot color types do not. Anthocyanins are important secondary metabolites in plants, protecting them from damage caused by strong light, heavy metals, and pathogens. Furthermore, they are important nutrients for human health. Molecular mechanisms underlying anthocyanin accumulation in purple carrot cultivars and loss of anthocyanin production in non-purple carrot cultivars remain unknown. The taproots of the three purple carrot cultivars were rich in anthocyanin, and levels increased during development. Conversely, the six non-purple carrot cultivars failed to accumulate anthocyanins in the underground part of taproots. Six novel structural genes, CA4H1, CA4H2, 4CL1, 4CL2, CHI1, and F3'H1, were isolated from purple carrots. The expression profiles of these genes, together with other structural genes known to be involved in anthocyanin biosynthesis, were analyzed in three purple and six non-purple carrot cultivars at the 60-day-old stage. PAL3/PAL4, CA4H1, and 4CL1 expression levels were higher in purple than in non-purple carrot cultivars. Expression of CHS1, CHI1, F3H1, F3'H1, DFR1, and LDOX1/LDOX2 was highly correlated with the presence of anthocyanin as these genes were highly expressed in purple carrot taproots but not or scarcely expressed in non-purple carrot taproots. This study isolated six novel structural genes involved in anthocyanin biosynthesis in carrots. Among the 13 analyzed structural genes, PAL3/PAL4, CA4H1, 4CL1, CHS1, CHI1, F3H1, F3'H1, DFR1, and LDOX1/LDOX2 may participate in anthocyanin biosynthesis in the taproots of purple carrot cultivars. CHS1, CHI1, F3H1, F3'H1, DFR1, and LDOX1/LDOX2 may lead to loss of light-independent anthocyanin production in orange and yellow carrots. These results suggest that

  19. Comprehensive analysis of gene expression profiles of the beet armyworm Spodoptera exigua larvae challenged with Bacillus thuringiensis Vip3Aa toxin.

    Science.gov (United States)

    Bel, Yolanda; Jakubowska, Agata K; Costa, Juliana; Herrero, Salvador; Escriche, Baltasar

    2013-01-01

    Host-pathogen interactions result in complex relationship, many aspects of which are not completely understood. Vip proteins, which are Bacillus thuringensis (Bt) insecticidal toxins produced during the vegetative stage, are selectively effective against specific insect pests. This new group of Bt proteins represents an interesting alternative to the classical Bt Cry toxins because current data suggests that they do not share the same mode of action. We have designed and developed a genome-wide microarray for the beet armyworm Spodoptera exigua, a serious lepidopteran pest of many agricultural crops, and used it to better understand how lepidopteran larvae respond to the treatment with the insecticidal protein Vip3Aa. With this approach, the goal of our study was to evaluate the changes in gene expression levels caused by treatment with sublethal doses of Vip3Aa (causing 99% growth inhibition) at 8 and 24 h after feeding. Results indicated that the toxin provoked a wide transcriptional response, with 19% of the microarray unigenes responding significantly to treatment. The number of up- and down-regulated unigenes was very similar. The number of genes whose expression was regulated at 8 h was similar to the number of genes whose expression was regulated after 24 h of treatment. The up-regulated sequences were enriched for genes involved in innate immune response and in pathogen response such as antimicrobial peptides (AMPs) and repat genes. The down-regulated sequences were mainly unigenes with homology to genes involved in metabolism. Genes related to the mode of action of Bt Cry proteins were found, in general, to be slightly overexpressed. The present study is the first genome-wide analysis of the response of lepidopteran insects to Vip3Aa intoxication. An insight into the molecular mechanisms and components related to Vip intoxication will allow designing of more effective management strategies for pest control.

  20. Comprehensive analysis of gene expression profiles of the beet armyworm Spodoptera exigua larvae challenged with Bacillus thuringiensis Vip3Aa toxin.

    Directory of Open Access Journals (Sweden)

    Yolanda Bel

    Full Text Available Host-pathogen interactions result in complex relationship, many aspects of which are not completely understood. Vip proteins, which are Bacillus thuringensis (Bt insecticidal toxins produced during the vegetative stage, are selectively effective against specific insect pests. This new group of Bt proteins represents an interesting alternative to the classical Bt Cry toxins because current data suggests that they do not share the same mode of action. We have designed and developed a genome-wide microarray for the beet armyworm Spodoptera exigua, a serious lepidopteran pest of many agricultural crops, and used it to better understand how lepidopteran larvae respond to the treatment with the insecticidal protein Vip3Aa. With this approach, the goal of our study was to evaluate the changes in gene expression levels caused by treatment with sublethal doses of Vip3Aa (causing 99% growth inhibition at 8 and 24 h after feeding. Results indicated that the toxin provoked a wide transcriptional response, with 19% of the microarray unigenes responding significantly to treatment. The number of up- and down-regulated unigenes was very similar. The number of genes whose expression was regulated at 8 h was similar to the number of genes whose expression was regulated after 24 h of treatment. The up-regulated sequences were enriched for genes involved in innate immune response and in pathogen response such as antimicrobial peptides (AMPs and repat genes. The down-regulated sequences were mainly unigenes with homology to genes involved in metabolism. Genes related to the mode of action of Bt Cry proteins were found, in general, to be slightly overexpressed. The present study is the first genome-wide analysis of the response of lepidopteran insects to Vip3Aa intoxication. An insight into the molecular mechanisms and components related to Vip intoxication will allow designing of more effective management strategies for pest control.

  1. Gene detection and toxin production evaluation of hemolysin BL of Bacillus cereus isolated from milk and dairy products marketed in Brazil

    Directory of Open Access Journals (Sweden)

    Andre L.S. Reis

    2013-12-01

    Full Text Available Bacillus cereusis an ubiquitous, spore-forming bacteria that can survive pasteurization and the majority of the heating processes used in the dairy industry. Besides, it is a pathogen responsible for different types of food poisoning. One type of foodborne disease caused by B.cereusis the diarrheal syndrome, which is caused by the ingestion of vegetative cells producing toxins in the small intestine. One virulence factor for the diarrheal syndrome is the toxin hemolysin BL (HBL, a three-component protein formed by the L1, L2 and B components. In order to evaluate the presence of diarrheal strains isolated from milk and dairy products, 63 B. cereus isolates were obtained from 260 samples of UHT milk, pasteurized milk and powdered milk, sold in commercial establishments and from different brands. The isolates were subjected to the Polymerase Chain Reaction (PCR for the detection of the encoding genes for the L1, L2 and B components and the toxin production capacity were evaluated with an immunoassay. A total of 23 [36.5%] isolates were identified carrying simultaneously the three tested genes, from which, 20 [86.9%] showed toxigenic capacity. 26 [41.3%] isolates did not carry any of genes tested and the other 14 [22.2%] were positive for one or two of them. The results showed a high toxigenic capacity among the B. cereus isolates able to produce the HBL, indicating a potential risk for consumers.

  2. Sequence analysis and identification of the pyrKDbF operon from Lactococcus lactis including a novel gene, pyrK, involved in pyrimidine biosynthesis

    DEFF Research Database (Denmark)

    Andersen, Paal Skytt; Martinussen, Jan; Hammer, Karin

    1996-01-01

    Three genes encoding enzymes involved in the biosynthesis of pyrimidines have been found to constitute an operon in Lactococcus lactis. Two of the genes are the well-known pyr genes pyrDb and pyrF, encoding dihydroorotate dehydrogenase and orotidine monophosphate decarboxylase, respectively....... The third gene encodes a protein which was shown to be necessary for the activity of the pyrDb-encoded dihydroorotate dehydrogenase; we propose to name the gene pyrK. The pyrK-encoded protein is homologous to a number of proteins which are involved in electron transfer. The lactococcal pyrKDbF operon...... is highly homologous to the corresponding part of the much-larger pyr operon of Bacillus subtilis. orf2, the pyrK homolog in B. subtilis, has also been shown to be necessary for pyrimidine biosynthesis (A.E. Kahler and R.L. Switzer, J. Bacteriol. 178:5013-5016, 1996). Four genes adjacent to the operon, i...

  3. Tissue-specific gene-expression patterns of genes associated with thymol/carvacrol biosynthesis in thyme (Thymus vulgaris L.) and their differential changes upon treatment with abiotic elicitors.

    Science.gov (United States)

    Majdi, Mohammad; Malekzadeh-Mashhady, Atefe; Maroufi, Asad; Crocoll, Christoph

    2017-06-01

    Thyme (Thymus vulgaris L.) is known to produce a variety of phenolic monoterpenes such as thymol and carvacrol. Thymol and carvacrol are health-promoting, biocide and antitoxin compounds and have been considered as the main constituents of essential oils in T. vulgaris. To improve our understanding of the regulation of monoterpene biosynthesis in thyme, the expression of genes related to thymol and carvacrol biosynthesis in different tissues and in response to abiotic elicitors was analyzed. Methyl jasmonate (MeJA), salicylic acid (SA), trans-cinnamic acid (tCA) and UV-C irradiation were applied to T. vulgare leaves and transcript levels of early (DXR) and late (TvTPS1, CYP71D178 and CYP71D180) biosynthetic genes of thymol and carvacrol were measured. The results showed that early step and late step genes in thymol/carvacrol biosynthesis are differentially regulated. DXR was not found to be exclusively expressed in glandular trichomes; in contrast, biosynthetic genes including γ-terpinene synthase (TvTPS1) and two cytochrome P450s, CYP71D178 and CYP71D180, were preferentially expressed in glandular secretory trichomes. The high expression of late biosynthetic genes in glandular trichomes, which also contain the highest concentration of thymol and carvacrol, suggests that glandular trichomes are the structure in which thymol/carvacrol biosynthesis and accumulation occur. Our results indicate that in addition to abiotic elicitors, developmental and spatial factors also play a key role in the biosynthesis of thymol and carvacrol, most likely relating to glandular trichome density and/or activity. Hence optimization of these factors could be considered as a useful strategy to achieve high yield of valuable compounds in T. vulgare or other closely related plant species. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. Candida albicans response regulator gene SSK1 regulates a subset of genes whose functions are associated with cell wall biosynthesis and adaptation to oxidative stress.

    Science.gov (United States)

    Chauhan, Neeraj; Inglis, Diane; Roman, Elvira; Pla, Jesus; Li, Dongmei; Calera, Jose A; Calderone, Richard

    2003-10-01

    Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30 degrees C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or

  5. Comparative Transcriptome Analysis of a Toxin-Producing Dinoflagellate Alexandrium catenella and Its Non-Toxic Mutant

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    2014-11-01

    Full Text Available The dinoflagellates and cyanobacteria are two major kingdoms of life producing paralytic shellfish toxins (PSTs, a large group of neurotoxic alkaloids causing paralytic shellfish poisonings around the world. In contrast to the well elucidated PST biosynthetic genes in cyanobacteria, little is known about the dinoflagellates. This study compared transcriptome profiles of a toxin-producing dinoflagellate, Alexandrium catenella (ACHK-T, and its non-toxic mutant form (ACHK-NT using RNA-seq. All clean reads were assembled de novo into a total of 113,674 unigenes, and 66,812 unigenes were annotated in the known databases. Out of them, 35 genes were found to express differentially between the two strains. The up-regulated genes in ACHK-NT were involved in photosynthesis, carbon fixation and amino acid metabolism processes, indicating that more carbon and energy were utilized for cell growth. Among the down-regulated genes, expression of a unigene assigned to the long isoform of sxtA, the initiator of toxin biosynthesis in cyanobacteria, was significantly depressed, suggesting that this long transcript of sxtA might be directly involved in toxin biosynthesis and its depression resulted in the loss of the ability to synthesize PSTs in ACHK-NT. In addition, 101 putative homologs of 12 cyanobacterial sxt genes were identified, and the sxtO and sxtZ genes were identified in dinoflagellates for the first time. The findings of this study should shed light on the biosynthesis of PSTs in the dinoflagellates.

  6. UPLC/Q-TOF MS-based metabolomics and qRT-PCR in enzyme gene screening with key role in triterpenoid saponin biosynthesis of Polygala tenuifolia.

    Directory of Open Access Journals (Sweden)

    Fusheng Zhang

    Full Text Available The dried root of Polygala tenuifolia, named Radix Polygalae, is a well-known traditional Chinese medicine. Triterpenoid saponins are some of the most important components of Radix Polygalae extracts and are widely studied because of their valuable pharmacological properties. However, the relationship between gene expression and triterpenoid saponin biosynthesis in P. tenuifolia is unclear.In this study, ultra-performance liquid chromatography (UPLC coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS-based metabolomic analysis was performed to identify and quantify the different chemical constituents of the roots, stems, leaves, and seeds of P. tenuifolia. A total of 22 marker compounds (VIP>1 were explored, and significant differences in all 7 triterpenoid saponins among the different tissues were found. We also observed an efficient reference gene GAPDH for different tissues in this plant and determined the expression level of some genes in the triterpenoid saponin biosynthetic pathway. Results showed that MVA pathway has more important functions in the triterpenoid saponin biosynthesis of P. tenuifolia. The expression levels of squalene synthase (SQS, squalene monooxygenase (SQE, and beta-amyrin synthase (β-AS were highly correlated with the peak area intensity of triterpenoid saponins compared with data from UPLC/Q-TOF MS-based metabolomic analysis.This finding suggested that a combination of UPLC/Q-TOF MS-based metabolomics and gene expression analysis can effectively elucidate the mechanism of triterpenoid saponin biosynthesis and can provide useful information on gene discovery. These findings can serve as a reference for using the overexpression of genes encoding for SQS, SQE, and/or β-AS to increase the triterpenoid saponin production of P. tenuifolia.

  7. Identification and Characterization of EctR1, a New Transcriptional Regulator of the Ectoine Biosynthesis Genes in the Halotolerant Methanotroph Methylomicrobium alcaliphilum 20Z▿ †

    OpenAIRE

    Mustakhimov, Ildar I.; Reshetnikov, Alexander S.; Glukhov, Anatoly S.; Khmelenina, Valentina N.; Kalyuzhnaya, Marina G.; Trotsenko, Yuri A.

    2009-01-01

    Genes encoding key enzymes of the ectoine biosynthesis pathway in the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z have been shown to be organized into an ectABC-ask operon. Transcription of the ect operon is initiated from two promoters, ectAp1 and ectAp2 (ectAp1p2), similar to the σ70-dependent promoters of Escherichia coli. Upstream of the gene cluster, an open reading frame (ectR1) encoding a MarR-like transcriptional regulator was identified. Investigation of the ...

  8. Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria x ananassa).

    NARCIS (Netherlands)

    Almeida, J.R.; Amico, d' E.; Preuss, A.; Carbone, F.; Vos, de C.H.

