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Sample records for k62 mutant strain

  1. Mutant prevention concentrations of pradofloxacin for susceptible and mutant strains of Escherichia coli with reduced fluoroquinolone susceptibility.

    Science.gov (United States)

    Marcusson, Linda L; Komp Lindgren, Patricia; Olofsson, Sara K; Hughes, Diarmaid; Cars, Otto

    2014-10-01

    Pharmacodynamic and mutant prevention properties of the fluoroquinolone pradofloxacin (PRA) were measured against a set of 17 Escherichia coli strains carrying no, one or two known mutations conferring reduced fluoroquinolone susceptibility. The strains included susceptible wild-types, isogenic constructed mutants, isogenic selected mutants and clinical isolates. The effectiveness of PRA was determined with regard to preventing the selection of resistant mutants, using static and changing concentrations of drug. Ciprofloxacin was used as a reference drug. Minimum inhibitory concentrations (MICs) and mutant prevention concentrations (MPCs) of PRA for the susceptible wild-type strains were in the range 0.012-0.016mg/L and 0.2-0.3mg/L, respectively, giving a mean±standard deviation mutant prevention index (MPI=MPC/MIC) of 17.7±1.1. The mean MPI PRA of the 14 mutant strains was 19.2±12, and the mean MPI across all 17 strains was 18.9±10.8. In an in vitro kinetic model in which PRA was diluted with a half-life of 7h to mimic in vivo conditions, an initial concentration of PRA of 1.6-2.4mg/L (8-10× MPC), giving a PRA AUC/MPC ratio of 73-92, and a T>MPC of 21-23h was sufficient to prevent the selection of resistant mutants from the three susceptible wild-type strains. Dosing to reduce selection for antibiotic resistance in veterinary therapy has a role in reducing the reservoir of resistant mutants. We conclude that a level of dosing that prevents the selection of resistant mutants during therapy should be achievable in vivo. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  2. Revealing Differences in Metabolic Flux Distributions between a Mutant Strain and Its Parent Strain Gluconacetobacter xylinus CGMCC 2955

    Science.gov (United States)

    Liu, Miao; Yang, Xiao-Ning; Zhu, Hui-Xia; Jia, Yuan-Yuan; Jia, Shi-Ru; Piergiovanni, Luciano

    2014-01-01

    A better understanding of metabolic fluxes is important for manipulating microbial metabolism toward desired end products, or away from undesirable by-products. A mutant strain, Gluconacetobacter xylinus AX2-16, was obtained by combined chemical mutation of the parent strain (G. xylinus CGMCC 2955) using DEC (diethyl sulfate) and LiCl. The highest bacterial cellulose production for this mutant was obtained at about 11.75 g/L, which was an increase of 62% compared with that by the parent strain. In contrast, gluconic acid (the main byproduct) concentration was only 5.71 g/L for mutant strain, which was 55.7% lower than that of parent strain. Metabolic flux analysis indicated that 40.1% of the carbon source was transformed to bacterial cellulose in mutant strain, compared with 24.2% for parent strain. Only 32.7% and 4.0% of the carbon source were converted into gluconic acid and acetic acid in mutant strain, compared with 58.5% and 9.5% of that in parent strain. In addition, a higher flux of tricarboxylic acid (TCA) cycle was obtained in mutant strain (57.0%) compared with parent strain (17.0%). It was also indicated from the flux analysis that more ATP was produced in mutant strain from pentose phosphate pathway (PPP) and TCA cycle. The enzymatic activity of succinate dehydrogenase (SDH), which is one of the key enzymes in TCA cycle, was 1.65-fold higher in mutant strain than that in parent strain at the end of culture. It was further validated by the measurement of ATPase that 3.53–6.41 fold higher enzymatic activity was obtained from mutant strain compared with parent strain. PMID:24901455

  3. Revealing differences in metabolic flux distributions between a mutant strain and its parent strain Gluconacetobacter xylinus CGMCC 2955.

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    Cheng Zhong

    Full Text Available A better understanding of metabolic fluxes is important for manipulating microbial metabolism toward desired end products, or away from undesirable by-products. A mutant strain, Gluconacetobacter xylinus AX2-16, was obtained by combined chemical mutation of the parent strain (G. xylinus CGMCC 2955 using DEC (diethyl sulfate and LiCl. The highest bacterial cellulose production for this mutant was obtained at about 11.75 g/L, which was an increase of 62% compared with that by the parent strain. In contrast, gluconic acid (the main byproduct concentration was only 5.71 g/L for mutant strain, which was 55.7% lower than that of parent strain. Metabolic flux analysis indicated that 40.1% of the carbon source was transformed to bacterial cellulose in mutant strain, compared with 24.2% for parent strain. Only 32.7% and 4.0% of the carbon source were converted into gluconic acid and acetic acid in mutant strain, compared with 58.5% and 9.5% of that in parent strain. In addition, a higher flux of tricarboxylic acid (TCA cycle was obtained in mutant strain (57.0% compared with parent strain (17.0%. It was also indicated from the flux analysis that more ATP was produced in mutant strain from pentose phosphate pathway (PPP and TCA cycle. The enzymatic activity of succinate dehydrogenase (SDH, which is one of the key enzymes in TCA cycle, was 1.65-fold higher in mutant strain than that in parent strain at the end of culture. It was further validated by the measurement of ATPase that 3.53-6.41 fold higher enzymatic activity was obtained from mutant strain compared with parent strain.

  4. Crystal structure of penicillin G acylase from the Bro1 mutant strain of Providencia rettgeri.

    OpenAIRE

    McDonough, M. A.; Klei, H. E.; Kelly, J. A.

    1999-01-01

    Penicillin G acylase is an important enzyme in the commercial production of semisynthetic penicillins used to combat bacterial infections. Mutant strains of Providencia rettgeri were generated from wild-type cultures subjected to nutritional selective pressure. One such mutant, Bro1, was able to use 6-bromohexanamide as its sole nitrogen source. Penicillin acylase from the Bro1 strain exhibited an altered substrate specificity consistent with the ability of the mutant to process 6-bromohexana...

  5. Comparative metabolic flux analysis of an Ashbya gossypii wild type strain and a high riboflavin-producing mutant strain.

    Science.gov (United States)

    Jeong, Bo-Young; Wittmann, Christoph; Kato, Tatsuya; Park, Enoch Y

    2015-01-01

    In the present study, we analyzed the central metabolic pathway of an Ashbya gossypii wild type strain and a riboflavin over-producing mutant strain developed in a previous study in order to characterize the riboflavin over-production pathway. (13)C-Metabolic flux analysis ((13)C-MFA) was carried out in both strains, and the resulting data were fit to a steady-state flux isotopomer model using OpenFLUX. Flux to pentose-5-phosphate (P5P) via the pentose phosphate pathway (PPP) was 9% higher in the mutant strain compared to the wild type strain. The flux from purine synthesis to riboflavin in the mutant strain was 1.6%, while that of the wild type strain was only 0.1%, a 16-fold difference. In addition, the flux from the cytoplasmic pyruvate pool to the extracellular metabolites, pyruvate, lactate, and alanine, was 2-fold higher in the mutant strain compared to the wild type strain. This result demonstrates that increased guanosine triphosphate (GTP) flux through the PPP and purine synthesis pathway (PSP) increased riboflavin production in the mutant strain. The present study provides the first insight into metabolic flux through the central carbon pathway in A. gossypii and sets the foundation for development of a quantitative and functional model of the A. gossypii metabolic network.

  6. Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

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    Guocai Li

    Full Text Available Neisseria gonorrhoeae (N. gonorrhoeae outer membrane protein reduction modifiable protein (Rmp has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

  7. Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

    Science.gov (United States)

    Li, Guocai; Xie, Rushan; Zhu, Xiaoping; Mao, Yanli; Liu, Shuangxi; Jiao, Hongmei; Yan, Hua; Xiong, Kun; Ji, Mingchun

    2014-01-01

    Neisseria gonorrhoeae (N. gonorrhoeae) outer membrane protein reduction modifiable protein (Rmp) has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

  8. [Composition of cell walls of 2 mutant strains of Streptomyces chrysomallus].

    Science.gov (United States)

    Zaretskaia, M Sh; Nefelova, M V; Baratova, L A; Polin, A N

    1984-12-01

    The cell walls and peptidoglycans of two mutant strains, Streptomyces chrysomallus var. carotenoides and Streptomyces chrysomallus var. macrotetrolidi, were studied. The strains are organisms producing carotenes and antibiotics of the macrotetrolide group. By the qualitative composition of the peptidoglycans the mutants belong to Streptomyces and are similar. Their glycan portion consists of equimolar quantities of N-acetyl glucosamine and muramic acid. The peptide subunit is presented by glutamic acid, L, L-diaminopimelic acid, glycine and alanine. The molar ratio of alanine is 1.2-1.3. The mutant strains differ in the content of carbohydrates, total phosphorus and phosphorus belonging to teichoic acids. Teichoic acids of the cell walls of the both strains are of the ribitolhosphate nature. The cell walls of the mutants contain polysaccharides differing from teichoic acids and consisting of glucose, galactose, arabinose and fucose. The influence of the cell wall composition of the mutant strains on their morphology and metabolism and comparison of the data relative to the mutant strains with those relative to the starting strain are discussed.

  9. Induction, isolation, and characterization of aspergillus niger mutant strains producing elevated levels of beta-galactosidase.

    OpenAIRE

    1981-01-01

    An Aspergillus niger mutant strain, VTT-D-80144, with an improvement of three- to fourfold in the production of extracellular beta-galactosidase was isolated after mutagenesis. The production of beta-galactosidase by this mutant was unaffected by fermentor size, and the enzyme was also suitable for immobilization.

  10. Comparison of mutant and parent strains of Clostridium acetobutylicum: butyrate uptake at different temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Soni, B.K. [Institute of Gas Technology, 1700 S. Mount Prospect Road, Des Plaines, IL 60018 (United States); Jain, M.K. [MBI International, P.O. Box 27609, 3900 Collins Road, Lansing, MI 48909 (United States)

    1997-10-01

    A high butanol producing mutant strain of Clostridium acetobutylicum ATCC 4259 was obtained by chemical mutagenesis. Both mutant and parent strains were evaluated for butyrate uptake using the culture effluents of solventogenic fermentor-2 of the two-fermentor continuous system. Batch incubation of fermentor-2 culture effluents at 37 C indicated lower butyrate uptake rates for mutant and parent strain, at 0.05 and 0.03 g l{sup -1} h{sup -1}, respectively. Increased butyrate uptake rates of 0.33 and 0.26 g l{sup -1} h{sup -1} for mutant and parent strain, respectively, were observed when effluents were batch incubated at lower temperature of 30 C. Butyrate conversion efficiency, at 5 {+-} 0.1 g l{sup -1} of externally added butyrate, were 98.8% and 96.9% for mutant and parent strain, respectively. Butyrate up to the externally added concentration of 11.4 g l{sup -1} did not inhibit butyrate uptake. The maximum butyrate consumption at a slightly reduced uptake rate was seen at 10.2 g l{sup -1} butyrate concentration at 27 C. Based on the results under different temperatures, the electron flow pattern has been computed and the mechanism for butyrate uptake has been hypothesized. (orig.) With 6 figs., 4 tabs., 26 refs.

  11. Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

    Science.gov (United States)

    King, Stephen J.; Dutcher, Susan K.

    1997-01-01

    To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. PMID:9008712

  12. Infection of the ferret stomach by isogenic flagellar mutant strains of Helicobacter mustelae.

    OpenAIRE

    Andrutis, K A; Fox, J G; Schauer, D B; Marini, R P; Li, X.; L. Yan; Josenhans, C; Suerbaum, S

    1997-01-01

    Helicobacter mustelae, like Helicobacter pylori, possesses two flagellin proteins, FlaA and FlaB. Isogenic mutant strains of H. mustelae have been constructed by disruption of the flaA or flaB gene with a kanamycin resistance cassette or by introduction of both a kanamycin and a chloramphenicol resistance gene to produce a double mutant. To determine whether one or both flagellin proteins are necessary for colonization and persistence of infection with H. mustelae, 19 ferrets, specific pathog...

  13. Construction of a doramectin producer mutant from an avermectin-overproducing industrial strain of Streptomyces avermitilis.

    Science.gov (United States)

    Zhao, Xuejin; Wang, Yuanxin; Wang, Shiwei; Chen, Zhi; Wen, Ying; Song, Yuan

    2009-12-01

    The avermectin analogue doramectin (CHC-B1), which is produced in mutants that have an altered biosynthesis pathway of avermectin, is one of the most effective agricultural pesticides and antiparasitics. We report here the construction of a bkdF olmA double-deletion mutant lacking one of the branched-chain alpha-keto acid dehydrogenase encoding genes (bkdF) and the oligomycin PKS encoding gene cluster (olmA) in Streptomyces avermitilis 76-05. We then characterized the production of various antibiotics in cultures of the deletion mutant. In a fermentation medium supplemented with cyclohexanecarboxylic acid, this double mutant produced doramectin and its analogues but no oligomycin. The mutant proved to be genetically stable, without any antibiotic resistance markers inserted into its chromosome, and could potentially become an industrial doramectin-producing strain after further improvement.

  14. Tryptophan provision by dietary supplementation of a Bacillus subtilis mutant strain in piglets

    DEFF Research Database (Denmark)

    Torres-Pitarch, A; Nielsen, B.; Canibe, Nuria

    2015-01-01

    Supplementing Bacillus (B.) subtilis mutants selected to overproduce a specific amino acid (AA) may be an alternative method to provide essential AA in pig diets. Two experiments on a B. subtilis strain selected to overproduce Trp were conducted using 8-kg pigs fed Trp-deficient diets for 20 d. B...... to counterbalance the Trp deficiency in any of the two experiments. No effect of B. subtilis supplementation to piglet diets was observed on the plasma AA profile. In conclusion, this mutant strain of B. subtilis was not able to compensate a Trp deficiency in the tested doses....

  15. Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina.

    Science.gov (United States)

    Druzhinina, Irina S; Schmoll, Monika; Seiboth, Bernhard; Kubicek, Christian P

    2006-03-01

    The ascomycete Hypocrea jecorina (Trichoderma reesei), an industrial producer of cellulases and hemicellulases, can efficiently degrade plant polysaccharides. However, the catabolic pathways for the resulting monomers and their relationship to enzyme induction are not well known. Here we used the Biolog Phenotype MicroArrays technique to evaluate the growth of H. jecorina on 95 carbon sources. For this purpose, we compared several wild-type isolates, mutants producing different amounts of cellulases, and strains transformed with a heterologous antibiotic resistance marker gene. The wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources. The cellulase mutants were relatively similar to their parental strains. Both in the mutant and in the transformed strains, the most significant changes occurred in utilization of xylitol, erythritol, D-sorbitol, D-ribose, D-galactose, L-arabinose, N-acetyl-D-glucosamine, maltotriose, and beta-methyl-glucoside. Increased production of cellulases was negatively correlated with the ability to grow on gamma-aminobutyrate, adonitol, and 2-ketogluconate; and positively correlated with that on d-sorbitol and saccharic acid. The reproducibility, relative simplicity, and high resolution (+/-10% of increase in mycelial density) of the phenotypic microarrays make them a useful tool for the characterization of mutant and transformed strains and for a global analysis of gene function.

  16. Sexual behavior of mutant strains of the medfly Ceratitis capitata (Diptera: Tephritidae

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    R.D Briceño

    2003-09-01

    Full Text Available Males of the mutant strains (blind, vestigal-winged of the Mediterranean fruit fly (medfly, Ceratits capitata (Wiedmann showed differences in behavior compared with control (mass-reared males. Mutant males made fewer mating attempts and achieved fewer matings than control males. Vestigal-winged females copulated less frequently with both mutants. Blind males climbed rather than jumped onto females and copulated in very low numbers compared with control and vestigal males. Blind females copulated normally with control, males and in very low numbers with both types of mutant malesMachos mutantes (ciegos, alas vestigiales de la mosca del mediterráneo Ceratitis capitata (Wiedmann mostraron diferencias en conducta comparados con los machos testigo (cría masiva. Los machos mutantes, realizaron menos intentos por aparearse y lograron menos apareamientos que los machos testigo. Las hembras con alas vestigiales, copularon menos con ambas clases de mutantes. Los machos ciegos, subieron en lugar de saltar sobre las hembras y copularon en números muy bajos comparados con los machos testigo y con los de alas vestigiales. Las hembras ciegas, copularon de forma normal con los machos testigo y en números muy bajos con ambos tipos de machos mutantes

  17. Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

    NARCIS (Netherlands)

    Huisman, Gjalt W.; WONINK, E; DEKONING, G; Preusting, Hans; Witholt, Bernard

    1992-01-01

    We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have ide

  18. Characterization and increment of amylase production in mutant strains of Iranian native Bacillus licheniformis

    Directory of Open Access Journals (Sweden)

    Mohsen Mobini-Dehkordi

    2017-03-01

    Results: In this study, two interesting mutant strains were isolated and named B.L.2.M.1 and B.L.2.M.2. Mutations caused many changes in bacteria such as cell growth speed and enzyme production content. Differences in cell growth, production of amylase and other characters were significant at 0.05 level (Pvalue

  19. A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus, lacking flagella, has unusual growth physiology.

    Science.gov (United States)

    Lewis, Derrick L; Notey, Jaspreet S; Chandrayan, Sanjeev K; Loder, Andrew J; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

    2015-03-01

    A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.

  20. [Ribosome engineering of streptomyces sp. FJ3 from Three Gorges reservoir area and metabolic product of the selected mutant strain].

    Science.gov (United States)

    Hai, Le; Huang, Yuqi; Liao, Guojian; Hu, Changhua

    2011-07-01

    To explore new resource from inactive actinomycete strains, we screened resistant mutant strains by ribosome engineering, and analyzed the products derived from the selected mutant strains. Three Gorges reservoir area-derived actinomycete strains including BD20, FJ3, WZ20 and FJ5 were used as initial strains, which showed no-antibacterial activities. The streptomycin-resistant (str(R)) mutants and rifampicin-resistant (rif(R)) mutants were screened by single colony isolation on streptomycin-containing plates and rifampicin-containing plates according to the method for obtaining drug-resistant mutants in ribosome engineering. The four initial strains and their str(R)-mutants and rif(R)-mutants were fermented in a liquid medium with the same composition. Mutants with anti-Staphylococcus aureus activity were obtained by paper chromatography. The components of fermentation broth were analyzed by high performance liquid chromatography (HPLC) and high performance liquid chromatography-mass spectrometry (LC-MS). Furthermore, FJ3 strain was identified by 16S rDNA and morphology. The minimal inhibitory concentration (MIC) of streptomycin and rifampicin for FJ3 was: 0.5 microg/mL and 110 microg/mL, respectively. Twenty-four strR-mutant strains and 20 rif(R)-mutant strains of FJ3 mutant strains were selected for bioassay. The result of the antibacterial activity screening demonstrated that six strains inhibited bacteria. Two strains (FJ3-2 and FJ3-6) were screened from the streptomycin-resistance mutants of inactive strain FJ3. The result of bioassay showed that the fermentation broth of FJ3-2 and FJ3-6 exhibited obvious anti-Staphylococcus aureus activity. The assay of paper chromatography showed that the active substance may be nucleic acid class antibiotic via using solvent system Doskochilova. Moreover, the results of HPLC and LC-MS exhibited that this substance may be thiolutin. Ribosome engineering for changing the secondary metabolic function of the inactive wild

  1. Construction and characterization of a H19 epitope point mutant of MDV CVI988/Rispens strain.

    Science.gov (United States)

    Cui, Z; Qin, A; Lee, L F; Wu, P; Kung, H J

    1999-01-01

    A recombinant virus, CVI/rpp38, was developed from the Marek's disease virus (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by transfection of CVI988/Rispens-infected chick embryo fibroblasts (CEFs) with plasmid pHA25 DNA containing pp38 gene from GA strain of MDV. Monoclonal antibody (MAb) H19 which reacts with pp38 from GA but not with that from CVI988 was used to screen for recombinant viruses in transfected cell culture plates by immunofluorescent assay (IFA). A positive plaque was isolated, propagated, and purified from cell-free virus particles after sonication of infected CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in IFA but also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CVI988 virus. DNA sequence of the mutant virus showed a substitution of G at position 320 by a resulting in a change of an amino acid residue from arginine to glutamine. Comparison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 and CVI988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified H19 epitope in pp38. This mutant is potentially useful to further explore the biological function of pp38 and its H19 epitope.