    2007-01-01

    The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria x ananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of

  9. Comparison of 454-ESTs from Huperzia serrata and Phlegmariurus carinatus reveals putative genes involved in lycopodium alkaloid biosynthesis and developmental regulation

    Directory of Open Access Journals (Sweden)

    Steinmetz André

    2010-09-01

    Full Text Available Abstract Background Plants of the Huperziaceae family, which comprise the two genera Huperzia and Phlegmariurus, produce various types of lycopodium alkaloids that are used to treat a number of human ailments, such as contusions, swellings and strains. Huperzine A, which belongs to the lycodine type of lycopodium alkaloids, has been used as an anti-Alzheimer's disease drug candidate. Despite their medical importance, little genomic or transcriptomic data are available for the members of this family. We used massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform to generate a substantial EST dataset for Huperzia serrata (H. serrata and Phlegmariurus carinatus (P. carinatus as representative members of the Huperzia and Phlegmariurus genera, respectively. H. serrata and P. carinatus are important plants for research on the biosynthesis of lycopodium alkaloids. We focused on gene discovery in the areas of bioactive compound biosynthesis and transcriptional regulation as well as genetic marker detection in these species. Results For H. serrata, 36,763 unique putative transcripts were generated from 140,930 reads totaling over 57,028,559 base pairs; for P. carinatus, 31,812 unique putative transcripts were generated from 79,920 reads totaling over 30,498,684 base pairs. Using BLASTX searches of public databases, 16,274 (44.3% unique putative transcripts from H. serrata and 14,070 (44.2% from P. carinatus were assigned to at least one protein. Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG orthology annotations revealed that the functions of the unique putative transcripts from these two species cover a similarly broad set of molecular functions, biological processes and biochemical pathways. In particular, a total of 20 H. serrata candidate cytochrome P450 genes, which are more abundant in leaves than in roots and might be involved in lycopodium alkaloid biosynthesis, were found based on the comparison of H

  10. A bistable gene switch for antibiotic biosynthesis: the butyrolactone regulon in Streptomyces coelicolor.

    Directory of Open Access Journals (Sweden)

    Sarika Mehra

    2008-07-01

    Full Text Available Many microorganisms, including bacteria of the class Streptomycetes, produce various secondary metabolites including antibiotics to gain a competitive advantage in their natural habitat. The production of these compounds is highly coordinated in a population to expedite accumulation to an effective concentration. Furthermore, as antibiotics are often toxic even to their producers, a coordinated production allows microbes to first arm themselves with a defense mechanism to resist their own antibiotics before production commences. One possible mechanism of coordination among individuals is through the production of signaling molecules. The gamma-butyrolactone system in Streptomyces coelicolor is a model of such a signaling system for secondary metabolite production. The accumulation of these signaling molecules triggers antibiotic production in the population. A pair of repressor-amplifier proteins encoded by scbA and scbR mediates the production and action of one particular gamma-butyrolactone, SCB1. Based on the proposed interactions of scbA and scbR, a mathematical model was constructed and used to explore the ability of this system to act as a robust genetic switch. Stability analysis shows that the butyrolactone system exhibits bistability and, in response to a threshold SCB1 concentration, can switch from an OFF state to an ON state corresponding to the activation of genes in the cryptic type I polyketide synthase gene cluster, which are responsible for production of the hypothetical polyketide. The switching time is inversely related to the inducer concentration above the threshold, such that short pulses of low inducer concentration cannot switch on the system, suggesting its possible role in noise filtering. In contrast, secondary metabolite production can be triggered rapidly in a population of cells producing the butyrolactone signal due to the presence of an amplification loop in the system. S. coelicolor was perturbed experimentally

  11. A systematic computational analysis of biosynthetic gene cluster evolution: lessons for engineering biosynthesis.

    Directory of Open Access Journals (Sweden)

    Marnix H Medema

    2014-12-01

    Full Text Available Bacterial secondary metabolites are widely used as antibiotics, anticancer drugs, insecticides and food additives. Attempts to engineer their biosynthetic gene clusters (BGCs to produce unnatural metabolites with improved properties are often frustrated by the unpredictability and complexity of the enzymes that synthesize these molecules, suggesting that genetic changes within BGCs are limited by specific constraints. Here, by performing a systematic computational analysis of BGC evolution, we derive evidence for three findings that shed light on the ways in which, despite these constraints, nature successfully invents new molecules: 1 BGCs for complex molecules often evolve through the successive merger of smaller sub-clusters, which function as independent evolutionary entities. 2 An important subset of polyketide synthases and nonribosomal peptide synthetases evolve by concerted evolution, which generates sets of sequence-homogenized domains that may hold promise for engineering efforts since they exhibit a high degree of functional interoperability, 3 Individual BGC families evolve in distinct ways, suggesting that design strategies should take into account family-specific functional constraints. These findings suggest novel strategies for using synthetic biology to rationally engineer biosynthetic pathways.

  12. BIOSYNTHESIS OF microRNAs AND THEIR ROLE IN GENE EXPRESSION PROFILING IN BREAST CANCER

    Directory of Open Access Journals (Sweden)

    Leonardo Barcelos de Paula

    2011-01-01

    Full Text Available The aggressive nature of breast cancer in young women may be related to the occurrence of mutations in the BRCA1/BRCA2 genes responsible for DNA repair. Despite of cases are associated with and without a family history of breast and ovarian cancer such changes are present in only a small percentage of cases, which corresponds to 80-10% of patients with familial breast cancer and 3.2-10.6% of women withbreast cancer non-familial (sporadic. The penetrance rate of this variability is not well understood today, but we know that reproductive factors, risks posed by particular mutations and other genetic modifiers The expression profile of miRNAs can also reveal changes in the regulatory processes that distinguish the appearance of cancer familial and sporadic breast cancer in young patients. miRNAs have been described as related to the aggressiveness of breast cancer and the sensitivity of human mammary tumor strains to antiestrogen. Such evidence indicates that the molecular mechanisms responsible for the aggressive behavior of breast carcinoma in young women has not been sufficiently clarified.

  13. De Novo Assembly, Functional Annotation and Comparative Analysis of Withania somnifera Leaf and Root Transcriptomes to Identify Putative Genes Involved in the Withanolides Biosynthesis

    Science.gov (United States)

    Gupta, Parul; Goel, Ridhi; Pathak, Sumya; Srivastava, Apeksha; Singh, Surya Pratap; Sangwan, Rajender Singh; Asif, Mehar Hasan; Trivedi, Prabodh Kumar

    2013-01-01

    Withania somnifera is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicine systems due to bioactive molecules known as withanolides. As genomic information regarding this plant is very limited, little information is available about biosynthesis of withanolides. To facilitate the basic understanding about the withanolide biosynthesis pathways, we performed transcriptome sequencing for Withania leaf (101L) and root (101R) which specifically synthesize withaferin A and withanolide A, respectively. Pyrosequencing yielded 8,34,068 and 7,21,755 reads which got assembled into 89,548 and 1,14,814 unique sequences from 101L and 101R, respectively. A total of 47,885 (101L) and 54,123 (101R) could be annotated using TAIR10, NR, tomato and potato databases. Gene Ontology and KEGG analyses provided a detailed view of all the enzymes involved in withanolide backbone synthesis. Our analysis identified members of cytochrome P450, glycosyltransferase and methyltransferase gene families with unique presence or differential expression in leaf and root and might be involved in synthesis of tissue-specific withanolides. We also detected simple sequence repeats (SSRs) in transcriptome data for use in future genetic studies. Comprehensive sequence resource developed for Withania, in this study, will help to elucidate biosynthetic pathway for tissue-specific synthesis of secondary plant products in non-model plant organisms as well as will be helpful in developing strategies for enhanced biosynthesis of withanolides through biotechnological approaches. PMID:23667511

  14. De novo assembly, functional annotation and comparative analysis of Withania somnifera leaf and root transcriptomes to identify putative genes involved in the withanolides biosynthesis.

    Directory of Open Access Journals (Sweden)

    Parul Gupta

    Full Text Available Withania somnifera is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicine systems due to bioactive molecules known as withanolides. As genomic information regarding this plant is very limited, little information is available about biosynthesis of withanolides. To facilitate the basic understanding about the withanolide biosynthesis pathways, we performed transcriptome sequencing for Withania leaf (101L and root (101R which specifically synthesize withaferin A and withanolide A, respectively. Pyrosequencing yielded 8,34,068 and 7,21,755 reads which got assembled into 89,548 and 1,14,814 unique sequences from 101L and 101R, respectively. A total of 47,885 (101L and 54,123 (101R could be annotated using TAIR10, NR, tomato and potato databases. Gene Ontology and KEGG analyses provided a detailed view of all the enzymes involved in withanolide backbone synthesis. Our analysis identified members of cytochrome P450, glycosyltransferase and methyltransferase gene families with unique presence or differential expression in leaf and root and might be involved in synthesis of tissue-specific withanolides. We also detected simple sequence repeats (SSRs in transcriptome data for use in future genetic studies. Comprehensive sequence resource developed for Withania, in this study, will help to elucidate biosynthetic pathway for tissue-specific synthesis of secondary plant products in non-model plant organisms as well as will be helpful in developing strategies for enhanced biosynthesis of withanolides through biotechnological approaches.

  15. The manipulation of gene expression and the biosynthesis of Vitamin C, E and folate in light-and dark-germination of sweet corn seeds.

    Science.gov (United States)

    Liu, Fengyuan; Xiang, Nan; Hu, Jian Guang; Shijuan, Yan; Xie, Lihua; Brennan, Charles Stephen; Huang, Wenjie; Guo, Xinbo

    2017-08-08

    This study investigates the potential interrelationship between gene expression and biosynthesis of vitamin C, E and folate in sweet corn sprouts. Germination of sweet corn kernels was conducted in light and dark environments to determine if this relationship was regulated by photo-illumination. Results indicated that light and dark environments affected the DHAR, TMT and GTPCH expression and that these genes were the predominant genes of vitamin C, E and folate biosynthesis pathways respectively during the germination. Levels of vitamin C and folate increased during the germination of sweet corn seeds while vitamin E had a declining manner. Sweet corn sprouts had higher vitamin C and E levels as well as relevant gene expression levels in light environment while illumination had little influence on the folate contents and the gene expression levels during the germination. These results indicate that there might be a collaborative relationship between vitamin C and folate regulation during sweet corn seed germination, while an inhibitive regulation might exist between vitamin C and E.

  16. Functional identification of genes responsible for the biosynthesis of 1-methoxy-indol-3-ylmethyl-glucosinolate in Brassica rapa ssp. chinensis.

    Science.gov (United States)

    Wiesner, Melanie; Schreiner, Monika; Zrenner, Rita

    2014-05-08

    Brassica vegetables contain a class of secondary metabolites, the glucosinolates (GS), whose specific degradation products determine the characteristic flavor and smell. While some of the respective degradation products of particular GS are recognized as health promoting substances for humans, recent studies also show evidence that namely the 1-methoxy-indol-3-ylmethyl GS might be deleterious by forming characteristic DNA adducts. Therefore, a deeper knowledge of aspects involved in the biosynthesis of indole GS is crucial to design vegetables with an improved secondary metabolite profile. Initially the leafy Brassica vegetable pak choi (Brassica rapa ssp. chinensis) was established as suitable tool to elicit very high concentrations of 1-methoxy-indol-3-ylmethyl GS by application of methyl jasmonate. Differentially expressed candidate genes were discovered in a comparative microarray analysis using the 2 × 104 K format Brassica Array and compared to available gene expression data from the Arabidopsis AtGenExpress effort. Arabidopsis knock out mutants of the respective candidate gene homologs were subjected to a comprehensive examination of their GS profiles and confirmed the exclusive involvement of polypeptide 4 of the cytochrome P450 monooxygenase subfamily CYP81F in 1-methoxy-indol-3-ylmethyl GS biosynthesis. Functional characterization of the two identified isoforms coding for CYP81F4 in the Brassica rapa genome was performed using expression analysis and heterologous complementation of the respective Arabidopsis mutant. Specific differences discovered in a comparative microarray and glucosinolate profiling analysis enables the functional attribution of Brassica rapa ssp. chinensis genes coding for polypeptide 4 of the cytochrome P450 monooxygenase subfamily CYP81F to their metabolic role in indole glucosinolate biosynthesis. These new identified Brassica genes will enable the development of genetic tools for breeding vegetables with improved GS composition

  17. Regulation of FA and TAG biosynthesis pathway genes in endosperms and embryos of high and low oil content genotypes of Jatropha curcas L.

    Science.gov (United States)

    Sood, Archit; Chauhan, Rajinder Singh

    2015-09-01

    The rising demand for biofuels has raised concerns about selecting alternate and promising renewable energy crops which do not compete with food supply. Jatropha (Jatropha curcas L.), a non-edible energy crop of the family euphorbiaceae, has the potential of providing biodiesel feedstock due to the presence of high proportion of unsaturated fatty acids (75%) in seed oil which is mainly accumulated in endosperm and embryo. The molecular basis of seed oil biosynthesis machinery has been studied in J. curcas, however, what genetic differences contribute to differential oil biosynthesis and accumulation in genotypes varying for oil content is poorly understood. We investigated expression profile of 18 FA and TAG biosynthetic pathway genes in different developmental stages of embryo and endosperm from high (42%) and low (30%) oil content genotypes grown at two geographical locations. Most of the genes showed relatively higher expression in endosperms of high oil content genotype, whereas no significant difference was observed in endosperms versus embryos of low oil content genotype. The promoter regions of key genes from FA and TAG biosynthetic pathways as well as other genes implicated in oil accumulation were analyzed for regulatory elements and transcription factors specific to oil or lipid accumulation in plants such as Dof, CBF (LEC1), SORLIP, GATA and Skn-1_motif etc. Identification of key genes from oil biosynthesis and regulatory elements specific to oil deposition will be useful not only in dissecting the molecular basis of high oil content but also improving seed oil content through transgenic or molecular breeding approaches. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  18. AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes

    Directory of Open Access Journals (Sweden)

    Ruifang Ma

    2017-08-01

    Full Text Available Lignans, such as lariciresinol and its derivatives, have been identified as effective antiviral ingredients in Isatis indigotica. Evidence suggests that the APETALA2/ethylene response factor (AP2/ERF family might be related to the biosynthesis of lignans in I. indigotica. However, the special role played by the AP2/ERF family in the metabolism and its underlying putative mechanism still need to be elucidated. One novel AP2/ERF gene, named Ii049, was isolated and characterized from I. indigotica in this study. The quantitative real-time PCR analysis revealed that Ii049 was expressed highest in the root and responded to methyl jasmonate, salicylic acid (SA and abscisic acid treatments to various degrees. Subcellular localization analysis indicated that Ii049 protein was localized in the nucleus. Knocking-down the expression of Ii049 caused a remarkable reduction of lignan/lignin contents and transcript levels of genes involved in the lignan/lignin biosynthetic pathway. Ii049 bound to the coupled element 1, RAV1AAT and CRTAREHVCBF2 motifs of genes IiPAL and IiCCR, the key structural genes in the lignan/lignin pathway. Furthermore, Ii049 was also essential for SA biosynthesis, and SA induced lignan accumulation in I. indigotica. Notably, the transgenic I. indigotica hairy roots overexpressing Ii049 showed high expression levels of lignan/lignin biosynthetic genes and SA content, resulting in significant accumulation of lignan/lignin. The best-engineered line (OVX049-10 produced 425.60 μg·g−1 lariciresinol, an 8.3-fold increase compared with the wild type production. This study revealed the function of Ii049 in regulating lignan/lignin biosynthesis, which had the potential to increase the content of valuable lignan/lignin in economically significant medicinal plants.

  19. Overexpression of Three Glucosinolate Biosynthesis Genes in Brassica napus Identifies Enhanced Resistance to Sclerotinia sclerotiorum and Botrytis cinerea.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Zhang

    Full Text Available Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with an extensive host range including Brassica crops. Glucosinolates (GSLs are an important group of secondary metabolites characteristic of the Brassicales order, whose degradation products are proving to be increasingly important in plant protection. Enhancing the defense effect of GSL and their associated degradation products is an attractive strategy to strengthen the resistance of plants by transgenic approaches. We generated the lines of Brassica napus with three biosynthesis genes involved in GSL metabolic pathway (BnMAM1, BnCYP83A1 and BnUGT74B1, respectively. We then measured the foliar GSLs of each transgenic lines and inoculated them with S. sclerotiorum and B. cinerea. Compared with the wild type control, over-expressing BnUGT74B1 in B. napus increased the aliphatic and indolic GSL levels by 1.7 and 1.5 folds in leaves respectively; while over-expressing BnMAM1 or BnCYP83A1 resulted in an approximate 1.5-fold higher only in the aliphatic GSL level in leaves. The results of plant inoculation demonstrated that BnUGT74B1-overexpressing lines showed less severe disease symptoms and tissue damage compared with the wild type control, but BnMAM1 or BnCYP83A1-overexpressing lines showed no significant difference in comparison to the controls. These results suggest that the resistance to S. sclerotiorum and B. cinerea in B. napus could be enhanced through tailoring the GSL profiles by transgenic approaches or molecular breeding, which provides useful information to assist plant breeders to design improved breeding strategies.

  20. Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles).

    Science.gov (United States)

    Trevisan-Silva, Dilza; Gremski, Luiza H; Chaim, Olga M; da Silveira, Rafael B; Meissner, Gabriel O; Mangili, Oldemir C; Barbaro, Katia C; Gremski, Waldemiro; Veiga, Silvio S; Senff-Ribeiro, Andrea

    2010-01-01

    Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom. 2009 Elsevier Masson SAS. All rights reserved.

  1. Polyamine toxins

    DEFF Research Database (Denmark)

    Strømgaard, Kristian; Jensen, Lars S; Vogensen, Stine B

    2005-01-01

    Polyamine toxins, isolated from spiders and wasps, have been used as pharmacological tools for the study of ionotropic receptors, but their use have so far been hampered by their lack of selectivity. In this mini-review, we describe how careful synthetic modification of native polyamine toxins ha...

  2. Parthenolide accumulation and expression of genes related to parthenolide biosynthesis affected by exogenous application of methyl jasmonate and salicylic acid in Tanacetum parthenium.