  2. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis

    Institute of Scientific and Technical Information of China (English)

    Cheng-gang CAI; Bing-gan LOU; Xiao-dong ZHENG

    2008-01-01

    A new feather-degrading bacterium was isolated from a local feather waste site and identified as Bacillus subtilis based on morphological, physiochemical, and phylogenetic characteristics. Screening for mutants with elevated keratinolytic activity using N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis resulted in a mutant strain KD-N2 producing keratinolytic activity about 2.5 times that of the wild-type strain. The mutant strain produced inducible keratinase in different substrates of feathers, hair, wool and silk under submerged cultivation. Scanning electron microscopy studies showed the degradation of feathers, hair and silk by the keratinase. The optimal conditions for keratinase production include initial pH of 7.5, inoculum size of 2% (v/v), age of inoculum of 16 h, and cultivation at 23 ℃. The maximum keratinolytic activity of KD-N2 was achieved after 30 h. Essential amino acids like threonine, valine, methionine as well as ammonia were produced when feathers were used as substrates. Strain KD-N2,therefore, shows great promise of finding potential applications in keratin hydrolysis and keratinase production.

  3. Production of a thermal stress resistant mutant Euglena gracilis strain using Fe-ion beam irradiation.

    Science.gov (United States)

    Yamada, Koji; Kazama, Yusuke; Mitra, Sharbanee; Marukawa, Yuka; Arashida, Ryo; Abe, Tomoko; Ishikawa, Takahiro; Suzuki, Kengo

    2016-08-01

    Euglena gracilis is a common phytoplankton species, which also has motile flagellate characteristics. Recent research and development has enabled the industrial use of E. gracilis and selective breeding of this species is expected to further expand its application. However, the production of E. gracilis nuclear mutants is difficult because of the robustness of its genome. To establish an efficient mutation induction procedure for E. gracilis, we employed Fe-ion beam irradiation in the RIKEN RI beam factory. A decrease in the survival rate was observed with the increase in irradiation dose, and the upper limit used for E. gracilis selective breeding was around 50 Gy. For a practical trial of Fe-ion irradiation, we conducted a screening to isolate high-temperature-tolerant mutants. The screening yielded mutants that proliferated faster than the wild-type strain at 32 °C. Our results demonstrate the effectiveness of heavy-ion irradiation on E. gracilis selective breeding.

  4. A Mutant Strain of a Surfactant-Producing Bacterium with Increased Emulsification Activity

    Institute of Scientific and Technical Information of China (English)

    Liu Qingmei; Yao Jianming; Pan Renrui; Yu Zengliang

    2005-01-01

    As reported in this paper, a strain of oil-degrading bacterium Sp- 5- 3 was determined to belong to Enterobacteriaceae, which would be useful for microbial enhanced oil recovery(MEOR). The aim of our study was to generate a mutant using low energy N+ beam implantation. With 10 keV of energy and 5.2 × 10TM N+/cm2 of dose - the optimum condition, a mutant,S - 34, was obtained, which had nearly a 5-fold higher surface and a 13-fold higher of emulsification activity than the wild type. The surface activity was measured by two methods, namely, a surface tension measuring instrument and a recording of the repulsive circle of the oil film; the emulsification activity was scaled through measuring the separating time of the oil-fermentation mixture. The metabolic acid was determined as methane by means of gas chromatography.

  5. Production of cellulase using a mutant strain of trichoderma reesei growing on lactose in batch culture

    Energy Technology Data Exchange (ETDEWEB)

    Chaudhuri, B.K. (Indian Inst. of Tech., New Delhi (India). Biochemical Engineering Research Centre); Sahai, V. (Indian Inst. of Tech., New Delhi (India). Biochemical Engineering Research Centre)

    1993-05-01

    The production of cellulases in batch culture was studied using a mutant strain of Trichoderma reesei C-5 growing on lactose. Growth kinetic parameters on 2% lactose were studied and the comparative results for growth and enzyme productivities at two different lactose levels are discussed. The cellulase synthesis rate depended on the lactose concentration in the medium. Although growth was favoured at a higher lactose level, the volumetric enzyme productivity did not increase in proportion and the specific enzyme productivity decreased to a certain extent, indicating that partial catabolic inhibition at higher lactose concentrations may be possible. However, it was noted that the mutant strain was highly derepressed and capable of synthesising active cellulases on lactose. (orig.)

  6. Regulation of nitrogen metabolism is altered in a glnB mutant strain of Rhizobium leguminosarum.

    Science.gov (United States)

    Amar, M; Patriarca, E J; Manco, G; Bernard, P; Riccio, A; Lamberti, A; Defez, R; Iaccarino, M

    1994-02-01

    We isolated a Rhizobium leguminosarum mutant strain altered in the glnB gene. This event, which has never been described in the Rhizobiaceae, is rare in comparison to mutants isolated in the contiguous gene, glnA. The glnB mutation removes the glnBA promoter but in vivo does not prevent glnA expression from its own promoter, which is not nitrogen regulated. The glnB mutant strain does not grow on nitrate as a sole nitrogen source and it is Nod+, Fix+. Two -24/-12 promoters, for the glnII and glnBA genes, are constitutively expressed in the glnB mutant, while two -35/-10-like promoters for glnA and ntrBC are unaffected. We propose that the glnB gene product, the PII protein, plays a negative role in the ability of NtrC to activate transcription from its target promoters and a positive role in the mechanism of nitrate utilization.

  7. Isolation, characterization and long term preservation of mutant strains of Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Baeza, Marcelo; Retamales, Patricio; Sepúlveda, Dionisia; Lodato, Patricia; Jiménez, Antonio; Cifuentes, Víctor

    2009-04-01

    The yeast Xanthophyllomyces dendrorhous is biotechnologically important due to its ability to produce the pigment astaxanthin, but is poorly understood at the genetic level. This is mainly because its preservation is difficult and many of the mutants obtained are unstable. The objectives of the present work were (i) the mutagenesis X. dendrorhous and, (ii) isolation of mutants with auxotrophic markers suitable for genetic studies of the carotenogenesis pathway and sexual cycle. Additionally, two kinds of preservation methods at the laboratory level were tested for the storage of strains. A collection of X. dendrorhous mutants affected in the production of carotenoid pigments or development of sexual structures and auxotrophic requirements were isolated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine and the antibiotic nystatin. From a detailed analysis about the requirements of auxotrophic mutants the ARG7, ARG3 and PRO3 loci can be defined in this yeast. Among the methods assayed for the long-term preservation of X. dendrorhous strains, the dehydrated gelatin drop method showed the highest recovery of viable yeast after storage for 65 months. No changes in auxotrophic properties and in macro or micro morphology were observed after applying the latter method.

  8. Butyric acid fermentation from pre-treated wheat straw by a mutant clostridium tyrobutyricum strain

    DEFF Research Database (Denmark)

    Baroi, George Nabin; Baumann, Ivan; Westermann, Peter;

    ’s platform for a variety of products for industrial use. Butyric acid is considered as a potential chemical building-block for the production of chemicals for e.g. polymeric compounds and the aim of this work was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces less acetic...... acid (higher selectivity), has a higher yield and a higher productivity of butyric acid from pre-treated lignocellulosic biomass. Pre-treated wheat straw was used as the main carbon source. After one year of serial adaptation and selection a mutant strain of C. tyrobutyricum was developed. This new...

  9. Cytological characterization of an Aspergillus Nidulans mutant from a strain with chromosomic duplication.

    Science.gov (United States)

    Giancoli, Agata Cristiane Huppert; de Azevedo, João Lúcio; Pizzirani-Kleiner, Aline Aparecida

    2010-01-01

    A development mutant, named V103, was obtained spontaneously from the A strain of A. nidulans. The A strain contains a duplicated segment of chromosome I that has undergone translocation to chromosome II (I II). It is mitotically unstable and generates phenotypically deteriorated types, some with enhanced stability. The deteriorated variants of A. nidulans show abnormal development, exhibiting slower colony growth, variations in colony pigmentation and changes in conidiophore structure. The alterations observed in the conidiophore include fewer metulae and phialides, further elongation and ramification of these structures, delayed nuclear migration and the presence of secondary conidiophores.

  10. Cytological characterization of an Aspergillus nidulans mutant from a strain with chromosomic duplication

    Directory of Open Access Journals (Sweden)

    Ágata Cristiane Huppert Giancoli

    2010-03-01

    Full Text Available A development mutant, named V103, was obtained spontaneously from the A strain of A. nidulans. The A strain contains a duplicated segment of chromosome I that has undergone translocation to chromosome II (I → II. It is mitotically unstable and generates phenotypically deteriorated types, some with enhanced stability. The deteriorated variants of A. nidulans show abnormal development, exhibiting slower colony growth, variations in colony pigmentation and changes in conidiophore structure. The alterations observed in the conidiophore include fewer metulae and phialides, further elongation and ramification of these structures, delayed nuclear migration and the presence of secondary conidiophores.

  11. Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

    DEFF Research Database (Denmark)

    Baumgarten, Thomas; Schlegel, Susan; Wagner, Samuel

    2017-01-01

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived...... from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains......, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3...

  12. Coenzyme Q10 production in a 150-l reactor by a mutant strain of Rhodobacter sphaeroides.

    Science.gov (United States)

    Kien, Nguyen Ba; Kong, In-Soo; Lee, Min-Gyu; Kim, Joong Kyun

    2010-05-01

    For the commercial production of CoQ(10), batch-type fermentations were attempted in a 150-l fermenter using a mutant strain of R. sphaeroides. Optimum temperature and initial aeration rate were found to be 30 degrees C and 2 vvm, respectively. Under optimum fermentation conditions, the maximum value of specific CoQ(10) content was achieved reproducibly as 6.34 mg/g DCW after 24 h, with 3.02 g/l of DCW. During the fermentation, aeration shift (from the adequate aeration at the early growth phase to the limited aeration in active cellular metabolism) was a key factor in CoQ(10) production for scale-up. A higher value of the specific CoQ(10) content (8.12 mg/g DCW) was achieved in fed-batch fermentation and comparable to those produced by the pilot-scale fed-batch fermentations of A. tumefaciens, which indicated that the mutant strain of R. sphaeroides used in this study was a potential high CoQ(10) producer. This is the first detailed study to demonstrate a pilot-scale production of CoQ(10) using a mutant strain of R. sphaeroides.