    Science.gov (United States)

    Majdi, Mohammad; Abdollahi, Mohammad Reza; Maroufi, Asad

    2015-11-01

    Up-regulation of germacrene A synthase and down-regulation of parthenolide hydroxylase genes play key role in parthenolide accumulation of feverfew plants treated with methyl jasmonate and salicylic acid. Parthenolide is an important sesquiterpene lactone due to its anti-migraine and anti-cancer properties. Parthenolide amount was quantified by high-performance liquid chromatography after foliar application of methyl jasmonate (100 µM) or salicylic acid (1.0 mM) on feverfew leaves in time course experiment (3-96 h). Results indicate that exogenous application of methyl jasmonate or salicylic acid activated parthenolide biosynthesis. Parthenolide content reached its highest amount at 24 h after methyl jasmonate or salicylic acid treatments, which were 3.1- and 1.96-fold higher than control plants, respectively. Parthenolide transiently increased due to methyl jasmonate or salicylic acid treatments until 24 h, but did not show significant difference compared with control plants at 48 and 96 h time points in both treatments. Also, the transcript levels of early pathway (upstream) genes of terpene biosynthesis including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and the biosynthetic genes of parthenolide including germacrene A synthase, germacrene A oxidase, costunolide synthase and parthenolide synthase were increased by methyl jasmonate and salicylic acid treatments, but with different intensity. The transcriptional levels of these genes were higher in methyl jasmonate-treated plants than salicylic acid-treated plants. Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is

  3. Comparative transcriptome analysis of genes involved in anthocyanin biosynthesis in the red and yellow fruits of sweet cherry (Prunus avium L..

    Directory of Open Access Journals (Sweden)

    Hairong Wei

    Full Text Available Fruit color is one of the most important economic traits of the sweet cherry (Prunus avium L.. The red coloration of sweet cherry fruit is mainly attributed to anthocyanins. However, limited information is available regarding the molecular mechanisms underlying anthocyanin biosynthesis and its regulation in sweet cherry.In this study, a reference transcriptome of P. avium L. was sequenced and annotated to identify the transcriptional determinants of fruit color. Normalized cDNA libraries from red and yellow fruits were sequenced using the next-generation Illumina/Solexa sequencing platform and de novo assembly. Over 66 million high-quality reads were assembled into 43,128 unigenes using a combined assembly strategy. Then a total of 22,452 unigenes were compared to public databases using homology searches, and 20,095 of these unigenes were annotated in the Nr protein database. Furthermore, transcriptome differences between the four stages of fruit ripening were analyzed using Illumina digital gene expression (DGE profiling. Biological pathway analysis revealed that 72 unigenes were involved in anthocyanin biosynthesis. The expression patterns of unigenes encoding phenylalanine ammonia-lyase (PAL, 4-coumarate-CoA ligase (4CL, chalcone synthase (CHS, chalcone isomerase (CHI, flavanone 3-hydroxylase (F3H, flavanone 3'-hydroxylase (F3'H, dihydroflavonol 4-reductase (DFR, anthocyanidin synthase (ANS and UDP glucose: flavonol 3-O-glucosyltransferase (UFGT during fruit ripening differed between red and yellow fruit. In addition, we identified some transcription factor families (such as MYB, bHLH and WD40 that may control anthocyanin biosynthesis. We confirmed the altered expression levels of eighteen unigenes that encode anthocyanin biosynthetic enzymes and transcription factors using quantitative real-time PCR (qRT-PCR.The obtained sweet cherry transcriptome and DGE profiling data provide comprehensive gene expression information that lends insights

  4. Comparative transcriptome analysis of genes involved in anthocyanin biosynthesis in the red and yellow fruits of sweet cherry (Prunus avium L.).

    Science.gov (United States)

    Wei, Hairong; Chen, Xin; Zong, Xiaojuan; Shu, Huairui; Gao, Dongsheng; Liu, Qingzhong

    2015-01-01

    Fruit color is one of the most important economic traits of the sweet cherry (Prunus avium L.). The red coloration of sweet cherry fruit is mainly attributed to anthocyanins. However, limited information is available regarding the molecular mechanisms underlying anthocyanin biosynthesis and its regulation in sweet cherry. In this study, a reference transcriptome of P. avium L. was sequenced and annotated to identify the transcriptional determinants of fruit color. Normalized cDNA libraries from red and yellow fruits were sequenced using the next-generation Illumina/Solexa sequencing platform and de novo assembly. Over 66 million high-quality reads were assembled into 43,128 unigenes using a combined assembly strategy. Then a total of 22,452 unigenes were compared to public databases using homology searches, and 20,095 of these unigenes were annotated in the Nr protein database. Furthermore, transcriptome differences between the four stages of fruit ripening were analyzed using Illumina digital gene expression (DGE) profiling. Biological pathway analysis revealed that 72 unigenes were involved in anthocyanin biosynthesis. The expression patterns of unigenes encoding phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavanone 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP glucose: flavonol 3-O-glucosyltransferase (UFGT) during fruit ripening differed between red and yellow fruit. In addition, we identified some transcription factor families (such as MYB, bHLH and WD40) that may control anthocyanin biosynthesis. We confirmed the altered expression levels of eighteen unigenes that encode anthocyanin biosynthetic enzymes and transcription factors using quantitative real-time PCR (qRT-PCR). The obtained sweet cherry transcriptome and DGE profiling data provide comprehensive gene expression information that lends insights into

  5. Occurrence, genotyping, shiga toxin genes and associated risk factors of E. coli isolated from dairy farms, handlers and milk consumers.

    Science.gov (United States)

    Awadallah, M A; Ahmed, H A; Merwad, A M; Selim, M A

    2016-11-01

    The objectives of the current study were to determine the occurrence and genotypes of E. coli in dairy farms, workers and milk consumers and to evaluate risk factors associated with contamination of milk in dairy farms. Molecular characterization of shiga toxin associated genes and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) finger printing of E. coli from different sources were also studied. Paired milk samples and rectal swabs from 125 dairy cows, rectal swabs from 82 calves and hand swabs from 45 dairy workers from five dairy farms were collected. In addition, 100 stool samples from 70 diarrheic and 30 healthy humans were collected and examined for the presence of E. coli. E. coli was isolated from milk (22.4%), dairy cattle feces (33.6%), calf feces (35.4%), dairy worker hand swabs (11.1%) and stools of milk consumers (2%, from diarrheic patients only). Only stx1 was identified in seven of 12 E. coli O125 isolated from different sources. High genetic diversity was determined (Simpson's index of diversity, D = 1) and E. coli O125 isolates were classified into 12 distinct profiles, E1-E12. The dendrogram analysis showed that two main clusters were generated. Mastitis in dairy cows was considered a risk factor associated with contamination of the produced milk with E. coli. The isolation of E. coli from rectal swabs of dairy cows and calves poses a zoonotic risk through consumption of unpasteurized contaminated dairy milk. Educational awareness should be developed to address risks related to consumption of raw milk. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. The prevalence of selected genes involved in the biosynthesis of trichothecenes assessed with the specific PCR tests in Fusarium spp. isolated from cereals in southern Poland.

    Science.gov (United States)

    Wolny-Koładka, Katarzyna A

    2015-01-01

    The analysis was conducted using 50 isolates of fungi of the genus Fusarium belonging to the species classified as major trichothecene mycotoxin producers: F. graminearum, F. culmorum, F. sporotrichioides, and F. poae. The tested fungi were isolated from ears of cereal crops in southern Poland during the two growing seasons (2011 and 2012). The aim of this study was to evaluate the prevalence of genes involved in the biosynthesis of trichothecene mycotoxins using the specific PCR tests. Molecular analyses indicated that the genes responsible for the production of trichothecenes (Tri3, Tri5, Tri7, Tri13) were abundant in the examined genetic material. The tested fungal isolates were characterized by a large diversity in terms of the number and composition of the possessed Tri genes. On the other hand, 14 of 50 isolates were found not to carry any of Tri genes.

  7. Diversity and impact of prokaryotic toxins on aquatic environments: a review.

    Science.gov (United States)

    Valério, Elisabete; Chaves, Sandra; Tenreiro, Rogério

    2010-10-01

    . Clostridium members are also spore-forming bacteria and can persist in hostile environmental conditions for long periods of time, contributing to their hazard grade. Similarly, Pseudomonas species are widespread in the environment. Since P. aeruginosa is an emergent opportunistic pathogen, its toxins may represent new hazards for humans and animals. This review presents an overview of the diversity of toxins produced by prokaryotic microorganisms associated with aquatic habitats and their impact on environment, life and health of humans and other animals. Moreover, important issues like the availability of these toxins in the environment, contamination sources and pathways, genes involved in their biosynthesis and molecular mechanisms of some representative toxins are also discussed.

  8. Diversity and Impact of Prokaryotic Toxins on Aquatic Environments: A Review

    Directory of Open Access Journals (Sweden)

    Rogério Tenreiro

    2010-10-01

    and irrigation water. Clostridium members are also spore-forming bacteria and can persist in hostile environmental conditions for long periods of time, contributing to their hazard grade. Similarly, Pseudomonas species are widespread in the environment. Since P. aeruginosa is an emergent opportunistic pathogen, its toxins may represent new hazards for humans and animals. This review presents an overview of the diversity of toxins produced by prokaryotic microorganisms associated with aquatic habitats and their impact on environment, life and health of humans and other animals. Moreover, important issues like the availability of these toxins in the environment, contamination sources and pathways, genes involved in their biosynthesis and molecular mechanisms of some representative toxins are also discussed.

  9. Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission.

    Science.gov (United States)

    Parra-Lobato, Maria C; Gomez-Jimenez, Maria C

    2011-08-01

    After fruit ripening, many fruit-tree species undergo massive natural fruit abscission. Olive (Olea europaea L.) is a stone-fruit with cultivars such as Picual (PIC) and Arbequina (ARB) which differ in mature fruit abscission potential. Ethylene (ET) is associated with abscission, but its role during mature fruit abscission remains largely uncharacterized. The present study investigates the possible roles of ET and polyamine (PA) during mature fruit abscission by modulating genes involved in the ET signalling and biosynthesis pathways in the abscission zone (AZ) of both cultivars. Five ET-related genes (OeACS2, OeACO2, OeCTR1, OeERS1, and OeEIL2) were isolated in the AZ and adjacent cells (AZ-AC), and their expression in various olive organs and during mature fruit abscission, in relation to interactions between ET and PA and the expression induction of these genes, was determined. OeACS2, OeACO2, and OeEIL2 were found to be the only genes that were up-regulated in association with mature fruit abscission. Using the inhibition of ET and PA biosynthesis, it is demonstrated that OeACS2 and OeEIL2 expression are under the negative control of PA while ET induces their expression in AZ-AC. Furthermore, mature fruit abscission depressed nitric oxide (NO) production present mainly in the epidermal cells and xylem of the AZ. Also, NO production was differentially responsive to ET, PA, and different inhibitors. Taken together, the results indicate that PA-dependent ET signalling and biosynthesis pathways participate, at least partially, during mature fruit abscission, and that endogenous NO and 1-aminocyclopropane-1-carboxylic acid maintain an inverse correlation, suggesting an antagonistic action of NO and ET in abscission signalling. © 2011 The Author(s).

  10. The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.

    Science.gov (United States)

    Cai, Wenlong; Goswami, Anwesha; Yang, Zhaoyong; Liu, Xiaodong; Green, Keith D; Barnard-Britson, Sandra; Baba, Satoshi; Funabashi, Masanori; Nonaka, Koichi; Sunkara, Manjula; Morris, Andrew J; Spork, Anatol P; Ducho, Christian; Garneau-Tsodikova, Sylvie; Thorson, Jon S; Van Lanen, Steven G

    2015-05-29

    A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Effect of Enzyme Inhibitors on Terpene Trilactones Biosynthesis and Gene Expression Profiling in Ginkgo biloba Cultured Cells.

    Science.gov (United States)

    Chen, Lijia; Tong, Hui; Wang, Mingxuan; Zhu, Jianhua; Zi, Jiachen; Song, Liyan; Yu, Rongmin

    2015-12-01

    The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism.

  12. Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis.

    Science.gov (United States)

    Jadaun, Jyoti Singh; Sangwan, Neelam S; Narnoliya, Lokesh K; Singh, Neha; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh

    2017-04-01

    Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, 1-deoxy-d-xylulose-5-phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system. © 2016 Scandinavian Plant Physiology Society.

  13. Aflatoxin biosynthesis: current frontiers.

    Science.gov (United States)

    Roze, Ludmila V; Hong, Sung-Yong; Linz, John E

    2013-01-01

    Aflatoxins are among the principal mycotoxins that contaminate economically important food and feed crops. Aflatoxin B1 is the most potent naturally occurring carcinogen known and is also an immunosuppressant. Occurrence of aflatoxins in crops has vast economic and human health impacts worldwide. Thus, the study of aflatoxin biosynthesis has become a focal point in attempts to reduce human exposure to aflatoxins. This review highlights recent advances in the field of aflatoxin biosynthesis and explores the functional connection between aflatoxin biosynthesis, endomembrane trafficking, and response to oxidative stress. Dissection of the regulatory mechanisms involves a complete comprehension of the aflatoxin biosynthetic process and the dynamic network of transcription factors that orchestrates coordinated expression of the target genes. Despite advancements in the field, development of a safe and effective multifaceted approach to solve the aflatoxin food contamination problem is still required.

  14. A Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis via regulating the behaviour of cholesterol in Drosophila.

    Science.gov (United States)

    Enya, Sora; Ameku, Tomotsune; Igarashi, Fumihiko; Iga, Masatoshi; Kataoka, Hiroshi; Shinoda, Tetsuro; Niwa, Ryusuke

    2014-10-10

    In insects, the precise timing of moulting and metamorphosis is strictly guided by ecdysteroids that are synthesised from dietary cholesterol in the prothoracic gland (PG). In the past decade, several ecdysteroidogenic enzymes, some of which are encoded by the Halloween genes, have been identified and characterised. Here, we report a novel Halloween gene, noppera-bo (nobo), that encodes a member of the glutathione S-transferase family. nobo was identified as a gene that is predominantly expressed in the PG of the fruit fly Drosophila melanogaster. We generated a nobo knock-out mutant, which displayed embryonic lethality and a naked cuticle structure. These phenotypes are typical for Halloween mutants showing embryonic ecdysteroid deficiency. In addition, the PG-specific nobo knock-down larvae displayed an arrested phenotype and reduced 20-hydroxyecdysone (20E) titres. Importantly, both embryonic and larval phenotypes were rescued by the administration of 20E or cholesterol. We also confirm that PG cells in nobo loss-of-function larvae abnormally accumulate cholesterol. Considering that cholesterol is the most upstream material for ecdysteroid biosynthesis in the PG, our results raise the possibility that nobo plays a crucial role in regulating the behaviour of cholesterol in steroid biosynthesis in insects.

  15. Characterization of a Bordetella pertussis Diaminopimelate (DAP) Biosynthesis Locus Identifies dapC, a Novel Gene Coding for an N-Succinyl-l,l-DAP Aminotransferase

    OpenAIRE

    Fuchs, Thilo M.; Schneider, Boris; Krumbach, Karin; Eggeling, Lothar; Gross, Roy

    2000-01-01

    The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli...

  16. Characterization of a Bordetella pertussis diaminopimelate (DAP) biosynthesis locus identifies dapC, a novel gene coding for an N-succinyl-L, L-DAP aminotransferase

    OpenAIRE

    Fuchs, T. M.; Schneider, B.; Krumbach, K.; Eggeling, L.; Gross, S. M.

    2000-01-01

    The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs), In line with the successful complementation of the E, coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E, coli...

  17. Transcriptome Analysis of Salicylic Acid Treatment in Rehmannia glutinosa Hairy Roots Using RNA-seq Technique for Identification of Genes Involved in Acteoside Biosynthesis

    Directory of Open Access Journals (Sweden)

    Fengqing Wang

    2017-05-01

    Full Text Available Rehmannia glutinosa is a common bulk medicinal material that has been widely used in China due to its active ingredients. Acteoside, one of the ingredients, has antioxidant, antinephritic, anti-inflammatory, hepatoprotective, immunomodulatory, and neuroprotective effects, is usually selected as a quality-control component for R. glutinosa herb in the Chinese Pharmacopeia. The acteoside biosynthesis pathway in R. glutinosa has not yet been clearly established. Herein, we describe the establishment of a genetic transformation system for R. glutinosa mediated by Agrobacterium rhizogenes. We screened the optimal elicitors that markedly increased acteoside accumulation in R. glutinosa hairy roots. We found that acteoside accumulation dramatically increased with the addition of salicylic acid (SA; the optimal SA dose was 25 μmol/L for hairy roots. RNA-seq was applied to analyze the transcriptomic changes in hairy roots treated with SA for 24 h in comparison with an untreated control. A total of 3,716, 4,018, and 2,715 differentially expressed transcripts (DETs were identified in 0 h-vs.-12 h, 0 h-vs.-24 h, and 12 h-vs.-24 h libraries, respectively. KEGG pathway-based analysis revealed that 127 DETs were enriched in “phenylpropanoid biosynthesis.” Of 219 putative unigenes involved in acteoside biosynthesis, 54 were found to be up-regulated at at least one of the time points after SA treatment. Selected candidate genes were analyzed by quantitative real-time PCR (qRT-PCR in hairy roots with SA, methyl jasmonate (MeJA, AgNO3 (Ag+, and putrescine (Put treatment. All genes investigated were up-regulated by SA treatment, and most candidate genes were weakly increased by MeJA to some degree. Furthermore, transcription abundance of eight candidate genes in tuberous roots of the high-acteoside-content (HA cultivar QH were higher than those of the low-acteoside-content (LA cultivar Wen 85-5. These results will pave the way for understanding the molecular

  18. Biosynthesis and chemical transformation of benzoxazinoids in rye during seed germination and the identification of a rye Bx6-like gene

    DEFF Research Database (Denmark)

    Tanwir, Fariha; Dionisio, Giuseppe; B. Adhikari, Khem

    2017-01-01

    forms of benzoxazinoids accumulated in roots rather than in shoots during seedling development, in particular reaching high levels of HMBOA-glc in roots. Chemical profiles of benzoxazinoid accumulation in the developing seedling reflected the combined effects of de novo biosynthesis of the compounds...... development in rye. Our results showed that ScBx genes had highest levels of expression at 24–30 h after germination, followed by a decrease at later stages. For ScBx1-ScBx5 genes expression was higher in shoots compared with root tissues and vice versa for ScBx6-like gene transcripts. Moreover, methylated...... as well as the turnover of compounds either pre-stored in the embryo or de novo biosynthesized. Bioinformatic analysis, together with the differential distribution of ScBx6-like transcripts in root and shoot tissues, suggested the presence of a ZmBx6 homolog encoding a 2-oxoglutarate dependent...

  19. Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of Lactococcus lactis.

    Science.gov (United States)

    Dong, Xiangrong; Wang, Yanping; Yang, Fengyuan; Zhao, Shanshan; Tian, Bing; Li, Tao

    2017-01-01

    Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress. Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g -1  dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain. The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.

  20. Expansion of banana (Musa acuminata) gene families involved in ethylene biosynthesis and signalling after lineage-specific whole-genome duplications.

    Science.gov (United States)

    Jourda, Cyril; Cardi, Céline; Mbéguié-A-Mbéguié, Didier; Bocs, Stéphanie; Garsmeur, Olivier; D'Hont, Angélique; Yahiaoui, Nabila

    2014-05-01

    Whole-genome duplications (WGDs) are widespread in plants, and three lineage-specific WGDs occurred in the banana (Musa acuminata) genome. Here, we analysed the impact of WGDs on the evolution of banana gene families involved in ethylene biosynthesis and signalling, a key pathway for banana fruit ripening. Banana ethylene pathway genes were identified using comparative genomics approaches and their duplication modes and expression profiles were analysed. Seven out of 10 banana ethylene gene families evolved through WGD and four of them (1-aminocyclopropane-1-carboxylate synthase (ACS), ethylene-insensitive 3-like (EIL), ethylene-insensitive 3-binding F-box (EBF) and ethylene response factor (ERF)) were preferentially retained. Banana orthologues of AtEIN3 and AtEIL1, two major genes for ethylene signalling in Arabidopsis, were particularly expanded. This expansion was paralleled by that of EBF genes which are responsible for control of EIL protein levels. Gene expression profiles in banana fruits suggested functional redundancy for several MaEBF and MaEIL genes derived from WGD and subfunctionalization for some of them. We propose that EIL and EBF genes were co-retained after WGD in banana to maintain balanced control of EIL protein levels and thus avoid detrimental effects of constitutive ethylene signalling. In the course of evolution, subfunctionalization was favoured to promote finer control of ethylene signalling. © 2014 CIRAD New Phytologist © 2014 New Phytologist Trust.

  1. Isolation and characterization of 14 additional genes specifying the anaerobic biosynthesis of cobalamin (vitamin B12) in Propionibacterium freudenreichii (P. shermanii).

    Science.gov (United States)

    Roessner, Charles A; Huang, Ke-Xue; Warren, Martin J; Raux, Evelyne; Scott, A Ian

    2002-06-01

    A search for genes encoding enzymes involved in cobalamin (vitamin B12) production in the commercially important organism Propionibacterium freudenreichii (P. shermanii) has resulted in the isolation of an additional 14 genes encoding enzymes responsible for 17 steps of the anaerobic B12 pathway in this organism. All of the genes believed to be necessary for the biosynthesis of adenosylcobinamide from uroporphyrinogen III have now been isolated except two (cbiA and an as yet unidentified gene encoding cobalt reductase). Most of the genes are contained in two divergent operons, one of which, in turn, is closely linked to the operon encoding the B12-dependent enzyme methylmalonyl-CoA mutase. The close linkage of the three genes encoding the subunits of transcarboxylase to the hemYHBXRL gene cluster is reported. The functions of the P. freudenreichii B12 pathway genes are discussed, and a mechanism for the regulation of cobalamin and propionic acid production by oxygen in this organism is proposed.

  2. A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans

    DEFF Research Database (Denmark)

    Stonebloom, Solomon; Ebert, Berit; Xiong, Guangyan

    2016-01-01

    BACKGROUND: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few...

  3. Disruption of transporters affiliated with enantio-pyochelin biosynthesis gene cluster of Pseudomonas protegens Pf-5 has pleiotropic effects

    Science.gov (United States)

    Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens) is a biocontrol bacterium that produces the siderophore enantio-pyochelin under conditions of iron starvation in a process that is often accompanied by the secretion of its biosynthesis intermediates, salicylic acid and dihydroaeruginoic ...

  4. De Novo Transcriptomes of Forsythia koreana Using a Novel Assembly Method: Insight into Tissue- and Species-Specific Expression of Lignan Biosynthesis-Related Gene.

    Directory of Open Access Journals (Sweden)

    Akira Shiraishi

    Full Text Available Forsythia spp. are perennial woody plants which are one of the most extensively used medicinal sources of Chinese medicines and functional diets owing to their lignan contents. Lignans have received widespread attention as leading compounds in the development of antitumor drugs and healthy diets for reducing the risks of lifestyle-related diseases. However, the molecular basis of Forsythia has yet to be established. In this study, we have verified de novo deep transcriptome of Forsythia koreana leaf and callus using the Illumina HiSeq 1500 platform. A total of 89 million reads were assembled into 116,824 contigs using Trinity, and 1,576 of the contigs displayed the sequence similarity to the enzymes responsible for plant specialized metabolism including lignan biosynthesis. Notably, gene ontology (GO analysis indicated the remarkable enrichment of lignan-biosynthetic enzyme genes in the callus transcriptome. Nevertheless, precise annotation and molecular phylogenetic analyses were hindered by partial sequences of open reading frames (ORFs of the Trinity-based contigs. To obtain more numerous contigs harboring a full-length ORF, we developed a novel overlapping layout consensus-based procedure, virtual primer-based sequence reassembly (VP-seq. VP-seq elucidated 709 full-length ORFs, whereas only 146 full-length ORFs were assembled by Trinity. The comparison of expression profiles of leaf and callus using VP-seq-based full-length ORFs revealed 50-fold upregulation of secoisolariciresinol dehydrogenase (SIRD in callus. Expression and phylogenetic cluster analyses predicted candidates for matairesinol-glucosylating enzymes. We also performed VP-seq analysis of lignan-biosynthetic enzyme genes in the transcriptome data of other lignan-rich plants, Linum flavum, Linum usitatissimum and Podophyllum hexandrum. The comparative analysis indicated both common gene clusters involved in biosynthesis upstream of matairesinol such as SIRD and plant lineage

  5. Chalcone synthase family genes have redundant roles in anthocyanin biosynthesis and in response to blue/UV-A light in turnip (Brassica rapa; Brassicaceae).

    Science.gov (United States)

    Zhou, Bo; Wang, Yu; Zhan, Yaguang; Li, Yuhua; Kawabata, Saneyuki

    2013-12-01

    The epidermis of Brassica rapa (turnip) cv. Tsuda contains light-induced anthocyanins, visible signs of activity of chalcone synthase (CHS), a key anthocyanin biosynthetic enzyme, which is encoded by the CHS gene family. To elucidate the regulation of this light-induced pigmentation, we isolated Brassica rapa CHS1-CHS6 (BrCHS1-CHS6) and characterized their cis-elements and expression patterns. Epidermises of light-exposed swollen hypocotyls (ESHS) were harvested to analyze transcription levels of BrCHS genes by real-time PCR. Different promoters for the genes were inserted into tobacco to examine pCHS-GUS activity by histochemistry. Yeast-one-hybridization was used to detect binding activity of BrCHS motifs to transcription factors. Transcript levels of BrCHS1, -4, and -5 and anthocyanin-biosynthesis-related genes F3H, DFR, and ANS were high, while those of BrCHS2, -3, and -6 were almost undetectable in pigmented ESHS. However, in leaves, CHS5, F3H, and ANS expression was higher than in nonpigmented ESHS, but transcription of DFR was not detected. In the analysis of BrCHS1 and BrCHS3 promoter activity, GUS activity was strong in pigmented flowers of BrPCHS1-GUS-transformed tobacco plants, but nearly absent in BrPCHS3-GUS-transformed plants. Transcript levels of regulators, BrMYB75 and BrTT8, were strongly associated with the anthocyanin content and were light-induced. Coregulated cis-elements were found in promoters of BrCHS1,-4, and -5, and BrMYB75 and BrTT8 had high binding activities to the BrCHS Unit 1 motif. The chalcone synthase gene family encodes a redundant set of light-responsive, tissue-specific genes that are expressed at different levels and are involved in flavonoid biosynthesis in Tsuda turnip.

  6. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

    Directory of Open Access Journals (Sweden)

    Smrati Mishra

    Full Text Available Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

  7. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

    Science.gov (United States)

    Mishra, Smrati; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh; Asha; Jadaun, Jyoti Singh; Sangwan, Neelam S

    2016-01-01

    Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

  8. MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to Bacillus thuringiensis Cry1Ac toxin in diamondback moth.

    Directory of Open Access Journals (Sweden)

    Zhaojiang Guo

    2015-04-01

    Full Text Available Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L., was previously mapped to a multigenic resistance locus (BtR-1. Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.

  9. Genetic shifts in methicillin-resistant Staphylococcus aureus epidemic clones and toxin gene profiles in Japan: comparative analysis among pre-epidemic, epidemic and post-epidemic phases.

    Science.gov (United States)

    Osaka, Shunsuke; Okuzumi, Katsuko; Koide, Shota; Tamai, Kiyoko; Sato, Tomoaki; Tanimoto, Koichi; Tomita, Haruyoshi; Suzuki, Masahiro; Nagano, Yukiko; Shibayama, Keigo; Arakawa, Yoshichika; Nagano, Noriyuki

    2018-03-01

    The decline in methicillin-resistant Staphylococcus aureus (MRSA) isolation rates has become a general observation worldwide, including Japan. We hypothesized that some genetic shift in MRSA might cause this phenomenon, and therefore we investigated the genetic profiles among MRSA clinical isolates obtained from three different epidemic phases in Japan. A total of 353 MRSA isolates were selected from 202 medical facilities in 1990 (pre-epidemic phase), 2004 (epidemic phase) and 2016 (post-epidemic phase). Molecular typing was performed by PCR detection of 22 genes using the polymerase chain reaction (PCR)-based ORF typing (POT) system, including an additional eight genes including small genomic islets and seven toxin genes. Isolates with a POT1 of score 93, identified as presumed clonal complex (pCC)5-staphylococcal cassette chromosome mec (SCCmec) type II including ST5-SCCmec type II New York/Japan clone, represented the major epidemic MRSA lineage in 1990 and 2004. In 2016, however, a marked decrease in isolates with a POT1 score of 93, along with changes in the epidemiology of toxin genes carried, was noted, where the carriers of tst genes including the tst-sec combination were markedly reduced, and those possessing the seb gene alone were markedly increased. Rather, isolates with a POT1 score of 106, including pCC1 or pCC8 among the isolates with SCCmec type IV, which often links to community-associated MRSA, were predominant. Interestingly, the pCC1 and pCC8 lineages were related to sea and tst-sec carriage, respectively. Over time, a transition in MRSA genetic profiles from a POT1 score of 93 in 1990 and 2004 to 106 in 2014 was found in Japan.

  10. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium botulinum strain PS-5

    Science.gov (United States)

    Botulinum neurotoxins (BoNTs) are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins (NAPs). The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presenc...

  11. Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli

    NARCIS (Netherlands)

    Noguera, P.; Posthuma-Trumpie, G.A.; Tuil, van M.; Wal, van der F.J.; Boer, de A.; Moers, A.P.H.A.; Amerongen, van A.

    2011-01-01

    The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay,

  12. Characterization of cDNA for PMT: a Partial Nicotine Biosynthesis-Related Gene Isolated from Indonesian Local Tobacco (Nicotiana tabacum cv. Sindoro1

    Directory of Open Access Journals (Sweden)

    SESANTI BASUKI

    2013-12-01

    Full Text Available Nicotine is the major alkaloid compound in cultivated tobacco (Nicotiana tabacum that could potentially be converted into carcinogenic compound (nor-nicotine. The PMT gene encoding putrescine N-methyltransferase (PMT is one of the two key genes that play a prominent role in nicotine biosynthesis. The aimed of this study was to isolate and characterize the cDNA sequence originated from Indonesian local tobacco cv. Sindoro1 (Ntpmt_Sindoro1. The results showed that the Ntpmt_Sindoro1 was 1124 bp in length. This cDNA fragment encodes for 374 amino acid residues. The predicted polypeptide from the cDNA is a hidrophilic protein, and has a predicted molecular weight of 40.95 kDa. The predicted amino acids sequence also showed high similarity to the PMT gene product Nicotiana sp. available in the GenBank data base. The amino acid sequences also exert conserved residues specifically exhibited only by PMT gene originated from N. tabacum. Clustering analysis revealed that Ntpmt_Sindoro1 belongs to the same clade as the PMT3 gene, a member of the N. tabacum PMT gene family. The Ntpmt_Sindoro1 cDNA sequence covering exon1-exon8 of the PMT gene fragment has been registered in the GenBank data base, under the accession number JX978277.

  13. The Tomato Hoffman's Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures.

    Science.gov (United States)

    Qiu, Zhengkun; Wang, Xiaoxuan; Gao, Jianchang; Guo, Yanmei; Huang, Zejun; Du, Yongchen

    2016-01-01

    Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. Many of the genes that mediate anthocyanin accumulation have been identified through studies of flowers and fruits; however, the mechanisms of genes involved in anthocyanin regulation in seedlings under low-temperature stimulus are less well understood. Genetic characterization of a tomato inbred line, FMTT271, which showed no anthocyanin pigmentation, revealed a mutation in a bHLH transcription factor (TF) gene, which corresponds to the ah (Hoffman's anthocyaninless) locus, and so the gene in FMTT271 at that locus was named ah. Overexpression of the wild type allele of AH in FMTT271 resulted in greater anthocyanin accumulation and increased expression of several genes in the anthocyanin biosynthetic pathway. The expression of AH and anthocyanin accumulation in seedlings was shown to be developmentally regulated and induced by low-temperature stress. Additionally, transcriptome analyses of hypocotyls and leaves from the near-isogenic lines seedlings revealed that AH not only influences the expression of anthocyanin biosynthetic genes, but also genes associated with responses to abiotic stress. Furthermore, the ah mutation was shown to cause accumulation of reactive oxidative species and the constitutive activation of defense responses under cold conditions. These results suggest that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses.

  14. The Tomato Hoffman's Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures.

    Directory of Open Access Journals (Sweden)

    Zhengkun Qiu

    Full Text Available Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. Many of the genes that mediate anthocyanin accumulation have been identified through studies of flowers and fruits; however, the mechanisms of genes involved in anthocyanin regulation in seedlings under low-temperature stimulus are less well understood. Genetic characterization of a tomato inbred line, FMTT271, which showed no anthocyanin pigmentation, revealed a mutation in a bHLH transcription factor (TF gene, which corresponds to the ah (Hoffman's anthocyaninless locus, and so the gene in FMTT271 at that locus was named ah. Overexpression of the wild type allele of AH in FMTT271 resulted in greater anthocyanin accumulation and increased expression of several genes in the anthocyanin biosynthetic pathway. The expression of AH and anthocyanin accumulation in seedlings was shown to be developmentally regulated and induced by low-temperature stress. Additionally, transcriptome analyses of hypocotyls and leaves from the near-isogenic lines seedlings revealed that AH not only influences the expression of anthocyanin biosynthetic genes, but also genes associated with responses to abiotic stress. Furthermore, the ah mutation was shown to cause accumulation of reactive oxidative species and the constitutive activation of defense responses under cold conditions. These results suggest that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses.