  13. Characterization of a mutant strain of a filamentous fungus Cladosporium phlei for the mass production of the secondary metabolite phleichrome.

    Science.gov (United States)

    Yi, Min-Hee; Kim, Jung-Ae; Kim, Jung-Mi; Park, Jin-Ah; Kim, Beom-Tae; Park, Seung-Moon; Yang, Moon-Sik; Hwang, Ki-Jun; Kim, Dae-Hyuk

    2011-08-01

    UV-mutagenesis was performed to obtain mutant strains that demonstrate altered production of phleichrome, a secondary metabolite of Cladosporium phlei. Among fifty mutants selected, based on the increased area and intensity of the purple pigment surrounding the colonies, the strain M0035 showed the highest production of phleichrome, more than seven fold over wild type. Plate cultures of the M0035 strain resulted in a total of 592 mg phleichrome consisting of 146 mg and 446 mg from the mycelia and agar media, respectively. The M0035 strain displayed a growth rate and a mycelial mass comparable to the parental strain but had significantly reduced asexual sporulation.

  14. In vitro characterization of Salmonella typhi mutant strains for live oral vaccines.

    Science.gov (United States)

    Dragunsky, E M; Rivera, E; Hochstein, H D; Levenbook, I S

    1990-06-01

    Several Salmonella typhi attenuated mutant strains, suggested as candidates for live oral vaccine, were examined for their characteristics in vitro in comparison with parental strains Ty2 and CDC10-80. Three methods were used: interaction of bacteria with the human monocyte-macrophage U937 cell line evaluated by microscopic examination, bacterial growth in the cell culture medium estimated by absorbance and bacterial resistance to human plasma assessed by the viable count technique. The most informative data were obtained in the test with U937 cells. Ty2 penetrated almost 100% of the cells, multiplied rapidly and caused death of the cells. CDC10-80 infected about 30% of the cells, multiplied slightly and did not kill the cells. The Ty2 mutant galE via EX462 behaved like CDC10-80. Bacteria of the galE Ty21a, Vi + Ty21a, 541 Ty and 543 Ty, found in only 3-4% of the cells, did not multiply within the cells and decreased in number with time. These findings correlate with the reported virulence of these strains for humans. With the second method, the rate of bacterial growth in cell culture medium did not differentiate Ty2, CDC10-80 and EX462. They grew at the same rate and faster than the remaining mutants. The plasma resistance test did not discriminate between EX462 and other mutants. These tests did not reveal any difference between Vi + Ty21a and Vi-Ty21a.

  15. Experimental infection of Mongolian gerbils with wild-type and mutant Helicobacter pylori strains.

    Science.gov (United States)

    Wirth, H P; Beins, M H; Yang, M; Tham, K T; Blaser, M J

    1998-10-01

    Experimental Helicobacter pylori infection was studied in Mongolian gerbils with fresh human isolates that carry or do not carry cagA (cagA-positive or cagA-negative, respectively), multiply passaged laboratory strains, wild-type strain G1.1, or isogenic ureA, cagA, or vacA mutants of G1.1. Animals were sacrificed 1 to 32 weeks after challenge, the stomach was removed from each animal for quantitative culture, urease test, and histologic testing, and blood was collected for antibody determinations. No colonization occurred after >/=20 in vitro passages of wild-type strain G1.1 or with the ureA mutant of G1.1. In contrast, infection occurred in animals challenged with wild-type G1.1 (99 of 101 animals) or the cagA (25 of 25) or vacA (25 of 29) mutant of G1.1. Infection with G1.1 persisted for at least 8 months. All 15 animals challenged with any of three fresh human cagA-positive isolates became infected, in contrast to only 6 (23%) of 26 animals challenged with one of four fresh human cagA-negative isolates (P < 0.001). Similar to infection in humans, H. pylori colonization of gerbils induced gastric inflammation and a systemic antibody response to H. pylori antigens. These data confirm the utility of gerbils as an animal model of H. pylori infection and indicate the importance of bacterial strain characteristics for successful infection.

  16. Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

    Science.gov (United States)

    Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

    2015-11-01

    Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection.

  17. Carotenoid production and gene expression in an astaxanthin-overproducing Xanthophyllomyces dendrorhous mutant strain.

    Science.gov (United States)

    Castelblanco-Matiz, Lina M; Barbachano-Torres, Alejandra; Ponce-Noyola, Teresa; Ramos-Valdivia, Ana C; Cerda García-Rojas, Carlos M; Flores-Ortiz, César M; Barahona-Crisóstomo, Salvador K; Baeza-Cancino, Marcelo E; Alcaíno-Gorman, Jennifer; Cifuentes-Guzmán, Víctor H

    2015-12-01

    The primary carotenoid synthesized by Xanthophyllomyces dendrorhous is astaxanthin, which is used as a feed additive in aquaculture. Cell growth kinetics and carotenoid production were correlated with the mRNA levels of the idi, crtE, crtYB, crtI, crtS and crtR genes, and the changes in gene sequence between the wild-type and a carotenoid overproducer XR4 mutant strain were identified. At the late stationary phase, the total carotenoid content in XR4 was fivefold higher than that of the wild-type strain. Additionally, the mRNA levels of crtE and crtS increased during the XR4 growth and were three times higher than the wild-type strain in the late stationary phase. Moreover, the nucleotide sequences of crtYB, crtI and crtR exhibited differences between the strains. Both the higher crtE and crtS transcript levels and the crtYB, crtI and crtR mutations can, at least in part, act to up-regulate the carotenoid biosynthesis pathway in the XR4 strain.

  18. Production of Ethanol by Indigenous Wild and Mutant Strain of Thermotolerant Kluyveromyces Marxianus Under Optimized Fermentation Conditions

    Directory of Open Access Journals (Sweden)

    Shaheen Aziz

    2009-12-01

    Full Text Available The maximum ethanol production and β-fructofuranocidase formation under fermentation studies were carried out in microprocessor controlled 23-L stainless steel fermenter at the following conditions: Temp = 40 ˚C (wild &mutant organism , pH= 5.5, carbon source (molasses=15% sugar nitrogen source (ammonium sulphate 0.75%, 300 RPM stirring speed and oxygen flow rate was 0.1 vvm. Mutant. Strain of thermotolerant Kluyveromyces marxianus M15 produced maximum production of ethanol at 48hr.All Kinetic Parameters have been studied for the utilization of substrate and production of maximum ethanol for both wild and mutant strains. It has been observed that wild strain was growing up to 550C while the mutant strain was growing up to 650C. In this comparison study, wild and mutant strain showed that Mutant-derived M15 was stronger over its parental culture due to its more thermal stability and production of ethanol at 65 oC at which wild organism could not grow.

  19. Characterization of a Mutant Listeria monocytogenes Strain Expressing Green Fluorescent Protein

    Institute of Scientific and Technical Information of China (English)

    Ling-Li JIANG; Hou-Hui SONG; Xue-Yan CHEN; Chun-Lin KE; Jing-Jing XU; Ning CHEN; Wei-Huan FANG

    2005-01-01

    To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene(hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature.Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogenes could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.

  20. The Breeding of a Pigment Mutant Strain of Steroid Hydroxylation Aspergillus Flavus by Low Energy Ion Implantation

    Institute of Scientific and Technical Information of China (English)

    YE Hui; MA Jingming; FENG Chun; CHENG Ying; ZHU Suwen; CHENG Beijiu

    2009-01-01

    In the process of the fermentation of steroid C11α-hydroxylgenation strain Aspergillus flavus AF-ANo208.a red pigment is derived.which will affect the isolation and purification of the target product.Low energy ion beam implantation is a new tool for breeding excellent mutant strains.In this study,the ion beam implantation experiments were performed by infusing two different ions:argon ion(Ar+)and nitrogen ion(N+).The results showed that the optimal ion implantation was N+ with an optimum dose of 2.08×1015 ions/cm2.with which the mutant strain AF-ANml6 that produced no red pigment was obtained.The strain had high genetic stability and kept the strong capacity of C11α-hydroxylgenation,which could be utilized in industrial fermentation.The difierences between the original strain and the mutant strain at a molecular level were analyzed by randomly amplified polymorphic DNA(RAPD).The results indicated that the frequency of variation Was 7.00%,which would establish the basis of application investigation into the breeding of pigment mutant strains by low energy ion implantation.

  1. Gas exchange in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133 and Its hydrogenase-deficient mutant strain NHM5.

    Science.gov (United States)

    Lindberg, Pia; Lindblad, Peter; Cournac, Laurent

    2004-04-01

    Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H(2)), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O(2), CO(2), N(2), and H(2) was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO(2) and O(2). The amount of H(2) produced per molecule of N(2) fixed was found to vary with light conditions, high light giving a greater increase in H(2) production than N(2) fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H(2) produced per molecule of N(2) fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO(2), caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 micro mol (mg of chlorophyll a)(-1) h(-1) to 9 micro mol (mg of chlorophyll a)(-1) h(-1) after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.

  2. Genomic sequence of a mutant strain of Caenorhabditis elegans with an altered recombination pattern

    Directory of Open Access Journals (Sweden)

    Marra Marco

    2010-02-01

    Full Text Available Abstract Background The original sequencing and annotation of the Caenorhabditis elegans genome along with recent advances in sequencing technology provide an exceptional opportunity for the genomic analysis of wild-type and mutant strains. Using the Illumina Genome Analyzer, we sequenced the entire genome of Rec-1, a strain that alters the distribution of meiotic crossovers without changing the overall frequency. Rec-1 was derived from ethylmethane sulfonate (EMS-treated strains, one of which had a high level of transposable element mobility. Sequencing of this strain provides an opportunity to examine the consequences on the genome of altering the distribution of meiotic recombination events. Results Using Illumina sequencing and MAQ software, 83% of the base pair sequence reads were aligned to the reference genome available at Wormbase, providing a 21-fold coverage of the genome. Using the software programs MAQ and Slider, we observed 1124 base pair differences between Rec-1 and the reference genome in Wormbase (WS190, and 441 between the mutagenized Rec-1 (BC313 and the wild-type N2 strain (VC2010. The most frequent base-substitution was G:C to A:T, 141 for the entire genome most of which were on chromosomes I or X, 55 and 31 respectively. With this data removed, no obvious pattern in the distribution of the base differences along the chromosomes was apparent. No major chromosomal rearrangements were observed, but additional insertions of transposable elements were detected. There are 11 extra copies of Tc1, and 8 of Tc2 in the Rec-1 genome, most likely the remains of past high-hopper activity in a progenitor strain. Conclusion Our analysis of high-throughput sequencing was able to detect regions of direct repeat sequences, deletions, insertions of transposable elements, and base pair differences. A subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome

  3. Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.

    OpenAIRE

    Wilson, K. J.; Anjaiah, V; Nambiar, P T; Ausubel, F M

    1987-01-01

    Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nod...

  4. Purification and characterisation of α-amylase produced by mutant strain of Aspergillus oryzae EMS-18.

    Science.gov (United States)

    Abdullah, Roheena; Ikram-ul-Haq

    2015-01-01

    α-Amylase produced by a mutant strain of Aspergillus oryzae EMS-18 has been purified to homogeneity as judged by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified by using 70% ammonium sulphate precipitation followed by anion exchange chromatography on DEAE-Sephadex column and gel filtration on Sephadex G-100. An enzyme purification factor of 9.5-fold was achieved with a final specific activity of 1987.7 U/mg protein and overall yield of 23.8%. The molecular weight of purified α-amylase was estimated to be 48 kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature and pH 40°C and 5.0, respectively. Except Ca(++) all other metal ions such as Mg, Mn, Na, Zn, Ni, Fe, Cu, Co and Ba were found to be inhibitory to enzyme activity.

  5. A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase. Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain.

    Science.gov (United States)

    Middelhoven, W J; van Eijk, J; van Renesse, R; Blijham, J M

    1978-01-01

    NAD-specific glutamate dehydrogenase (GDH-B) was induced in a wild-type strain derived of alpha-sigma 1278b by alpha-amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing alpha-amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate. Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases area lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.

  6. Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

    Directory of Open Access Journals (Sweden)

    Annemarie Kramer

    Full Text Available The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

  7. Induced drought tolerance through wild and mutant bacterial strain Pseudomonas simiae in mung bean (Vigna radiata L.).

    Science.gov (United States)

    Kumari, Sarita; Vaishnav, Anukool; Jain, Shekhar; Varma, Ajit; Choudhary, Devendra Kumar

    2016-01-01

    The present study focused on the overproducing mutant of a plant growth promoting rhizobacterium (PGPR) Pseudomonas simiae strain AU (MTCC-12057) for significant drought tolerance in mung bean plants. Five mutants namely AU-M1, AU-M2, AU-M3, AU-M4 and AU-M5 were made after treatment of wild type strain with N-methyl-N-nitro-N-nitrosoguanidine. Mutant strain AU-M4 was recorded for enhanced ACC deaminase (ACC-D) activity, indole acetic acid (IAA) production and inorganic phosphate (Pi) solubilization compared to wild strain and other four mutant strains under drought condition. AU-M4 showed higher phosphate solubilization index (8.17) together with higher ACC-D activity (98 nmol/mg/h) and IAA concentration (69.35 µg/ml) compared with the wild type P. simiae strain AU ACC-D activity (79 nmol/mg/h) and IAA concentration (38.98 µg/ml) respectively. In this report, we investigated the effect of both wild and mutant type bacterial strain on mung bean plants under drought stress. Results showed that mutant AU-M4 and wild type strain AU inoculated plants exhibited superior tolerance against drought stress, as shown by their enhanced plant biomass (fresh weight), higher water content, higher proline accumulation and lower osmotic stress injury. Mutant AU-M4 and wild strain AU inoculated plants reduced the ethylene level by 59 and 45% respectively, compared to the control under stress condition. Furthermore, bacterial inoculated plants showed enhanced induced systemic drought tolerance by reducing stomata size and net photosynthesis resulting higher water content in mung bean plants that may help in survival of plants during drought condition. To mitigate the effects of drought stress, use of PGPR will be needed to ensure sufficient production of food from crop plants. Taking current leads available, concerted future research is needed in this area, particularly on field evaluation with application of potential microorganisms.

  8. Phenotypic and genotypic characterization of mutants of the virginiamycin producing strain 899 and its relatedness to the type strain of Streptomyces virginiae.

    Science.gov (United States)

    Lanoot, B; Vancanneyt, M; Hoste, B; Cnockaert, M C; Piecq, M; Gosselé, F; Swings, J

    2005-01-01

    A representative set of 19 mutants, with a known genealogy, of the virginiamycin producing strain Streptomyces virginiae 899 was investigated phenotypically and genotypically. Colour of the aerial and substrate mycelium were very variable both among spontaneous variants and those obtained after induced mutagenesis. At genotypic level, all mutants showed nearly identical BOX patterns, not reflecting the phenotypic heterogeneity observed. More than 40 years of forced mutational pressure did not cause huge chromosomal distortions but was most likely limited to base substitutions. The species S. virginiae, including besides producers of virginiamycin the type strain and non-type strains producing other bioactive compounds, is genomically heterogeneous on the basis of BOX-PCR fingerprinting and DNA-DNA hybridizations. The virginiamycin producing strain 899 does not belong to the species S. virginiae despite its phenotypic similarity to the latter.

  9. Mutant human myocilin induces strain specific differences in ocular hypertension and optic nerve damage in mice.

    Science.gov (United States)

    McDowell, Colleen M; Luan, Tomi; Zhang, Zhang; Putliwala, Tasneem; Wordinger, Robert J; Millar, J Cameron; John, Simon W M; Pang, Iok-Hou; Clark, Abbot F

    2012-07-01

    Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 10(7) pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

    Energy Technology Data Exchange (ETDEWEB)

    Chu, K.-W. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chan, Shirley K.W. [Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chow, King L. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China) and Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China)]. E-mail: bokchow@ust.hk

    2005-09-30

    Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring.

  11. Functional analysis of an feoB mutant in Clostridium perfringens strain 13.

    Science.gov (United States)

    Awad, Milena M; Cheung, Jackie K; Tan, Joanne E; McEwan, Alastair G; Lyras, Dena; Rood, Julian I

    2016-10-01

    Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type; effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections.

  12. Structural characterization of bioengineered α-D-glucans produced by mutant glucansucrase GTF180 enzymes of lactobacillus reuteri strain 180

    NARCIS (Netherlands)

    Leeuwen, S.S. van; Kralj, S.; Eeuwema, W.; Gerwig, G.J.; Dijkhuizen, L.; Kamerling, J.P.

    2009-01-01

    Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith degradati

  13. Comparison Study on Colonization of hilA Mutant and Parent Strains of Salmonella enteritidis in Vertically Infected Broiler Chickens

    Directory of Open Access Journals (Sweden)

    MohammadSadegh Madadi

    2015-10-01

    Full Text Available Background: Salmonella actively stimulates its own uptake into the epithelial cells by inducing cytoskeleton rearrangements and membrane ruffling triggered by some proteins secreted by Salmonella into the cytosol of the epithelial cells via a type III secretion system (TTSS encoded bygenes of the Salmonella pathogenicity island 1 (SPI-1. hilA is a transcriptional activator encoded on Salmonella Pathogenicity Island 1 (SPI-1 genes.Methods: To assess the importance of hilA in a simulation modeling of vertical infection and shedding of S. enteritidis in broiler chickens a long-term experiment was designed. Two groups of 200 fertile eggs were inoculated with 20 colony forming units (CFU of hilA mutant of S. enteritidis or its parent strain just prior to incubation. Thirty five birds of each group were housed in separate rooms. On days 2, 4, 7, 14, 21, 28 and 35 of age, cloacal swabs from live birds as well as samples from internal organs (intestinal tract, liver and spleen were evaluated by bacteriological or molecular methods.Results: In most of sampling days colonization and invasion of parent strain S. enteritidis in intestine (especially ceaca and internal organs of chickens were higher with compared to its hilA mutant but this mutant strain could still colonize in intestinal tract and even invade liver or spleen.Conclusion: Colonization of hilA mutant of S. enteritidis indicated that hilA gene is only one part of the modulators in Salmonella invasion mechanism. The ability of hilA mutant to multiply and persist in host internal organs including ceaca may promise further research for potential of hilA mutant to prevent the initial colonization of the intestinal tract by a virulent S. enteritidis strain

  14. The effect of gamma irradiation on astaxanthin synthetase encoding gene in two mutant strains of Phaffia rhodozyma.

    Directory of Open Access Journals (Sweden)

    Naeimeh Najafi

    2013-09-01

    Full Text Available Astaxanthin, an orange-red carotenoid pigment, acts as a protective agent against oxidative damage to cells in vivo. The astaxanthin synthetase gene (crtS size consists of 3995 bp. This gene has been suggested to catalyse β-carotene to astaxanthin in Phaffia rhodozyma. The aim of this research was to find any possible changes in this gene in two mutant strains, Gam1 and Gam2 (with high astaxanthin pigment production, previously created by gamma irradiation.The astaxanthin synthetase gene sequence of Phaffia rhodozyma in the NCBI Gene bank was used to design primer. In Gam1, this gene was amplified using primers Asta F1, Asta R2, Asta F3, Asta R4. In Gam2, primers asta F1, asta R4 were used to amplify the gene. The amplified fragments were 8 sequenced using primers Asta F1, Asta R1, Asta F2, Asta R2, Asta F3, Asta R3 and Asta F4, Asta R4. Astaxanthin synthetase gene from two mutant strains, Gam1 and Gam2 were amplified using PCR. The amplified products were sequenced and aligned using the ClustalW software.The comparison of this gene showed 98% and 99% similarities between the reference sequence and Gam1 and Gam2 mutant strains, respectively, whereas the comparison of this gene in Gam1 and Gam2 mutant strains showed 97% similarity. However, the deduced proteins showed 78% and 83% between the reference protein obtained from the wild type and Gam1 and Gam2, respectively. This similarity was 75% between the mutant strains.