  15. The Tomato Hoffman’s Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures

    Science.gov (United States)

    Gao, Jianchang; Guo, Yanmei; Huang, Zejun; Du, Yongchen

    2016-01-01

    Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. Many of the genes that mediate anthocyanin accumulation have been identified through studies of flowers and fruits; however, the mechanisms of genes involved in anthocyanin regulation in seedlings under low-temperature stimulus are less well understood. Genetic characterization of a tomato inbred line, FMTT271, which showed no anthocyanin pigmentation, revealed a mutation in a bHLH transcription factor (TF) gene, which corresponds to the ah (Hoffman's anthocyaninless) locus, and so the gene in FMTT271 at that locus was named ah. Overexpression of the wild type allele of AH in FMTT271 resulted in greater anthocyanin accumulation and increased expression of several genes in the anthocyanin biosynthetic pathway. The expression of AH and anthocyanin accumulation in seedlings was shown to be developmentally regulated and induced by low-temperature stress. Additionally, transcriptome analyses of hypocotyls and leaves from the near-isogenic lines seedlings revealed that AH not only influences the expression of anthocyanin biosynthetic genes, but also genes associated with responses to abiotic stress. Furthermore, the ah mutation was shown to cause accumulation of reactive oxidative species and the constitutive activation of defense responses under cold conditions. These results suggest that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses. PMID:26943362

  16. Isolation and characterization of a R2R3-MYB transcription factor gene related to anthocyanin biosynthesis in the spathes of Anthurium andraeanum (Hort.).

    Science.gov (United States)

    Li, Chonghui; Qiu, Jian; Yang, Guangsui; Huang, Surong; Yin, Junmei

    2016-10-01

    A R2R3-MYB gene AaMYB2 was isolated from Anthurium andraeanum (Hort.) and was functionally characterized to be a positive transcriptional regulator for anthocyanin biosynthesis in the spathes and leaves. Spathe coloration is an important Anthurium andraeanum (Hort.) characteristic, which is mainly contributed by anthocyanins. R2R3-MYB transcription factors (TFs) are important regulators of anthocyanin biosynthesis in plants. Here we describe the identification and characterization of AaMYB2, a member in subgroup 6 of the R2R3-MYB TFs family, which correlated with anthocyanin biosynthesis in A. andraeanum. AaMYB2 was a nuclear-localization protein with positive transcriptional activity, and prominently expressed in the red spathes. Ectopic expression of AaMYB2 in tobacco led to anthocyanin accumulation and up-regulation of the early and late anthocyanin pathway genes, particularly NtDFR, NtANS, and NtUFGT, and the endogenous TF genes NtAn2 and NtAn1 in leaves. In the developing red spathes of 'Tropical' and 'Vitara', the expression of AaMYB2 was closely linked to anthocyanin accumulation, and co-expressed with AaCHS, AaF3H, and AaANS, the latter two of which were regarded as the potential targets of the R locus encoding a TF controlling spathe colors inheritance in anthurium. In addition, the transcription level of AaMYB2 in various cultivars with different color phenotypes showed that AaMYB2 was drastically expressed in the spathes from the red, pink, and purple cultivars, but hardly detected in the spathes from the white and green ones. Besides, AaMYB2 also showed higher expression in newly developmental leaves when anthocyanin was actively biosynthesized. Taken together, AaMYB2 positively related to anthocyanin biosynthesis in anthurium spathes and leaves, and appeared to regulate the expression of AaF3H, AaANS, and possibly AaCHS.

  17. TetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to botR.

    Science.gov (United States)

    Marvaud, J C; Eisel, U; Binz, T; Niemann, H; Popoff, M R

    1998-12-01

    The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) in Clostridium botulinum. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a helix-turn-helix motif and shows 29% identity with other putative regulatory genes in Clostridium, i.e., uviA from C. perfringens and txeR from C. difficile. We report for the first time the transformation of C. tetani by electroporation, which permitted us to investigate the function of tetR. Overexpression of tetR in C. tetani induced an increase in TeTx production and in the level of the corresponding mRNA. This indicates that TetR is a transcriptional activator of the TeTx gene. Overexpression of botR/A (60% identity with TetR at the amino acid level) in C. tetani induced an increase in TeTx production comparable to that for overexpression of tetR. However, botR/C (50% identity with TetR at the amino acid level) was less efficient. This supports that TetR positively regulates the TeTx gene in C. tetani and that a conserved mechanism of regulation of the neurotoxin genes is involved in C. tetani and C. botulinum.

  18. Oxylipin biosynthesis genes positively regulate programmed cell death during compatible infections with the synergistic pair potato virus X-potato virus Y and Tomato spotted wilt virus.

    Science.gov (United States)

    García-Marcos, Alberto; Pacheco, Remedios; Manzano, Aranzazu; Aguilar, Emmanuel; Tenllado, Francisco

    2013-05-01

    One of the most severe symptoms caused by compatible plant-virus interactions is systemic necrosis, which shares common attributes with the hypersensitive response to incompatible pathogens. Although several studies have identified viral symptom determinants responsible for systemic necrosis, mechanistic models of how they contribute to necrosis in infected plants remain scarce. Here, we examined the involvement of different branches of the oxylipin biosynthesis pathway in the systemic necrosis response caused either by the synergistic interaction of Potato virus X with Potato virus Y (PVX-PVY) or by Tomato spotted wilt virus (TSWV) in Nicotiana benthamiana. Silencing either 9-lipoxygenase (LOX), 13-LOX, or α-dioxygenase-1 (α-DOX-1) attenuated the programmed cell death (PCD)-associated symptoms caused by infection with either PVX-PVY or TSWV. In contrast, silencing of the jasmonic acid perception gene, COI1 (Coronatine insensitive 1), expedited cell death during infection with compatible viruses. This correlated with an enhanced expression of oxylipin biosynthesis genes and dioxygenase activity in PVX-PVY-infected plants. Moreover, the Arabidopsis thaliana double lox1 α-dox-1 mutant became less susceptible to TSWV infection. We conclude that oxylipin metabolism is a critical component that positively regulates the process of PCD during compatible plant-virus interactions but does not play a role in restraining virus accumulation in planta.

  19. [Carriage of Streptococcus pyogenes in primary school children: M-protein types, pyrogenic toxin genes, and investigation of the clonal relationships between the isolates].

    Science.gov (United States)

    Otlu, Barış; Karakurt, Cemşit; Bayındır, Yaşar; Kayabaş, Üner; Yakupoğulları, Yusuf; Gözükara Bağ, Harika

    2015-07-01

    M-protein and pyrogenic toxins are the most important virulence factors of Streptococcus pyogenes, and they play significant role in the pathophysiology of acute rheumatoid fever and scarlet fever, respectively. In this study, the pharyngeal carriage of S.pyogenes of the primary school children, clonal relationship of the strains, M-protein types, and the presence of pyrogenic toxin genes were aimed to be investigated. A total of 668 throat cultures obtained from children (age range: 6-16 years) in two primary schools in our region, were included in the study. The clonal relationships of the isolated group A streptococci (GAS) strains were investigated by DiversiLab assay (BioMérieux, France), and the clonal relatedness was confirmed by pulsed-field gel electrophoresis (PFGE) method. M-protein (emm) typing was performed by DNA sequencing as suggested by Centers for Disease Control and Prevention (CDC). The genes encoding pyrogenic toxins, speA and speC, were investigated by an in-house multiplex polymerase chain reaction (PCR) method. S.pyogenes was isolated from 134 (20.05%) of the throat samples. The GAS carriage rate of the students aged ≥10 was statistically higher than those 7-9 years age group (%22 vs %16.4, pprotein gene could be characterized only among 123 isolates by DNA sequencing, and 20 different emm types were detected. The most frequent emm type was emm1 (n=38, 30.9%) followed by emm12 (n=18, 14.6%), emm89 (n=10, 8.1%), emm118 (n=9, 7.3%), and emm4 (n=7, 5.7%). Pyrogenic toxin genes were found in 25 (18.6%) of the isolates, including speA in 11 isolates (8.2%) and speC in 12 isolates (8.9%) and both genes were detected in 2 isolates (1.5%). Sixty-two different Rep (Repetitive extragenic palindromic)-PCR profiles were detected in 134 S.pyogenes isolates by DiversiLab method. Thirteen different clusters were formed by a total of clonally related 36 isolates revealing a strain clustering ratio of 26.9%. Clonal relationship of all isolates in the same

  20. Botulinum toxin

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    Nigam P

    2010-01-01

    Full Text Available Botulinum toxin, one of the most poisonous biological substances known, is a neurotoxin produced by the bacterium Clostridium botulinum. C. botulinum elaborates eight antigenically distinguishable exotoxins (A, B, C 1 , C 2 , D, E, F and G. All serotypes interfere with neural transmission by blocking the release of acetylcholine, the principal neurotransmitter at the neuromuscular junction, causing muscle paralysis. The weakness induced by injection with botulinum toxin A usually lasts about three months. Botulinum toxins now play a very significant role in the management of a wide variety of medical conditions, especially strabismus and focal dystonias, hemifacial spasm, and various spastic movement disorders, headaches, hypersalivation, hyperhidrosis, and some chronic conditions that respond only partially to medical treatment. The list of possible new indications is rapidly expanding. The cosmetological applications include correction of lines, creases and wrinkling all over the face, chin, neck, and chest to dermatological applications such as hyperhidrosis. Injections with botulinum toxin are generally well tolerated and side effects are few. A precise knowledge and understanding of the functional anatomy of the mimetic muscles is absolutely necessary to correctly use botulinum toxins in clinical practice.

  1. Evaluation of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the identification of Campylobacter strains isolated from diarrheal patients in Japan.

    Science.gov (United States)

    Kabir, S M Lutful; Kikuchi, Ken; Asakura, Masahiro; Shiramaru, Sachi; Tsuruoka, Naoki; Goto, Aeko; Hinenoya, Atsushi; Yamasaki, Shinji

    2011-01-01

    We have developed a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection and identification of Campylobacter jejuni, C. coli, and C. fetus. The applicability of this assay was evaluated with 325 Campylobacter strains isolated from diarrheal patients in Japan and the results were compared with those obtained by other genetic methods, including hipO gene detection and 16S rRNA gene sequencing. Of the 325 strains analyzed, 314 and 11 were identified as C. jejuni and C. coli, respectively, by combination of hipO gene detection and 16S rRNA gene sequencing. When the multiplex PCR assay was employed, 309, 310, and 314 strains were identified as C. jejuni on the basis of cdtA, cdtB, and cdtC gene-specific primers, respectively. Similarly, 11, 11, and 10 strains were identified as C. coli on the basis of cdtA, cdtB, and cdtC gene-specific primers, respectively. Sequence analysis of the cdt gene region of 6 strains (5 C. jejuni and 1 C. coli) which did not yield specific PCR products in any of the cdt gene-based multiplex PCR assays revealed deletions or mutations of the cdt genes. Pulsed-field gel electrophoresis indicated that C. jejuni and C. coli strains were genetically diverse. Taken together, these findings suggest that the cdtC gene-based multiplex PCR seems to be a particularly simple and rapid method for differentiating between species of Campylobacter strains, such as C. jejuni and C. coli. However, combination of these multiplex PCR assays will allow more accurate identification.

  2. Hydra actinoporin-like toxin-1, an unusual hemolysin from the nematocyst venom of Hydra magnipapillata which belongs to an extended gene family.

    Science.gov (United States)

    Glasser, Eliezra; Rachamim, Tamar; Aharonovich, Dikla; Sher, Daniel

    2014-12-01

    Cnidarians rely on their nematocysts and the venom injected through these unique weaponry systems to catch prey and protect themselves from predators. The development and physiology of the nematocysts of Hydra magnipapillata, a classic model organism, have been intensively studied, yet the composition and biochemical activity of their venom components are mostly unknown. Here, we show that hydra actinoporin-like toxins (HALTs), which have previously been associated with Hydra nematocysts, belong to a multigene family comprising six genes, which have diverged from a single common ancestor. All six genes are expressed in a population of Hydra magnipapillata. When expressed recombinantly, HALT-1 (Δ-HYTX-Hma1a), an actinoporin-like protein found in the stenoteles (the main penetrating nematocysts used in prey capture), reveals hemolytic activity, albeit about two-thirds lower than that of the anemone actinoporin equinatoxin II (EqTII, Δ-AITX-Aeq1a). HALT-1 also differs from EqTII in the size of its pores, and likely does not utilize sphingomyelin as a membrane receptor. We describe features of the HALT-1 sequence which may contribute to this difference in activity, and speculate on the role of this unusual family of pore-forming toxins in the ecology of Hydra. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Disruption of Escherichia coli Nissle 1917 K5 capsule biosynthesis, through loss of distinct kfi genes, modulates interaction with intestinal epithelial cells and impact on cell health.

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    Jonathan Nzakizwanayo

    Full Text Available Escherichia coli Nissle 1917 (EcN is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants, prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB, or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC, were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.

  4. Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus.

    Science.gov (United States)

    Paolocci, Francesco; Robbins, Mark P; Madeo, Laura; Arcioni, Sergio; Martens, Stefan; Damiani, Francesco

    2007-01-01

    Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

  5. Viable but non-culturable state and toxin gene expression of enterohemorrhagic Escherichia coli O157 under cryopreservation.

    Science.gov (United States)

    Liu, Junyan; Zhou, Rong; Li, Lin; Peters, Brian M; Li, Bing; Lin, Chii-Wann; Chuang, Tsung-Liang; Chen, Dingqiang; Zhao, Xihong; Xiong, Zhiyong; Xu, Zhenbo; Shirtliff, Mark E

    2017-04-01

    As major food-borne pathogens worldwide, Escherichia coli are capable of toxin production directly causing severe human disease. However, routine methods are incapable of detecting viable but non-culturable (VBNC) bacteria in food products and raw materials, leading to false-negative identification. In this study, VBNC E. coli O157 strains were acquired after cryopreservation at -20 °C, with and without freeze-thawing; morphology was observed to be of shorter rod-shape, and toxin expression remained at relatively high levels. PMA-PCR assay for VBNC detection was also validated. Therefore, these results suggest that VBNC E. coli O157 strains may represent a strong threat to public health and food safety. Copyright © 2016 Institut Pasteur. All rights reserved.

  6. The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides 1O2-resistance genes in resistance to the photoactivated toxin cercosporin.

    Science.gov (United States)

    Beseli, Aydin; Goulart da Silva, Marilia; Daub, Margaret E

    2015-01-01

    The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production.

    Science.gov (United States)

    Ihekwaba, Adaoha E C; Mura, Ivan; Walshaw, John; Peck, Michael W; Barker, Gary C

    2016-11-01

    Clostridium botulinum produces botulinum neurotoxins (BoNTs), highly potent substances responsible for botulism. Currently, mathematical models of C. botulinum growth and toxigenesis are largely aimed at risk assessment and do not include explicit genetic information beyond group level but integrate many component processes, such as signalling, membrane permeability and metabolic activity. In this paper we present a scheme for modelling neurotoxin production in C. botulinum Group I type A1, based on the integration of diverse information coming from experimental results available in the literature. Experiments show that production of BoNTs depends on the growth-phase and is under the control of positive and negative regulatory elements at the intracellular level. Toxins are released as large protein complexes and are associated with non-toxic components. Here, we systematically review and integrate those regulatory elements previously described in the literature for C. botulinum Group I type A1 into a population dynamics model, to build the very first computational model of toxin production at the molecular level. We conduct a validation of our model against several items of published experimental data for different wild type and mutant strains of C. botulinum Group I type A1. The result of this process underscores the potential of mathematical modelling at the cellular level, as a means of creating opportunities in developing new strategies that could be used to prevent botulism; and potentially contribute to improved methods for the production of toxin that is used for therapeutics.

  8. Functional validation of putative toxin-antitoxin genes from the Gram-positive pathogen Streptococcus pneumoniae: phd-doc is the fourth bona-fide operon.