  15. DNA polymorphism and total protein in mutants of Metarhizium anisopliae var. Anisopliae (Metsch.) Sorokin strain E9

    OpenAIRE

    Freire,Laurineide Lopes de Carvalho; Costa,Ana Bolena Lima da; Góes,Larissa Brandão; Oliveira,Neiva Tinti de

    2001-01-01

    Five mutants (MaE10, MaE27, MaE24, MaE41 e MaE49) of Metarhizium anisopliae wild strain E9 were analysed for DNA profile through the RAPD technique and for changes in total protein content by spectrophotometry, polyacrylamide gel electrophoresis and densitometry. The pattern of RAPD markers showed genetic polymorphism among the strains: out of twenty primers seven were selected, producing 113 bands. Forty seven bands were present in all strains (41.6% of monomorphic bands) and 66 showed polym...

  16. Assessing benzene-induced toxicity on wild type Euglena gracilis Z and its mutant strain SMZ.

    Science.gov (United States)

    Peng, Cheng; Arthur, Dionne M; Sichani, Homa Teimouri; Xia, Qing; Ng, Jack C

    2013-11-01

    Benzene is a representative member of volatile organic compounds and has been widely used as an industrial solvent. Groundwater contamination of benzene may pose risks to human health and ecosystems. Detection of benzene in the groundwater using chemical analysis is expensive and time consuming. In addition, biological responses to environmental exposures are uninformative using such analysis. Therefore, the aim of this study was to employ a microorganism, Euglena gracilis (E. gracilis) as a putative model to monitor the contamination of benzene in groundwater. To this end, we examined the wild type of E. gracilis Z and its mutant form, SMZ in their growth rate, morphology, chlorophyll content, formation of reactive oxygen species (ROS) and DNA damage in response to benzene exposure. The results showed that benzene inhibited cell growth in a dose response manner up to 48 h of exposure. SMZ showed a greater sensitivity compared to Z in response to benzene exposure. The difference was more evident at lower concentrations of benzene (0.005-5 μM) where growth inhibition occurred in SMZ but not in Z cells. We found that benzene induced morphological changes, formation of lipofuscin, and decreased chlorophyll content in Z strain in a dose response manner. No significant differences were found between the two strains in ROS formation and DNA damage by benzene at concentrations affecting cell growth. Based on these results, we conclude that E. gracilis cells were sensitive to benzene-induced toxicities for certain endpoints such as cell growth rate, morphological change, depletion of chlorophyll. Therefore, it is a potentially suitable model for monitoring the contamination of benzene and its effects in the groundwater.

  17. Behavioral characterization of a mutant mouse strain lacking D-amino acid oxidase activity.

    Science.gov (United States)

    Zhang, Min; Ballard, Michael E; Basso, Ana M; Bratcher, Natalie; Browman, Kaitlin E; Curzon, Pete; Konno, Ryuichi; Meyer, Axel H; Rueter, Lynne E

    2011-02-02

    D-amino acid oxidase (DAO), an enzyme that degrades d-serine, has been suggested as a susceptibility factor for schizophrenia. Here we sought to understand more about the behavioral consequence of lacking DAO and the potential therapeutic implication of DAO inhibition by characterizing a mouse strain (ddY/DAO(-)) lacking DAO activity. We found that the mutant mice showed enhanced prepulse inhibition responses (PPI). Intriguingly, DAO-/- mice had increased sensitivity to the PPI-disruptive effect induced by the competitive NMDA antagonist, SDZ 220-581. In the 24-h inhibitory avoidance test, DAO-/- mice were not different from DAO+/+ mice during the recall. In Barnes Maze, we found that DAO-/- mice had a shortened latency to enter the escape tunnel. Interestingly, although these mice were hypoactive when tested in a protected open field, they showed a profound increase of activity on the edge of the unprotected open field of the Barnes Maze even with the escape tunnel removed. This increased edge activity does not appear to be related to a reduced level of anxiety given that there were no significant genotype effects on the measures of anxiety-like behaviors in two standard animal models of anxiety, elevated plus maze and novelty suppressed feeding. Our data suggest that DAO-/- mice might have altered functioning of NMDARs. However, these results provide only modest support for manipulations of DAO activity as a potential therapeutic approach to treat schizophrenia.

  18. Residual virulence and immunogenicity of CGV26 and CGV2631 B. melitensis Rev. 1 deletion mutant strains in sheep after subcutaneous or conjunctival vaccination.

    Science.gov (United States)

    Guilloteau, Laurence A; Laroucau, Karine; Olivier, Michel; Grillo, Maria Jesus; Marin, Clara M; Verger, Jean-Michel; Blasco, Jose-Maria

    2006-04-24

    The CGV26 and CGV2631 strains are novel engineered Brucella melitensis Rev.1 mutant strains deleted for the bp26 gene or for both bp26 and omp31 genes, respectively, coding for proteins of diagnostic significance. The residual virulence and immunogenicity of both mutants were compared to the parental Rev.1 strain in sheep after subcutaneous or conjunctival vaccination. The deletion of the bp26 gene or both bp26 and omp31 genes had no significant effect on the intracellular survival of the Rev.1 strain in ovine macrophage cultures. The kinetics of infection induced by both mutants in sheep was similar to the Rev.1 strain, and inoculation by the subcutaneous route produced wider and more generalized infections than the conjunctival route. All strains were cleared from lymph nodes and organs within 3 months after inoculation. The CGV26 and CGV2631 mutants induced both specific systemic antibody response and lymphoproliferation in sheep. The kinetics of the responses induced by the mutants was quite similar to that of the parental Rev.1 strain, except for the intensity of the lymphoproliferative response, which was attenuated for the CGV2631 mutant. In conclusion, the residual virulence of both CGV26 and CGV2631 mutants in sheep was similar to that of the parental Rev.1 vaccine strain. These mutants induced also significant specific antibody and cell-mediated immunity in sheep and are suitable to be evaluated as potential vaccine candidates against B. melitensis and B. ovis infections in sheep.

  19. Analysis of genetic relationship in mutant silkworm strains of Bombyx mori using inter simple sequence repeat (ISSR) markers

    Institute of Scientific and Technical Information of China (English)

    Dhanikachalam Velu; Kangayam M. Ponnuvel; Murugiah Muthulakshmi; Randhir K. Sinha; Syed M.H. Qadri

    2008-01-01

    Amplified inter simple sequence repeats (ISSR) markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat (SSR) motifs were used in this study. A total of 113 markers were produced among 20 mutant swains, of which 73.45% were found to be polymorphic. In selected mutant genetic stocks, the average number of observed allele was (1.7080±0.4567), effective alleles (1.5194±0.3950) and genetic diversity (Ht) (0.2901±0.0415). The dendrogram produced using the unweighted pair group method with arithmetic means (UPGMA) and cluster analysis made using Nei's genetic distance resulted in the formation of one major group containing 6 groups separated 20 mutant silkworm strains. Therefore, ISSR amplification is a valuable method for determining the genetic variability among mutant silkworm swains. This efficient molecular marker would be useful for characterizing a considerable number of silkworm swains maintained at the germplasm center.

  20. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents.

    Science.gov (United States)

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; Reis, Ana Beatriz de Bragança Dos; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-05-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

  1. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    Science.gov (United States)

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  2. Mutant strains of Spirulina (Arthrospira) platensis to increase the efficiency of micro-ecological life support systems

    Science.gov (United States)

    Brown, Igor

    The European Micro-Ecological Life Support System Alternative (MELiSSA) is an advanced idea for organizing a bioregenerative system for long term space flights and extraterrestrial settlements (Hendrickx, De Wever et al., 2005). Despite the hostility of both lunar and Martian environments to unprotected life, it seems possible to cultivate photosynthetic bacteria using closed bioreactors illuminated and heated by solar energy. Such reactors might be employed in critical processes, e.g. air revitalization, foodcaloric and protein source, as well as an immunomodulators production. The MELiSSA team suggested cyanobacterium Spirulina as most appropriate agent to revitalize air and produce a simple "fast" food. This is right suggestion because Spirulina was recently shown to be an oxygenic organism with the highest level of O2 production per unit mass (Ananyev et al., 2005). Chemical composition of Spirulina includes proteins (55Aiming to make Spirulina cultivation in life support systems like MELiSSA more efficient, we selected Spirulina mutant strains with increased fraction of methionine in the biomass of this cyanobacterium and compared the effect of parental wild strain of Spirulina and its mutants on the tendency of such experimental illnesses as radiationinduced lesions and hemolythic anemia. Results: It was found that mutant strains 198B and 27G contain higher quantities of total protein, essential amino acids, c-phycocyanin, allophycocyanin and chlorophyll a than parental wild strain of S. platensis. The strain 198B is also characterized with increased content of carotenoids. Revealed biochemical peculiarities of mutant strains suggest that these strains can serve as an additional source of essential amino acids as well as phycobiliproteins and carotenoids for the astronauts. Feeding animals suffering from radiation-induced lesions, c-phycocyanin, extracted from strain 27G, led to a correction in deficient dehydrogenase activity and energy-rich phosphate levels

  3. [Study of the transcriptional and transpositional activities of the Tirant retrotransposon in Drosophila melanogaster strains mutant for the flamenco locus].

    Science.gov (United States)

    Nefedova, L N; Urusov, F A; Romanova, N I; Shmel'kova, A O; Kim, A I

    2012-11-01

    Transpositions of the gypsy retrotransposon in the Drosophila melanogaster genome are controlled by the flamenco locus, which is represented as an accumulation of defective copies of transposable elements. In the present work, genetic control by the flamenco locus of the transcriptional and transpositional activities of the Tirant retrotransposon from the gypsy group was studied. Tissue-specific expression of Tirant was detected in the tissues of ovaries in a strain mutant for the flamenco locus. Tirant was found to be transpositionally active in isogenic D. melanogaster strains mutant for the flamenco locus. The sites of two new insertions have been localized by the method of subtractive hybridization. It has been concluded from the results obtained that the flamenco locus is involved in the genetic control of Tirant transpositions.

  4. Multi-omics approach to study global changes in a triclosan-resistant mutant strain of Acinetobacter baumannii ATCC 17978.