    Science.gov (United States)

    Chan, Wai Ting; Yeo, Chew Chieng; Sadowy, Ewa; Espinosa, Manuel

    2014-01-01

    Bacterial toxin-antitoxin (TAs) loci usually consist of two genes organized as an operon, where their products are bound together and inert under normal conditions. However, under stressful circumstances the antitoxin, which is more labile, will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. Previously based on in silico analyses, we proposed that Streptococcus pneumoniae, a pathogenic Gram-positive bacterium, may harbor between 4 and 10 putative TA loci depending on the strains. Here we have chosen the pneumococcal strain Hungary(19A)-6 which contains all possible 10 TA loci. In addition to the three well-characterized operons, namely relBE2, yefM-yoeB, and pezAT, we show here the functionality of a fourth operon that encodes the pneumococcal equivalent of the phd-doc TA. Transcriptional fusions with gene encoding Green Fluorescent Protein showed that the promoter was slightly repressed by the Phd antitoxin, and exhibited almost background values when both Phd-Doc were expressed together. These findings demonstrate that phd-doc shows the negative self-regulatory features typical for an authentic TA. Further, we also show that the previously proposed TAs XreA-Ant and Bro-XreB, although they exhibit a genetic organization resembling those of typical TAs, did not appear to confer a functional behavior corresponding to bona fide TAs. In addition, we have also discovered new interesting bioinformatics results for the known pneumococcal TAs RelBE2 and PezAT. A global analysis of the four identified toxins-antitoxins in the pneumococcal genomes (PezAT, RelBE2, YefM-YoeB, and Phd-Doc) showed that RelBE2 and Phd-Doc are the most conserved ones. Further, there was good correlation among TA types, clonal complexes and sequence types in the 48 pneumococcal strains analyzed.

  9. De Novo RNA Sequencing and Expression Analysis of Aconitum carmichaelii to Analyze Key Genes Involved in the Biosynthesis of Diterpene Alkaloids

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    Megha Rai

    2017-12-01

    Full Text Available Aconitum carmichaelii is an important medicinal herb used widely in China, Japan, India, Korea, and other Asian countries. While extensive research on the characterization of metabolic extracts of A. carmichaelii has shown accumulation of numerous bioactive metabolites including aconitine and aconitine-type diterpene alkaloids, its biosynthetic pathway remains largely unknown. Biosynthesis of these secondary metabolites is tightly controlled and mostly occurs in a tissue-specific manner; therefore, transcriptome analysis across multiple tissues is an attractive method to identify the molecular components involved for further functional characterization. In order to understand the biosynthesis of secondary metabolites, Illumina-based deep transcriptome profiling and analysis was performed for four tissues (flower, bud, leaf, and root of A. carmichaelii, resulting in 5.5 Gbps clean RNA-seq reads assembled into 128,183 unigenes. Unigenes annotated as possible rate-determining steps of an aconitine-type biosynthetic pathway were highly expressed in the root, in accordance with previous reports describing the root as the accumulation site for these metabolites. We also identified 21 unigenes annotated as cytochrome P450s and highly expressed in roots, which represent candidate unigenes involved in the diversification of secondary metabolites. Comparative transcriptome analysis of A. carmichaelii with A. heterophyllum identified 20,232 orthogroups, representing 30,633 unigenes of A. carmichaelii, gene ontology enrichment analysis of which revealed essential biological process together with a secondary metabolic process to be highly enriched. Unigenes identified in this study are strong candidates for aconitine-type diterpene alkaloid biosynthesis, and will serve as useful resources for further validation studies.

  10. Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels

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    Genia eLücking

    2015-10-01

    Full Text Available The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like mega-plasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the nonribosomal peptide synthetase (NRPS, gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional Level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

  11. Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis.

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    Suzana Tiemi Ivamoto

    Full Text Available Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs. These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR. Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs and 134 putative transcriptionally active transposable elements (TE sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.

  12. Modification of membrane properties and fatty acids biosynthesis-related genes in Escherichia coli and Staphylococcus aureus: Implications for the antibacterial mechanism of naringenin.

    Science.gov (United States)

    Wang, Lang-Hong; Zeng, Xin-An; Wang, Man-Sheng; Brennan, Charles S; Gong, Deming

    2018-02-01

    In this work, modifications of cell membrane fluidity, fatty acid composition and fatty acid biosynthesis-associated genes of Escherichia coli ATCC 25922 (E. coli) and Staphylococcus aureus ATCC 6538 (S. aureus), during growth in the presence of naringenin (NAR), one of the natural antibacterial components in citrus plants, was investigated. Compared to E. coli, the growth of S. aureus was significantly inhibited by NAR in low concentrations. Combination of gas chromatography-mass spectrometry with fluorescence polarization analysis revealed that E. coli and S. aureus cells increased membrane fluidity by altering the composition of membrane fatty acids after exposure to NAR. For example, E. coli cells produced more unsaturated fatty acids (from 18.5% to 43.3%) at the expense of both cyclopropane and saturated fatty acids after growth in the concentrations of NAR from 0 to 2.20mM. For S. aureus grown with NAR at 0 to 1.47mM, the relative proportions of anteiso-branched chain fatty acids increased from 37.2% to 54.4%, whereas iso-branched and straight chain fatty acids decreased from 30.0% and 33.1% to 21.6% and 23.7%, respectively. Real time q-PCR analysis showed that NAR at higher concentrations induced a significant down-regulation of fatty acid biosynthesis-associated genes in the bacteria, with the exception of an increased expression of fabA gene. The minimum inhibitory concentration (MIC) of NAR against these two bacteria was determined, and both of bacteria underwent morphological changes after exposure to 1.0 and 2.0 MIC. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. A Root-Preferential DFR-Like Gene Encoding Dihydrokaempferol Reductase Involved in Anthocyanin Biosynthesis of Purple-Fleshed Sweet Potato

    Science.gov (United States)

    Liu, Xiaoqiang; Xiang, Min; Fan, Yufang; Yang, Chunxian; Zeng, Lingjiang; Zhang, Qitang; Chen, Min; Liao, Zhihua

    2017-01-01

    Purple-fleshed sweet potato is good for health due to rich anthocyanins in tubers. Although the anthocyanin biosynthetic pathway is well understood in up-ground organs of plants, the knowledge on anthocyanin biosynthesis in underground tubers is limited. In the present study, we isolated and functionally characterized a root-preferential gene encoding dihydrokaempferol reductase (IbDHKR) from purple-fleshed sweet potato. IbDHKR showed highly similarity with the reported dihydroflavonol reductases in other plant species at the sequence levels and the NADPH-binding motif and the substrate-binding domain were also found in IbDHKR. The tissue profile showed that IbDHKR was expressed in all the tested organs, but with much higher level in tuber roots. The expression level of IbDHKR was consistent with the anthocyanin content in sweet potato organs, suggesting that tuber roots were the main organs to synthesize anthocyanins. The recombinant 44 kD IbDHKR was purified and fed by three different dihydroflavonol substrates including dihydrokaempferol (DHK), dihydroquerctin, and dihydromyrecetin. The substrate feeding assay indicated that only DHK could be accepted as substrate by IbDHKR, which was reduced to leucopelargonidin confirmed by LC-MS. Finally, IbDHKR was overexpressed in transgenic tobacco. The IbDHKR-overexpression tobacco corolla was more highly pigmented and contained higher level of anthocyanins than the wild-type tobacco corolla. In summary, IbDHKR was a root-preferential gene involved in anthocyanin biosynthesis and its encoding protein, specifically catalyzing DHK reduction to yield leucopelargonidin, was a candidate gene for engineering anthocyanin biosynthetic pathway. PMID:28293252

  14. Repression by H-NS of genes required for the biosynthesis of the Vibrio cholerae biofilm matrix is modulated by the second messenger cyclic diguanylic acid.

    Science.gov (United States)

    Ayala, Julio C; Wang, Hongxia; Silva, Anisia J; Benitez, Jorge A

    2015-08-01

    Expression of Vibrio cholerae genes required for the biosynthesis of exopolysacchide (vps) and protein (rbm) components of the biofilm matrix is enhanced by cyclic diguanylate (c-di-GMP). In a previous study, we reported that the histone-like nucleoid structuring (H-NS) protein represses the transcription of vpsA, vpsL and vpsT. Here we demonstrate that the regulator VpsT can disrupt repressive H-NS nucleoprotein complexes at the vpsA and vpsL promoters in the presence of c-di-GMP, while H-NS could disrupt the VpsT-promoter complexes in the absence of c-di-GMP. Chromatin immunoprecipitation-Seq showed a remarkable trend for H-NS to cluster at loci involved in biofilm development such as the rbmABCDEF genes. We show that the antagonistic relationship between VpsT and H-NS regulates the expression of the rbmABCDEF cluster. Epistasis analysis demonstrated that VpsT functions as an antirepressor at the rbmA/F, vpsU and vpsA/L promoters. Deletion of vpsT increased H-NS occupancy at these promoters while increasing the c-di-GMP pool had the opposite effect and included the vpsT promoter. The negative effect of c-di-GMP on H-NS occupancy at the vpsT promoter required the regulator VpsR. These results demonstrate that c-di-GMP activates the transcription of genes required for the biosynthesis of the biofilm matrix by triggering a coordinated VpsR- and VpsT-dependent H-NS antirepression cascade. © 2015 John Wiley & Sons Ltd.

  15. Cell-specific expression of tryptophan decarboxylase and 10-hydroxygeraniol oxidoreductase, key genes involved in camptothecin biosynthesis in Camptotheca acuminata Decne (Nyssaceae

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    Santamaria Anna

    2010-04-01

    Full Text Available Abstract Background Camptotheca acuminata is a major natural source of the terpenoid indole alkaloid camptothecin (CPT. At present, little is known about the cellular distribution of the biosynthesis of CPT, which would be useful knowledge for developing new strategies and technologies for improving alkaloid production. Results The pattern of CPT accumulation was compared with the expression pattern of some genes involved in CPT biosynthesis in C. acuminata [i.e., Ca-TDC1 and Ca-TDC2 (encoding for tryptophan decarboxylase and Ca-HGO (encoding for 10-hydroxygeraniol oxidoreductase]. Both CPT accumulation and gene expression were investigated in plants at different degrees of development and in plantlets subjected to drought-stress. In all organs, CPT accumulation was detected in epidermal idioblasts, in some glandular trichomes, and in groups of idioblast cells localized in parenchyma tissues. Drought-stress caused an increase in CPT accumulation and in the number of glandular trichomes containing CPT, whereas no increase in epidermal or parenchymatous idioblasts was observed. In the leaf, Ca-TDC1 expression was detected in some epidermal cells and in groups of mesophyll cells but not in glandular trichomes; in the stem, it was observed in parenchyma cells of the vascular tissue; in the root, no expression was detected. Ca-TDC2 expression was observed exclusively in leaves of plantlets subjected to drought-stress, in the same sites described for Ca-TDC1. In the leaf, Ca-HGO was detected in all chlorenchyma cells; in the stem, it was observed in the same sites described for Ca-TDC1; in the root, no expression was detected. Conclusions The finding that the sites of CPT accumulation are not consistently the same as those in which the studied genes are expressed demonstrates an organ-to-organ and cell-to-cell translocation of CPT or its precursors.

  16. A Root-Preferential DFR-Like Gene Encoding Dihydrokaempferol Reductase Involved in Anthocyanin Biosynthesis of Purple-Fleshed Sweet Potato.

    Science.gov (United States)

    Liu, Xiaoqiang; Xiang, Min; Fan, Yufang; Yang, Chunxian; Zeng, Lingjiang; Zhang, Qitang; Chen, Min; Liao, Zhihua

    2017-01-01

    Purple-fleshed sweet potato is good for health due to rich anthocyanins in tubers. Although the anthocyanin biosynthetic pathway is well understood in up-ground organs of plants, the knowledge on anthocyanin biosynthesis in underground tubers is limited. In the present study, we isolated and functionally characterized a root-preferential gene encoding dihydrokaempferol reductase ( IbDHKR ) from purple-fleshed sweet potato. IbDHKR showed highly similarity with the reported dihydroflavonol reductases in other plant species at the sequence levels and the NADPH-binding motif and the substrate-binding domain were also found in IbDHKR. The tissue profile showed that IbDHKR was expressed in all the tested organs, but with much higher level in tuber roots. The expression level of IbDHKR was consistent with the anthocyanin content in sweet potato organs, suggesting that tuber roots were the main organs to synthesize anthocyanins. The recombinant 44 kD IbDHKR was purified and fed by three different dihydroflavonol substrates including dihydrokaempferol (DHK), dihydroquerctin, and dihydromyrecetin. The substrate feeding assay indicated that only DHK could be accepted as substrate by IbDHKR, which was reduced to leucopelargonidin confirmed by LC-MS. Finally, IbDHKR was overexpressed in transgenic tobacco. The IbDHKR-overexpression tobacco corolla was more highly pigmented and contained higher level of anthocyanins than the wild-type tobacco corolla. In summary, IbDHKR was a root-preferential gene involved in anthocyanin biosynthesis and its encoding protein, specifically catalyzing DHK reduction to yield leucopelargonidin, was a candidate gene for engineering anthocyanin biosynthetic pathway.

  17. Transcriptional profiling of type II toxin-antitoxin genes of Helicobacter pylori under different environmental conditions: identification of HP0967-HP0968 system

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    MIGUEL A DE LA CRUZ

    2016-11-01

    Full Text Available Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin-antitoxin systems have emerged as important virulence factors in many pathogenic bacteria. Three type II toxin-antitoxin (TA systems have previously been identified in the genome of H. pylori 26695: HP0315-HP0316, HP0892-HP0893, and HP0894-HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967-HP0968, which is encoded by the bicistronic operon hp0968-hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although the hp0968-hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968-hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968-hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa.

  18. Response of last instar Helicoverpa armigera larvae to Bt toxin ingestion: changes in the development and in the CYP6AE14, CYP6B2 and CYP9A12 gene expression.

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    Pilar Muñoz

    Full Text Available Bt crops are able to produce Cry proteins, which were originally present in Bacillus thuringiensis bacteria. Although Bt maize is very efficient against corn borers, Spanish crops are also attacked by the earworm H. armigera, which is less susceptible to Bt maize. Many mechanisms could be involved in this low susceptibility to the toxin, including the insect's metabolic resistance to toxins due to cytochrome P450 monooxygenases. This paper examines the response of last instar H. armigera larvae to feeding on a diet with Bt and non-Bt maize leaves in larval development and in the gene expression of three P450 cytochromes: CYP6AE14, CYP6B2 and CYP9A12. Larvae fed on sublethal amounts of the Bt toxin showed reduced food ingestion and reduced growth and weight, preventing most of them from achieving the critical weight and pupating; additionally, after feeding for one day on the Bt diet the larvae showed a slight increase in juvenile hormone II in the hemolymp. Larvae fed on the non-Bt diet showed the highest CYP6AE14, CYP6B2 and CYP9A12 expression one day after feeding on the non-Bt diet, and just two days later the expression decreased abruptly, a finding probably related to the developmental programme of the last instar. Moreover, although the response of P450 genes to plant allelochemicals and xenobiotics has been related in general to overexpression in the resistant insect, or induction of the genes when feeding takes place, the expression of the three genes studied was suppressed in the larvae feeding on the Bt toxin. The unexpected inhibitory effect of the Cry1Ab toxin in the P450 genes of H. armigera larvae should be thoroughly studied to determine whether this response is somehow related to the low susceptibility of the species to the Bt toxin.

  19. A Gene Cluster for Biosynthesis of Mannosylerythritol Lipids Consisted of 4-O-β-D-Mannopyranosyl-(2R,3S)-Erythritol as the Sugar Moiety in a Basidiomycetous Yeast Pseudozyma tsukubaensis.

    Science.gov (United States)

    Saika, Azusa; Koike, Hideaki; Fukuoka, Tokuma; Yamamoto, Shuhei; Kishimoto, Takahide; Morita, Tomotake

    2016-01-01

    Mannosylerythritol lipids (MELs) belong to the glycolipid biosurfactants and are produced by various fungi. The basidiomycetous yeast Pseudozyma tsukubaensis produces diastereomer type of MEL-B, which contains 4-O-β-D-mannopyranosyl-(2R,3S)-erythritol (R-form) as the sugar moiety. In this respect it differs from conventional type of MELs, which contain 4-O-β-D-mannopyranosyl-(2S,3R)-erythritol (S-form) as the sugar moiety. While the biosynthetic gene cluster for conventional type of MELs has been previously identified in Ustilago maydis and Pseudozyma antarctica, the genetic basis for MEL biosynthesis in P. tsukubaensis is unknown. Here, we identified a gene cluster involved in MEL biosynthesis in P. tsukubaensis. Among these genes, PtEMT1, which encodes erythritol/mannose transferase, had greater than 69% identity with homologs from strains in the genera Ustilago, Melanopsichium, Sporisorium and Pseudozyma. However, phylogenetic analysis placed PtEMT1p in a separate clade from the other proteins. To investigate the function of PtEMT1, we introduced the gene into a P. antarctica mutant strain, ΔPaEMT1, which lacks MEL biosynthesis ability owing to the deletion of PaEMT1. Using NMR spectroscopy, we identified the biosynthetic product as MEL-A with altered sugar conformation. These results indicate that PtEMT1p catalyzes the sugar conformation of MELs. This is the first report of a gene cluster for the biosynthesis of diastereomer type of MEL.

  20. Prevalence of enterotoxins and toxin gene profiles of Staphylococcus aureus isolates recovered from a bakery involved in a second staphylococcal food poisoning occurrence.