    Science.gov (United States)

    Fernando, Dinesh M; Chong, Patrick; Singh, Manu; Spicer, Victor; Unger, Mark; Loewen, Peter C; Westmacott, Garrett; Kumar, Ayush

    2017-01-01

    Acinetobacter baumannii AB042, a triclosan-resistant mutant strain, was examined for modulated gene expression using whole-genome sequencing, transcriptomics and proteomics in order to understand the mechanism of triclosan resistance as well as its impact on A. baumannii. Data revealed modulated expression of the fatty acid metabolism pathway, co-factors known to play a role in the synthesis of fatty acids, as well as several transcriptional regulators. The membrane composition of the mutant revealed a decrease in C18 with a corresponding increase in C16 fatty acids compared with the parent strain A. baumannii ATCC 17978. These data indicate that A. baumannii responds to triclosan by altering the expression of genes involved in fatty acid metabolism, antibiotic resistance and amino acid metabolism.

  5. Transcriptional changes in the nuc-2A mutant strain of Neurospora crassa cultivated under conditions of phosphate shortage.

    Science.gov (United States)

    Gras, Diana E; Silveira, Henrique C S; Peres, Nalu T A; Sanches, Pablo R; Martinez-Rossi, Nilce M; Rossi, Antonio

    2009-01-01

    The molecular mechanism that controls the response to phosphate shortage in Neurospora crassa involves four regulatory genes -nuc-2, preg, pgov, and nuc-1. Phosphate shortage is sensed by the nuc-2 gene, the product of which inhibits the functioning of the PREG-PGOV complex. This allows the translocation of the transcriptional factor NUC-1 into the nucleus, which activates the transcription of phosphate-repressible phosphatases. The nuc-2A mutant strain of N. crassa carries a loss-of-function mutation in the nuc-2 gene, which encodes an ankyrin-like repeat protein. In this study, we identified transcripts that are downregulated in the nuc-2A mutant strain. Functional grouping of these expressed sequence tags allowed the identification of genes that play essential roles in different cellular processes such as transport, transcriptional regulation, signal transduction, metabolism, protein synthesis, protein fate, and development. These results reveal novel aspects of the phosphorus-sensing network in N. crassa.

  6. Characterization of oxidative phosphorylation enzymes in Euglena gracilis and its white mutant strain W(gm)ZOflL.

    Science.gov (United States)

    Krnáčová, Katarína; Rýdlová, Ivana; Vinarčíková, Michaela; Krajčovič, Juraj; Vesteg, Matej; Horváth, Anton

    2015-03-12

    The enzymes involved in Euglena oxidative phosphorylation (OXPHOS) were characterized in this study. We have demonstrated that Euglena gracilis strain Z and its stable bleached non-photosynthetic mutant strain WgmZOflL both possess fully functional OXPHOS apparatus as well as pathways requiring terminal alternative oxidase(s) and alternative mitochondrial NADH-dehydrogenase(s). Light (or dark) and plastid (non)functionality seem to have little effect on oxygen consumption, the activities of the enzymes involved in OXPHOS and the action of respiration inhibitors in Euglena. This study also demonstrates biochemical properties of complex III (cytochrome c reductase) in Euglena.

  7. Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

    DEFF Research Database (Denmark)

    Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek

    2015-01-01

    growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant....... For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated...

  8. Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis.

    Science.gov (United States)

    Cantrell, Sally A; Leavell, Michael D; Marjanovic, Olivera; Iavarone, Anthony T; Leary, Julie A; Riley, Lee W

    2013-10-01

    The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen's persistence.

  9. Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

    Science.gov (United States)

    Ye, Xudong; Chen, Yurong; Wan, Yuechun; Hong, Yun-Jeong; Ruebelt, Martin C; Gilbertson, Larry A

    2016-03-01

    KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells.

  10. [Analysis of the structure and expression of the DIP1 gene in Drosophila melanogaster strains mutant for the flamenco gene].

    Science.gov (United States)

    Nefedova, L N; Potanova, M V; Romanova, N I; Kim, A I

    2009-02-01

    DIP1 gene transcription was analyzed with the use of RT-PCR in three Drosophila melanogaster strains with the flamenco- phenotype (flam(SS), flam(MS), and flam(Ore)) and in one flamenco+ strain at the stages of embryos (0-24 h), third-instar larvae, and adult flies. The mutant strains flam(SS) and flam(Ore) lack an active copy of transposon gypsy. Theflam(MS) strain was obtained by introducing an active copy of gypsy in flies of theflam(SS) strain and is characterized by a high rate of gypsy transpositions. The experiments showed that at least five forms of DIP1 gene transcripts are produced. The form of cDNA corresponding to CDS DIP1-d was discovered only in embryos. It was found that DIP1 gene transcription depends on the age of flies: at the larval stage the level of transcription is significantly reduced. However, no reduction of gene transcription is observed in theflam(Ore) strain. These results suggest that the flamenco- phenotype may be associated with an alteration of DIP1 gene transcription, as in differentflamenco- strains the DIP1 gene expression is changed differently.

  11. A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 29133

    Energy Technology Data Exchange (ETDEWEB)

    Lindberg, P.; Lindblad, P. [Uppsala Univ. (Sweden). Dept. of Physiological Botany; Schuetz, K.; Happe, T. [Universitaet Bonn (Germany). Botanisches Inst.

    2002-12-01

    The hupL gene, encoding the uptake hydrogenase large subunit, in Nostoc sp. strain ATCC 29133, a strain lacking a bidirectional hydrogenase, was inactivated by insertional mutagenesis. Recombinant strains were isolated and analysed, and one hupL{sup -} strain, NHM5, was selected for further study. Cultures of NHM5 were grown under nitrogen-fixing conditions and H{sub 2} evolution under air was observed using an H{sub 2} electrode. (Author)

  12. Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits.

    Science.gov (United States)

    Senior, A E; Langman, L; Cox, G B; Gibson, F

    1983-02-15

    To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.

  13. In Vitro and In Vivo Characterization of a Bordetella bronchiseptica Mutant Strain with a Deep Rough Lipopolysaccharide Structure

    Science.gov (United States)

    Sisti, Federico; Fernández, Julieta; Rodríguez, María Eugenia; Lagares, Antonio; Guiso, Nicole; Hozbor, Daniela Flavia

    2002-01-01

    Bordetella bronchiseptica is closely related to Bordetella pertussis, which produces respiratory disease primarily in mammals other than humans. However, its importance as a human pathogen is being increasingly recognized. Although a large amount of research on Bordetella has been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) during B. bronchiseptica infection is less understood. To get a better insight into this matter, we constructed and characterized the behavior of an LPS mutant with the deepest possible rough phenotype. We generated the defective mutant B. bronchiseptica LP39 on the waaC gene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although in B. bronchiseptica LP39 the production of the principal virulence determinants adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin persisted, the quantity of the two latter factors was diminished, with the levels of pertactin being the most greatly affected. Furthermore, the LPS of B. bronchiseptica LP39 did not react with sera obtained from mice that had been infected with the parental strain, indicating that this defective LPS is immunologically different from the wild-type LPS. In vivo experiments demonstrated that the ability to colonize the respiratory tract is reduced in the mutant, being effectively cleared from lungs within 5 days, whereas the parental strain survived at least for 30 days. In vitro experiments have demonstrated that, although B. bronchiseptica LP39 was impaired for adhesion to human epithelial cells, it is still able to survive within the host cells as efficiently as the parental strain. These results seem to indicate that the deep rough form of B. bronchiseptica LPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from human B. bronchiseptica chronic

  14. Functionality of cryopreserved juvenile ovaries from mutant mice in different genetic background strains after allotransplantation

    NARCIS (Netherlands)

    Huang, K.Y.; Groot, de S.A.; Woelders, H.; Horst, van der G.T.J.; Themmen, A.P.N.; Colenbrander, B.; Fentener van Vlissingen, J.M.

    2010-01-01

    The rapid expansion of mutant mouse colonies for biomedical research has resulted in lack of space at laboratory animal facilities and increasing risks of losing precious lines. These challenges require cheap and effective methods in addition to freezing embryos and sperm to archive the expanding mu

  15. Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

    NARCIS (Netherlands)

    Baumgarten, Thomas; Schlegel, Susan; Wagner, Samuel; Löw, Mirjam; Eriksson, Jonas; Bonde, Ida; Herrgård, Markus J; Heipieper, Hermann J; Nørholm, Morten H H; Slotboom, Dirk Jan; de Gier, Jan-Willem

    2017-01-01

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from

  16. [Separation of the biosynthesis products of a mutant strain of Actinomyces chrysomallus var. carotenoides and the identification of actinomycin antibiotics].

    Science.gov (United States)

    Sverdlova, A N; Nefelova, M V; Silaev, A B

    1979-01-01

    An orange antibiotically active substance isolated from the mycelium of a mutant strain of Actinomyces chrysomallus var. carotenoides was identified as a mixture of actinomycins according to its light absorption spectra, circular dichroism spectra, IR spectra and chromatographic comparison with the standard samples. A scheme for successive extraction of the biologically active substances from the mycelium resulting in isolation of a fraction enriched with antibiotic substances and a fraction enriched with pigments is presented. A method for separation and purification of 3 groups of biologically active substances from the mycelium enriched extract was developed.

  17. Restoration of growth by manganese in a mutant strain of Escherichia coli lacking most known iron and manganese uptake systems

    DEFF Research Database (Denmark)

    Taudte, Nadine; German, Nadezhda; Zhu, Yong-Guan

    2016-01-01

    The interplay of manganese and iron homeostasis and oxidative stress in Escherichia coli can give important insights into survival of bacteria in the phagosome and under differing iron or manganese bioavailabilities. Here, we characterized a mutant strain devoid of all know iron/manganese......-uptake systems relevant for growth in defined medium. Based on these results an exit strategy enabling the cell to cope with iron depletion and use of manganese as an alternative for iron could be shown. Such a strategy would also explain why E. coli harbors some iron- or manganese-dependent iso......-enzymes such as superoxide dismutases or ribonucleotide reductases. The benefits for gaining a means for survival would be bought with the cost of less efficient metabolism as indicated in our experiments by lower cell densities with manganese than with iron. In addition, this strain was extremely sensitive to the metalloid...

  18. Industrial robustness: understanding the mechanism of tolerance for the Populus hydrolysate-tolerant mutant strain of Clostridium thermocellum.