    Science.gov (United States)

    Hait, J; Tallent, S; Melka, D; Keys, C; Bennett, R

    2014-09-01

    The study objective was to characterize and analyse the distribution of enterotoxins and genes encoding enterotoxins in Staphylococcus aureus strains recovered from the 601 environment and ingredient samples obtained during multiple inspections of a bakery implicated in two separate staphylococcal food poisoning incidents. Staphylococcus aureus isolates were evaluated using serological assays for identification of classical staphylococcal enterotoxins (SEs) SEA-SEE and polymerase chain reaction for the detection of newly described SE and SE-like enterotoxin genes seg-seu. Pulsed field gel electrophoresis identified thirteen pattern types. During these investigations, a total of 585 environmental swabs and 16 raw ingredient samples were collected by investigators, 85 of which were confirmed to contain Staph. aureus; of those isolates, 95·3% (81/85) harboured enterotoxin genes and 4·7% (4/85) carried newly described SE and SE-like enterotoxin genes in the absence of classical enterotoxins. Our research demonstrates the prevalence and diversity of classical SEs and the probable underestimated impact of nonclassical SE and SE-like enterotoxins role in domestic staphylococcal food poisoning outbreaks. Given the abundance of SEs and SE-like toxins, these findings illustrate the utilization of PCR for enterotoxin gene identification and its significance in outbreak investigations. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

  1. Glutathione biosynthesis and activity of dependent enzymes in food grade lactic acid bacteria harboring multidomain bifunctional fusion gene (gshF).

    Science.gov (United States)

    Pophaly, Sarang Dilip; Poonam; Pophaly, Saurabh Dilip; Kapila, Suman; Nanda, Dhiraj Kumar; Tomar, Sudhir Kumar; Singh, Rameshwar

    2017-04-12

    To assess glutathione (GSH) biosynthesis ability and activity of dependent enzymes in food grade lactic acid bacteria and correlating with genomic information on glutathione system in LAB. Whole genome sequences of twenty-six food grade LAB were screened for presence/absence of set of genes involved in de novo glutathione system. Multiple strains of S. thermophilus (37), Lb. casei (37), Lb. rhamnosus (4), Lb. paracasei (8) Lb. plantarum (23) & Lb. fermentum (22) were screened for biochemical evidence of GSH system. Multiple sequence analysis of GshF sequences was carried out for comparing the genomic signatures between GSH producing and non-producing species. Streptococcus thermophilus was found to have de novo glutathione biosynthesis as well as import ability. Lactobacillus spp. were negative for GSH synthesis but could import it from the medium. All the species exhibited prolific glutathione reductase and peroxidase activity. Sequence analysis revealed absence of key amino acid residues as well as a truncated N-terminal region in lactobacilli. The study provides a comprehensive view on status of an important antioxidative system (the glutathione system) in LAB and is expected to serve as a primer for future work on mechanistic role of GSH in the group. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  2. Characterization of Flavan-3-ols and Expression of MYB and Late Pathway Genes Involved in Proanthocyanidin Biosynthesis in Foliage of Vitis bellula

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    Ke-Gang Li

    2013-03-01

    Full Text Available Proanthocyanidins (PAs are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC and high performance liquid chromatography-photodiode array detector (HPLC-PAD show the formation of (+-catechin, (−-epicatechin, (+-gallocatechin and (−-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are

  3. Characterization of Flavan-3-ols and Expression of MYB and Late Pathway Genes Involved in Proanthocyanidin Biosynthesis in Foliage of Vitis bellula

    Science.gov (United States)

    Zhu, Yue; Peng, Qing-Zhong; Du, Ci; Li, Ke-Gang; Xie, De-Yu

    2013-01-01

    Proanthocyanidins (PAs) are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC) and high performance liquid chromatography-photodiode array detector (HPLC-PAD) show the formation of (+)-catechin, (−)-epicatechin, (+)-gallocatechin and (−)-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are involved in

  4. Interspecies and Intraspecies Analysis of Trehalose Contents and the Biosynthesis Pathway Gene Family Reveals Crucial Roles of Trehalose in Osmotic-Stress Tolerance in Cassava

    Science.gov (United States)

    Han, Bingying; Fu, Lili; Zhang, Dan; He, Xiuquan; Chen, Qiang; Peng, Ming; Zhang, Jiaming

    2016-01-01

    Trehalose is a nonreducing α,α-1,1-disaccharide in a wide range of organisms, and has diverse biological functions that range from serving as an energy source to acting as a protective/signal sugar. However, significant amounts of trehalose have rarely been detected in higher plants, and the function of trehalose in the drought-tolerant crop cassava (Manihot esculenta Crantz) is unclear. We measured soluble sugar concentrations of nine plant species with differing levels of drought tolerance and 41 cassava varieties using high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD). Significantly high amounts of trehalose were identified in drought-tolerant crops cassava, Jatropha curcas, and castor bean (Ricinus communis). All cassava varieties tested contained high amounts of trehalose, although their concentrations varied from 0.23 to 1.29 mg·g−1 fresh weight (FW), and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. Moreover, the trehalose concentrations in cassava leaves increased 2.3–5.5 folds in response to osmotic stress simulated by 20% PEG 6000. Through database mining, 24 trehalose pathway genes, including 12 trehalose-6-phosphate synthases (TPS), 10 trehalose-6-phosphate phosphatases (TPP), and two trehalases were identified in cassava. Phylogenetic analysis indicated that there were four cassava TPS genes (MeTPS1–4) that were orthologous to the solely active TPS gene (AtTPS1 and OsTPS1) in Arabidopsis and rice, and a new TPP subfamily was identified in cassava, suggesting that the trehalose biosynthesis activities in cassava had potentially been enhanced in evolutionary history. RNA-seq analysis indicated that MeTPS1 was expressed at constitutionally high level before and after osmotic stress, while other trehalose pathway genes were either up-regulated or down-regulated, which may explain why cassava accumulated high level of trehalose under

  5. Interspecies and Intraspecies Analysis of Trehalose Contents and the Biosynthesis Pathway Gene Family Reveals Crucial Roles of Trehalose in Osmotic-Stress Tolerance in Cassava

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    Bingying Han

    2016-07-01

    Full Text Available Trehalose is a nonreducing α,α-1,1-disaccharide in a wide range of organisms, and has diverse biological functions that range from serving as an energy source to acting as a protective/signal sugar. However, significant amounts of trehalose have rarely been detected in higher plants, and the function of trehalose in the drought-tolerant crop cassava (Manihot esculenta Crantz is unclear. We measured soluble sugar concentrations of nine plant species with differing levels of drought tolerance and 41 cassava varieties using high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD. Significantly high amounts of trehalose were identified in drought-tolerant crops cassava, Jatropha curcas, and castor bean (Ricinus communis. All cassava varieties tested contained high amounts of trehalose, although their concentrations varied from 0.23 to 1.29 mg·g−1 fresh weight (FW, and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. Moreover, the trehalose concentrations in cassava leaves increased 2.3–5.5 folds in response to osmotic stress simulated by 20% PEG 6000. Through database mining, 24 trehalose pathway genes, including 12 trehalose-6-phosphate synthases (TPS, 10 trehalose-6-phosphate phosphatases (TPP, and two trehalases were identified in cassava. Phylogenetic analysis indicated that there were four cassava TPS genes (MeTPS1–4 that were orthologous to the solely active TPS gene (AtTPS1 and OsTPS1 in Arabidopsis and rice, and a new TPP subfamily was identified in cassava, suggesting that the trehalose biosynthesis activities in cassava had potentially been enhanced in evolutionary history. RNA-seq analysis indicated that MeTPS1 was expressed at constitutionally high level before and after osmotic stress, while other trehalose pathway genes were either up-regulated or down-regulated, which may explain why cassava accumulated high level of trehalose

  6. Transcriptome analysis of buds and leaves using 454 pyrosequencing to discover genes associated with the biosynthesis of active ingredients in Lonicera japonica Thunb.

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    Liu He

    Full Text Available BACKGROUND: Lonicera japonica Thunb. is a plant used in traditional Chinese medicine known for its anti-inflammatory, anti-oxidative, anti-carcinogenic, and antiviral pharmacological properties. The major active secondary metabolites of this plant are chlorogenic acid (CGA and luteoloside. While the biosynthetic pathways of these metabolites are relatively well known, the genetic information available for this species, especially the biosynthetic pathways of its active ingredients, is limited. METHODOLOGY/PRINCIPAL FINDINGS: We obtained one million reads (average length of 400 bp in a whole sequence run using a Roche/454 GS FLX titanium platform. Altogether, 85.69% of the unigenes covering the entire life cycle of the plant were annotated and 325 unigenes were assigned to secondary metabolic pathways. Moreover, 2039 unigenes were predicted as transcription factors. Nearly all of the possible enzymes involved in the biosynthesis of CGA and luteoloside were discovered in L. japonica. Three hydroxycinnamoyl transferase genes, including two hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes and one hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT gene featuring high similarity to known genes from other species, were cloned. The HCT gene was discovered for the first time in L. japonica. In addition, 188 candidate cytochrome P450 unigenes and 245 glycosyltransferase unigenes were found in the expressed sequence tag (EST dataset. CONCLUSION: This study provides a high quality EST database for L. japonica by 454 pyrosequencing. Based on the EST annotation, a set of putative genes involved in CGA and luteoloside biosynthetic pathways were discovered. The database serves as an important source of public information on genetic markers, gene expression, genomics, and functional genomics in L. japonica.

  7. Genome-Wide Analysis of the Biosynthesis and Deactivation of Gibberellin-Dioxygenases Gene Family in Camellia sinensis (L. O. Kuntze

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    Cheng Pan

    2017-09-01

    Full Text Available Gibberellins (GAs, a class of diterpenoid phytohormones, play a key role in regulating diverse processes throughout the life cycle of plants. Bioactive GA levels are rapidly regulated by Gibberellin-dioxygenases (GAox, which are involved in the biosynthesis and deactivation of gibberellin. In this manuscript, a comprehensive genome-wide analysis was carried out to find all GAox in Camellia sinensis. For the first time in a tea plant, 14 CsGAox genes, containing two domains, DIOX_N (PF14226 and 2OG-FeII_Oxy, were identified (PF03171. These genes all belong to 2-oxoglutarate-dependent dioxygenases (2-ODD, including four CsGA20ox (EC: 1.14.11.12, three CsGA3ox (EC: 1.14.11.15, and seven CsGA2ox (EC: 1.14.11.13. According to the phylogenetic classification as in Arabidopsis, the CsGAox genes spanned five subgroups. Each CsGAox shows tissue-specific expression patterns, although these vary greatly. Some candidate genes, which may play an important role in response to external abiotic stresses, have been identified with regards to patterns, such as CsGA20ox2, CsGA3ox2, CsGA3ox3, CsGA2ox1, CsGA2ox2, and CsGA2ox4. The bioactive GA levels may be closely related to the GA20ox, GA3ox and GA2ox genes. In addition, the candidate genes could be used as marker genes for abiotic stress resistance breeding in tea plants.

  8. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba

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    Qiangwen Chen

    2017-01-01

    Full Text Available Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs, are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT and mevalonate kinase (MVK as core enzymes. In this study, two full-length (cDNAs encoding AACT (GbAACT, GenBank Accession No. KX904942 and MVK (GbMVK, GenBank Accession No. KX904944 were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  9. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba.

    Science.gov (United States)

    Chen, Qiangwen; Yan, Jiaping; Meng, Xiangxiang; Xu, Feng; Zhang, Weiwei; Liao, Yongling; Qu, Jinwang

    2017-01-02

    Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba . Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C -acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT ( GbAACT , GenBank Accession No. KX904942) and MVK ( GbMVK , GenBank Accession No. KX904944) were cloned from G. biloba . The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  10. Variants in estrogen-biosynthesis genes CYP17 and CYP19 and breast cancer risk: a family-based genetic association study

    International Nuclear Information System (INIS)

    Ahsan, Habibul; Whittemore, Alice S; Chen, Yu; Senie, Ruby T; Hamilton, Steven P; Wang, Qiao; Gurvich, Irina; Santella, Regina M

    2005-01-01

    Case-control studies have reported inconsistent results concerning breast cancer risk and polymorphisms in genes that control endogenous estrogen biosynthesis. We report findings from the first family-based association study examining associations between female breast cancer risk and polymorphisms in two key estrogen-biosynthesis genes CYP17 (T→C promoter polymorphism) and CYP19 (TTTA repeat polymorphism). We conducted the study among 278 nuclear families containing one or more daughters with breast cancer, with a total of 1123 family members (702 with available constitutional DNA and questionnaire data and 421 without them). These nuclear families were selected from breast cancer families participating in the Metropolitan New York Registry, one of the six centers of the National Cancer Institute's Breast Cancer Family Registry. We used likelihood-based statistical methods to examine allelic associations. We found the CYP19 allele with 11 TTTA repeats to be associated with breast cancer risk in these families. We also found that maternal (but not paternal) carrier status of CYP19 alleles with 11 repeats tended to be associated with breast cancer risk in daughters (independently of the daughters' own genotype), suggesting a possible in utero effect of CYP19. We found no association of a woman's breast cancer risk either with her own or with her mother's CYP17 genotype. This family-based study indicates that a woman's personal and maternal carrier status of CYP19 11 TTTA repeat allele might be related to increased breast cancer risk. However, because this is the first study to report an association between CYP19 11 TTTA repeat allele and breast cancer, and because multiple comparisons have been made, the associations should be interpreted with caution and need confirmation in future family-based studies

  11. Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246.

    Science.gov (United States)

    Ma, Zhe; Geng, Jianing; Yi, Li; Xu, Bin; Jia, Ruoyu; Li, Yue; Meng, Qingshu; Fan, Hongjie; Hu, Songnian

    2013-06-07

    Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is an important pathogen causing swine streptococcosis in China. Pathogenicity islands (PAIs) of S. zooepidemicus have been transferred among bacteria through horizontal gene transfer (HGT) and play important roles in the adaptation and increased virulence of S. zooepidemicus. The present study used comparative genomics to examine the different pathogenicities of S. zooepidemicus. Genome of S. zooepidemicus ATCC35246 (Sz35246) comprises 2,167,264-bp of a single circular chromosome, with a GC content of 41.65%. Comparative genome analysis of Sz35246, S. zooepidemicus MGCS10565 (Sz10565), Streptococcus equi. ssp. equi. 4047 (Se4047) and S. zooepidemicus H70 (Sz70) identified 320 Sz35246-specific genes, clustered into three toxin-antitoxin (TA) systems PAIs and one restriction modification system (RM system) PAI. These four acquired PAIs encode proteins that may contribute to the overall pathogenic capacity and fitness of this bacterium to adapt to different hosts. Analysis of the in vivo and in vitro transcriptomes of this bacterium revealed differentially expressed PAI genes and non-PAI genes, suggesting that Sz35246 possess mechanisms for infecting animals and adapting to a wide range of host environments. Analysis of the genome identified potential Sz35246 virulence genes. Genes of the Fim III operon were presumed to be involved in breaking the host-restriction of Sz35246. Genome wide comparisons of Sz35246 with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. Four specific PAIs, which were judged to have been transferred into Sz35246 genome through HGT, were identified for the first time. Further analysis of the TA and RM systems in the PAIs will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines.

  12. Two LcbHLH transcription factors interacting with LcMYB1 in regulating late structural genes of anthocyanin biosynthesis in Nicotiana and Litchi chinensis during anthocyanin accumulation

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    Biao eLai

    2016-02-01

    Full Text Available Anthocyanin biosynthesis requires the MYB-bHLH-WD40 protein complex to activate the late biosynthetic genes. LcMYB1 was thought to act as key regulator in anthocyanin biosynthesis of litchi. However, basic helix-loop-helix proteins (bHLHs as partners have not been identified yet. The present study describes the functional characterization of three litchi bHLH candidate anthocyanin regulators, LcbHLH1, LcbHLH2 and LcbHLH3. Although these three litchi bHLHs phylogenetically clustered with bHLH proteins involved in anthcoyanin biosynthesis in other plant, only LcbHLH1 and LcbHLH3 were found to localize in the nucleus and physically interact with LcMYB1. The transcription levels of all these bHLHs were not coordinated with anthocyanin accumulation in different tissues and during development. However, when co-infiltrated with LcMYB1, both LcbHLH1 and LcbHLH3 enhanced anthocyanin accumulation in tobacco leaves with LcbHLH3 being the best inducer. Significant accumulation of anthocyanins in leaves transformed with the combination of LcMYB1 and LcbHLH3 were noticed, And this was associated with the up-regulation of two tobacco endogenous bHLH regulators, NtAn1a and NtAn1b, and late structural genes, like NtDFR and NtANS. Significant activity of the ANS promoter was observed in transient expression assays either with LcMYB1-LcbHLH1 or LcMYB1-LcbHLH3, while only minute activity was detected after transformation with only LcMYB1. In contrast, no activity was measured after induction with the combination of LcbHLH2 and LcMYB1. Higher DFR expression was also oberseved in paralleling with higher anthocyanins in co-transformed lines. LcbHLH1 and LcbHLH3 are essential partner of LcMYB1 in regulating the anthocyanin production in tobacco and probably also in litchi. The LcMYB1-LcbHLH complex enhanced anthocyanin accumulation may associate with activating the transcription of DFR and ANS.

  13. Gene expression analyses in tomato near isogenic lines provide evidence for ethylene and abscisic acid biosynthesis fine-tuning during arbuscular mycorrhiza development.

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    Fracetto, Giselle Gomes Monteiro; Peres, Lázaro Eustáquio Pereira; Lambais, Marcio Rodrigues

    2017-07-01

    Plant responses to the environment and microorganisms, including arbuscular mycorrhizal fungi, involve complex hormonal interactions. It is known that abscisic acid (ABA) and ethylene may be involved in the regulation of arbuscular mycorrhiza (AM) and that part of the detrimental effects of ABA deficiency in plants is due to ethylene overproduction. In this study, we aimed to determine whether the low susceptibility to mycorrhizal colonization in ABA-deficient mutants is due to high levels of ethylene and whether AM development is associated with changes in the steady-state levels of transcripts of genes involved in the biosynthesis of ethylene and ABA. For that, tomato (Solanum lycopersicum) ethylene overproducer epinastic (epi) mutant and the ABA-deficient notabilis (not) and sitiens (sit) mutants, in the same Micro-Tom (MT) genetic background, were inoculated with Rhizophagus clarus, and treated with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG). The development of AM, as well as the steady-state levels of transcripts involved in ethylene (LeACS2, LeACO1 and LeACO4) and ABA (LeNCED) biosynthesis, was determined. The intraradical colonization in epi, not and sit mutants was significantly reduced compared to MT. The epi mutant completely restored the mycorrhizal colonization to the levels of MT with the application of 10 µM of AVG, probably due to the inhibition of the ACC synthase gene expression. The steady-state levels of LeACS2 and LeACO4 transcripts were induced in mycorrhizal roots of MT, whereas the steady-state levels of LeACO1 and LeACO4 transcripts were significantly induced in sit, and the steady-state levels of LeNCED transcripts were significantly induced in all genotypes and in mycorrhizal roots of epi mutants treated with AVG. The reduced mycorrhizal colonization in sit mutants seems not to be limited by ethylene production via ACC oxidase regulation. Both ethylene overproduction and ABA deficiency impaired AM fungal

  14. Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

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    Carvalho, Luiz Jcb; Agustini, Marco Av; Anderson, James V; Vieira, Eduardo A; de Souza, Claudia Rb; Chen, Songbi; Schaal, Barbara A; Silva, Joseane P

    2016-06-10

    Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total β-carotene, containing all-E-, 9-Z-, and 13-Z-β-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no α-carotene was observed, variable amounts of a α-ring derived xanthophyll, lutein, was detected; with greater accumulation of α-ring xanthophylls than of β-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total β-carotene accumulation. Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low

  15. The effect of drought stress on the expression of key genes involved in the biosynthesis of phenylpropanoids and essential oil components in basil (Ocimum basilicum L.).

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    Abdollahi Mandoulakani, Babak; Eyvazpour, Elham; Ghadimzadeh, Morteza

    2017-07-01

    Basil (Ocimum basilicum L.), a medicinal plant of the Lamiaceae family, is used in traditional medicine; its essential oil is a rich source of phenylpropanoids. Methylchavicol and methyleugenol are the most important constituents of basil essential oil. Drought stress is proposed to enhance the essential oil composition and expression levels of the genes involved in its biosynthesis. In the current investigation, an experiment based on a completely randomized design (CRD) with three replications was conducted in the greenhouse to study the effect of drought stress on the expression level of four genes involved in the phenylpropanoid biosynthesis pathway in O. basilicum c.v. Keshkeni luvelou. The genes studied were chavicol O-methyl transferase (CVOMT), eugenol O-methyl transferase (EOMT), cinnamate 4-hydroxylase (C4H), 4-coumarate coA ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD). The effect of drought stress on the essential oil compounds and their relationship with the expression levels of the studied genes were also investigated. Plants were subjected to levels of 100%, 75%, and 50% of field capacity (FC) at the 6-8 leaf stage. Essential oil compounds were identified by gas chromatography/mass spectrometry (GC-MS) at flowering stage and the levels of gene expression were determind by real time PCR in plant leaves at the same stage. Results showed that drought stress increased the amount of methylchavicol, methyleugenol, β-Myrcene and α-bergamotene. The maximum amount of these compounds was observed at 50% FC. Real-time PCR analysis revealed that severe drought stress (50% FC) increased the expression level of CVOMT and EOMT by about 6.46 and 46.33 times, respectively, whereas those of CAD relatively remained unchanged. The expression level of 4CL and C4H reduced under drought stress conditions. Our results also demonstrated that changes in the expression levels of CVOMT and EOMT are significantly correlated with methylchavicol (r = 0.94, P ≤ 0

  16. Identification of genes involved in the biosynthesis of the third and fourth sugars of the Methanococcus maripaludis archaellin N-linked tetrasaccharide.

    Science.gov (United States)

    Ding, Yan; Jones, Gareth M; Uchida, Kaoru; Aizawa, Shin-Ichi; Robotham, Anna; Logan, Susan M; Kelly, John; Jarrell, Ken F

    2013-09-01

    N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes--mmp1084, mmp1085, mmp1086, and mmp1087--were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δmmp1086Δmmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δmmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δmmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV.

  17. A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari.

    Science.gov (United States)

    Kamei, Kazumasa; Kawabata, Hiroki; Asakura, Masahiro; Samosornsuk, Worada; Hinenoya, Atsushi; Nakagawa, Shinsaku; Yamasaki, Shinji

    2016-05-20

    In this study, we devised a multiplex PCR assay based on the gene of cytolethal distending toxin (cdt) B subunit to simultaneously detect and discriminate Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari. Species-specific PCR products were successfully obtained from all 38 C. jejuni, 12 C. fetus, 39 C. coli, 22 C. upsaliensis, 24 C. hyointestinalis, and 7 C. lari strains tested. On the other hand, no specific PCR products were obtained from other campylobacters and bacterial species tested (41 strains in total). The proposed multiplex PCR assay is a valuable tool for detection and descrimination of 6 major Campylobacter species, that are associated with gastrointestinal diseases in humans.

  18. Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.).

    Science.gov (United States)

    Deshpande, Ashish B; Chidley, Hemangi G; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

    2017-06-01

    Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Salicylic acid-induced changes in physiological parameters and genes of the flavonoid biosynthesis pathway in Artemisia vulgaris and Dendranthema nankingense during aphid feeding.

    Science.gov (United States)

    Sun, Y; Xia, X L; Jiang, J F; Chen, S M; Chen, F D; Lv, G S

    2016-02-19

    Phloem-feeding aphids cause serious damage to plants. The mechanisms of plant-aphid interactions are only partially understood and involve multiple pathways, including phytohormones. In order to investigate whether salicylic acid (SA) is involved and how it plays a part in the defense response to the aphid Macrosiphoniella sanbourni, physiological changes and gene expression profiles in response to aphid inoculation with or without SA pretreatment were compared between the aphid-resistant Artemisia vulgaris 'Variegata' and the susceptible chrysanthemum, Dendranthema nankingense. Changes in levels of reactive oxygen species, malondialdehyde (MDA), and flavonoids, and in the expression of genes involved in flavonoid biosynthesis, including PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), CHI (chalcone isomerase), F3H (flavanone 3-hydroxylase), F3'H (flavanone 3'-hydroxylase), and DFR (dihydroflavonol reductase), were investigated. Levels of hydrogen peroxide, superoxide anions, MDA, and flavonoids, and their related gene expression, increased after aphid infestation and SA pretreatment followed by aphid infestation; the aphid-resistant A. vulgaris exhibited a more rapid response than the aphid-susceptible D. nankingense to SA treatment and aphid infestation. Taken together, our results suggest that SA could be used to increase aphid resistance in the chrysanthemum.

  20. Stability of toxin gene proportion in red-pigmented populations of the cyanobacterium Planktothrix during 29 years of re-oligotrophication of Lake Zürich

    Directory of Open Access Journals (Sweden)

    Ostermaier Veronika

    2012-12-01

    Full Text Available Abstract Background Harmful algal blooms deteriorate the services of aquatic ecosystems. They are often formed by cyanobacteria composed of genotypes able to produce a certain toxin, for example, the hepatotoxin microcystin (MC, but also of nontoxic genotypes that either carry mutations in the genes encoding toxin synthesis or that lost those genes during evolution. In general, cyanobacterial blooms are favored by eutrophication. Very little is known about the stability of the toxic/nontoxic genotype composition during trophic change. Results Archived samples of preserved phytoplankton on filters from aquatic ecosystems that underwent changes in the trophic state provide a so far unrealized possibility to analyze the response of toxic/nontoxic genotype composition to the environment. During a period of 29 years of re-oligotrophication of the deep, physically stratified Lake Zürich (1980 to 2008, the population of the stratifying cyanobacterium Planktothrix was at a minimum during the most eutrophic years (1980 to 1984, but increased and dominated the phytoplankton during the past two decades. Quantitative polymerase chain reaction revealed that during the whole observation period the proportion of the toxic genotype was strikingly stable, that is, close to 100%. Inactive MC genotypes carrying mutations within the MC synthesis genes never became abundant. Unexpectedly, a nontoxic genotype, which lost its MC genes during evolution, and which could be shown to be dominant under eutrophic conditions in shallow polymictic lakes, also co-occurred in Lake Zürich but was never abundant. As it is most likely that this nontoxic genotype contains relatively weak gas vesicles unable to withstand the high water pressure in deep lakes, it is concluded that regular deep mixing selectively reduced its abundance through the destruction of gas vesicles. Conclusions The stability in toxic genotype dominance gives evidence for the adaptation to deep mixing of a

  1. Control of tylosin biosynthesis in Streptomyces fradiae.

    Science.gov (United States)

    Cundliffe, Eric

    2008-09-01

    Tylosin biosynthesis is controlled in cascade fashion by multiple transcriptional regulators, acting positively or negatively, in conjunction with a signalling ligand that acts as a classical inducer. The roles of regulatory gene products have been characterized by a combination of gene expression analysis and fermentation studies, using engineered strains of S. fradiae in which specific genes were inactivated or overexpressed. Among various novel features of the regulatory model, involvement of the signalling ligand is not essential for tylosin biosynthesis.

  2. Genomics study of the exposure effect of Gymnodinium catenatum, a paralyzing toxin producer, on Crassostrea gigas' defense system and detoxification genes.

    Directory of Open Access Journals (Sweden)

    Norma García-Lagunas

    Full Text Available BACKGROUND: Crassostrea gigas accumulates paralytic shellfish toxins (PST associated with red tide species as Gymnodinium catenatum. Previous studies demonstrated bivalves show variable feeding responses to toxic algae at physiological level; recently, only one study has reported biochemical changes in the transcript level of the genes involved in C. gigas stress response. PRINCIPAL FINDINGS: We found that 24 h feeding on toxic dinoflagellate cells (acute exposure induced a significant decrease in clearance rate and expression level changes of the genes involved in antioxidant defense (copper/zinc superoxide dismutase, Cu/Zn-SOD, cell detoxification (glutathione S-transferase, GST and cytochrome P450, CPY450, intermediate immune response activation (lipopolysaccharide and beta glucan binding protein, LGBP, and stress responses (glutamine synthetase, GS in Pacific oysters compared to the effects with the non-toxic microalga Isochrysis galbana. A sub-chronic exposure feeding on toxic dinoflagellate cells for seven and fourteen days (30×10³ cells mL⁻¹ showed higher gene expression levels. A significant increase was observed in Cu/Zn-SOD, GST, and LGBP at day 7 and a major increase in GS and CPY450 at day 14. We also observed that oysters fed only with G. catenatum (3×10³ cells mL⁻¹ produced a significant increase on the transcription level than in a mixed diet (3×10³ cells mL⁻¹ of G. catenatum+0.75×10⁶ cells mL⁻¹ I. galbana in all the analyzed genes. CONCLUSIONS: Our results provide gene expression data of PST producer dinoflagellate G. catenatum toxic effects on C. gigas, a commercially important bivalve. Over expressed genes indicate the activation of a potent protective mechanism, whose response depends on both cell concentration and exposure time against these toxic microalgae. Given the importance of dinoflagellate blooms in coastal environments, these results provide a more comprehensive overview of how oysters respond to

  3. Genomics study of the exposure effect of Gymnodinium catenatum, a paralyzing toxin producer, on Crassostrea gigas' defense system and detoxification genes.

    Science.gov (United States)

    García-Lagunas, Norma; Romero-Geraldo, Reyna; Hernández-Saavedra, Norma Y

    2013-01-01

    Crassostrea gigas accumulates paralytic shellfish toxins (PST) associated with red tide species as Gymnodinium catenatum. Previous studies demonstrated bivalves show variable feeding responses to toxic algae at physiological level; recently, only one study has reported biochemical changes in the transcript level of the genes involved in C. gigas stress response. We found that 24 h feeding on toxic dinoflagellate cells (acute exposure) induced a significant decrease in clearance rate and expression level changes of the genes involved in antioxidant defense (copper/zinc superoxide dismutase, Cu/Zn-SOD), cell detoxification (glutathione S-transferase, GST and cytochrome P450, CPY450), intermediate immune response activation (lipopolysaccharide and beta glucan binding protein, LGBP), and stress responses (glutamine synthetase, GS) in Pacific oysters compared to the effects with the non-toxic microalga Isochrysis galbana. A sub-chronic exposure feeding on toxic dinoflagellate cells for seven and fourteen days (30×10³ cells mL⁻¹) showed higher gene expression levels. A significant increase was observed in Cu/Zn-SOD, GST, and LGBP at day 7 and a major increase in GS and CPY450 at day 14. We also observed that oysters fed only with G. catenatum (3×10³ cells mL⁻¹ produced a significant increase on the transcription level than in a mixed diet (3×10³ cells mL⁻¹ of G. catenatum+0.75×10⁶ cells mL⁻¹ I. galbana) in all the analyzed genes. Our results provide gene expression data of PST producer dinoflagellate G. catenatum toxic effects on C. gigas, a commercially important bivalve. Over expressed genes indicate the activation of a potent protective mechanism, whose response depends on both cell concentration and exposure time against these toxic microalgae. Given the importance of dinoflagellate blooms in coastal environments, these results provide a more comprehensive overview of how oysters respond to stress generated by toxic dinoflagellate exposure.

  4. De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation.

    Science.gov (United States)

    Sun, Haiyue; Liu, Yushan; Gai, Yuzhuo; Geng, Jinman; Chen, Li; Liu, Hongdi; Kang, Limin; Tian, Youwen; Li, Yadong

    2015-09-02

    Cranberries (Vaccinium macrocarpon Ait.), renowned for their excellent health benefits, are an important berry crop. Here, we performed transcriptome sequencing of one cranberry cultivar, from fruits at two different developmental stages, on the Illumina HiSeq 2000 platform. Our main goals were to identify putative genes for major metabolic pathways of bioactive compounds and compare the expression patterns between white fruit (W) and red fruit (R) in cranberry. In this study, two cDNA libraries of W and R were constructed. Approximately 119 million raw sequencing reads were generated and assembled de novo, yielding 57,331 high quality unigenes with an average length of 739 bp. Using BLASTx, 38,460 unigenes were identified as putative homologs of annotated sequences in public protein databases, including NCBI NR, NT, Swiss-Prot, KEGG, COG and GO. Of these, 21,898 unigenes mapped to 128 KEGG pathways, with the metabolic pathways, secondary metabolites, glycerophospholipid metabolism, ether lipid metabolism, starch and sucrose metabolism, purine metabolism, and pyrimidine metabolism being well represented. Among them, many candidate genes were involved in flavonoid biosynthesis, transport and regulation. Furthermore, digital gene expression (DEG) analysis identified 3,257 unigenes that were differentially expressed between the two fruit developmental stages. In addition, 14,473 simple sequence repeats (SSRs) were detected. Our results present comprehensive gene expression information about the cranberry fruit transcriptome that could facilitate our understanding of the molecular mechanisms of fruit development in cranberries. Although it will be necessary to validate the functions carried out by these genes, these results could be used to improve the quality of breeding programs for the c