    Directory of Open Access Journals (Sweden)

    Jessica L Linville

    Full Text Available BACKGROUND: An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v. CONCLUSION/SIGNIFICANCE: The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the

  19. Optimisation of nutritional requirements for dopamine synthesis by calcium alginate-entrapped mutant strain of Aspergillus oryzae EMS-6.

    Science.gov (United States)

    Ali, Sikander; Nawaz, Wajeeha

    2017-02-01

    The optimisation of nutritional requirements for dopamine (DA) synthesis by calcium alginate-entrapped mutant variant of Aspergillus oryzae EMS-6 using submerged fermentation technique was investigated. A total of 13 strains were isolated from soil. Isolate I-2 was selected as a better producer of DA and improved by exposing with ethyl methylsulphonate (EMS). EMS-6 was selected as it exhibited 43 μg/mL DA activity. The mutant variable was further treated with low levels of l-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of mutant variant were entrapped in calcium alginate beads for stable product formation. EMS-6 gave maximum DA activity (124 μg/mL) when supplemented with 0.1% peptone and 0.2% sucrose, under optimised parameters viz. pH 3, temperature of 55 °C and incubation time of 70 min. The study involves the high profile of DA activity and is needed, as DA is capable to control numerous neurogenic disorders.

  20. [A recessive mutant causing testicular/ovarian teratoma in rats (Tera strain)].

    Science.gov (United States)

    Miwa, M; Kojima, M; Ohtani, T; Tsuji, K

    1987-04-01

    A hereditary testicular/ovarian teratoma strain (Tera) of rats was developed from the Csk: Wistar-Imamichi strain. As the teratoma consisted of tridermic tissues such as bone, epithelium and neural tissue, it was diagnosed as triphyllomatous teratoma. The frequency of the teratoma was about 25% in either sex, with no sexual difference. Accordingly, the heredity of the teratoma appeared to be an autosomal single recessive trait (symbol, tera).

  1. Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

    Directory of Open Access Journals (Sweden)

    Claudia Ehlers

    2011-01-01

    Full Text Available A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP. The nonreplicating allelic exchange vector (pRS345 carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazei Δ154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation.

  2. Study on the molecular basis of glucoamylase overproduc-tion of a mutant strain Aspergillus niger T21

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The molecular basis for increasing of the glucoamylase (GLA) production of an Aspergillus niger mutant T21 was investigated . Northern blot analysis showed that the amount of glaA specific mRNA of A . niger T21 was about 20 times higher than that of its start strain A . niger AS 3.795. The two glaA promoter fusions (PglaA)-uidAs were respectively introduced into A . niger. Analysis of GUS activity of the transformants revealed that the PglaA activity of the strain T21 is about 3 times stronger than that of the strain AS 3.795. It is considered to be one of the reasons for the increase of glaA transcriptional level in the strain T21. However, comparing with the 20 times increase in the amount of glaA mRNA the alteration of trans regulation should be the most important reason for that. The results of deletion analysis of 5′-cis region of A . niger T21 glaA gene indicated that the region from - 408 to - 513 bp upstream of ATG is responsible for the high level expression of glaA.

  3. KEY COMPARISON: Final report on CCQM-K62: Nutrients in infant/adult formula—Vitamins

    Science.gov (United States)

    Sharpless, Katherine E.; Rimmer, Catherine A.; Phinney, Karen W.; Nelson, Bryant C.; Duewer, David L.; Wise, Stephen A.; Kim, Byungjoo; Liu, Jun; Huang, Ting; Zhang, Wei

    2010-01-01

    Key comparison CCQM-K62 was designed to enable demonstration of the equivalence in capabilities for measurement of vitamins in a food matrix. A milk-based fortified human infant/adult formula was selected as the matrix based upon material availability and relevance. Because vitamins were added to the CCQM-K62 study material in a single form and at levels significantly higher than those that would be naturally occurring in the milk base, the ability of a laboratory to measure the study vitamins is only indicative of a laboratory's ability to measure vitamins in fortified foods. Target analytes were selected for study because of the ready availability of suitable standard materials and the range of their chemical properties: folic acid (vitamin B9) is a single water-soluble molecular entity that typically occurs at low levels and can be unstable, niacin (vitamin B3) is a single stable molecular entity and is typically present at higher concentrations than the other water-soluble vitamins, vitamin A has multiple molecular forms (including retinol and retinyl palmitate), is fat-soluble and typically occurs at relatively high levels. Results for participants measuring only folic acid or niacin are only indicative of their ability to make that measurement; results for participants measuring both folic acid and niacin are indicative of a laboratory's ability to measure folic acid, thiamine, niacin, and riboflavin in fortified foods but not vitamin C or other water-soluble vitamins. The ability to measure vitamin A (reported as retinol equivalents) in this material is also indicative of the participant's ability to measure vitamin E (as alpha-tocopherol and alpha-tocopheryl acetate) but is not indicative of the ability to measure vitamins D and K, which typically occur at much lower concentrations. The relative degrees of equivalence of the reported measurements for all three analytes in CCQM-K62 were within 10%; however, since only two results were submitted for niacin

  4. Fluoroquinolone and Quinazolinedione Activities against Wild-Type and Gyrase Mutant Strains of Mycobacterium smegmatis▿

    Science.gov (United States)

    Malik, Muhammad; Marks, Kevin R.; Mustaev, Arkady; Zhao, Xilin; Chavda, Kalyan; Kerns, Robert J.; Drlica, Karl

    2011-01-01

    Quinazolinediones (diones) are fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV. To assess activity against mycobacteria, C-8-methoxy dione derivatives were compared with cognate fluoroquinolones by using cultured Mycobacterium smegmatis. Diones exhibited higher MIC values than fluoroquinolones; however, MICs for fluoroquinolone-resistant gyrA mutants, normalized to the MIC for wild-type cells, were lower. Addition of a 3-amino group to the 2,4-dione core increased relative activity against mutants, while alteration of the 8-methoxy group to a methyl or of the 2,4-dione core to a 1,3-dione core lowered activity against mutants. A GyrA G89C bacterial variant was strikingly susceptible to most of the diones tested; in contrast, low susceptibility to fluoroquinolones was observed. Many of the bacteriostatic differences between diones and fluoroquinolones were explained by interactions at the N terminus of GyrA helix IV revealed by recently published X-ray structures of drug-topoisomerase-DNA complexes. When lethal activity was normalized to the MIC in order to minimize the effects of drug uptake, efflux, and ternary complex formation, a 3-amino-2,4-dione exhibited killing activity comparable to that of a cognate fluoroquinolone. Surprisingly, the lethal activity of the dione was inhibited less by chloramphenicol than that of the cognate fluoroquinolone. This observation adds the 2,4-dione structural motif to the list of structural features known to impart lethality to fluoroquinolone-like compounds in the absence of protein synthesis, a phenomenon that is not explained by X-ray structures of drug-enzyme-DNA complexes. PMID:21383100

  5. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants

    DEFF Research Database (Denmark)

    Baum, Cathrin; Haslinger-Löffler, Bettina; Westh, Henrik;

    2009-01-01

    Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing......) provides a rapid and reliable method for epidemiological studies. Rarely, non-spa-typeable S. aureus strains are encountered. The reason for this is not known. In this study, we characterized eight non-spa-typeable bacteremia isolates. Sequencing of the entire spa locus was successful for five strains...

  6. Funduscopy in adult zebrafish and its application to isolate mutant strains with ocular defects.

    Directory of Open Access Journals (Sweden)

    Markus Tschopp

    Full Text Available Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals.A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out.Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas.In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible.

  7. Global transcriptional analysis of spontaneous sakacin P-resistant mutant strains of Listeria monocytogenes during growth on different sugars.

    Directory of Open Access Journals (Sweden)

    Girum Tadesse Tessema

    Full Text Available Subclass IIa bacteriocins have strong antilisterial activity and can control the growth of Listeria monocytogenes in food. However, L. monocytogenes may develop resistance towards such bacteriocins. In this follow-up study, the transcriptomes of a high level (L502-1 and a low level (L502-6 spontaneous sakacin P-resistant mutant strain of L. monocytogenes were compared to the wild-type (L502. The growth of the resistant strains was reduced on mannose but not affected on cellobiose and the transcriptomics was performed during growth on these sugars. The mannose phosphotransferase system (PTS encoded by the mptACD operon (mpt is known for transporting mannose and also act as a receptor to class IIa bacteriocins. The mpt was repressed in L502-1 and this is in accordance with abolition of the bacteriocin receptor with resistance to class IIa bacteriocins. In contrast, the mpt was induced in L502-6. Despite the induction of the mpt, L502-6 showed 1,000 times more resistance phenotype and reduced growth on mannose suggesting the mannose-PTS may not be functional in L502-6. The microarray data suggests the presence of other transcriptional responses that may be linked to the sakacin P resistance phenotype particularly in L502-6. Most of commonly regulated genes encode proteins involved in transport and energy metabolism. The resistant strains displayed shift in general carbon catabolite control possibly mediated by the mpt. Our data suggest that the resistant strains may have a reduced virulence potential. Growth sugar- and mutant-specific responses were also revealed. The two resistant strains also displayed difference in stability of the sakacin P resistance phenotype, growth in the presence of both the lytic bacteriophage P100 and activated charcoal. Taken together, the present study showed that a single time exposure to the class IIa bacteriocin sakacin P may elicit contrasting phenotypic and transcriptome responses in L. monocytogenes possibly

  8. Modelling Hepatitis B Virus Antiviral Therapy and Drug Resistant Mutant Strains

    Science.gov (United States)

    Bernal, Julie; Dix, Trevor; Allison, Lloyd; Bartholomeusz, Angeline; Yuen, Lilly

    Despite the existence of vaccines, the Hepatitis B virus (HBV) is still a serious global health concern. HBV targets liver cells. It has an unusual replication process involving an RNA pre-genome that the reverse transcriptase domain of the viral polymerase protein translates into viral DNA. The reverse transcription process is error prone and together with the high replication rates of the virus, allows the virus to exist as a heterogeneous population of mutants, known as a quasispecies, that can adapt and become resistant to antiviral therapy. This study presents an individual-based model of HBV inside an artificial liver, and associated blood serum, undergoing antiviral therapy. This model aims to provide insights into the evolution of the HBV quasispecies and the individual contribution of HBV mutations in the outcome of therapy.

  9. Biogenesis and Ultrastructure of Carboxysomes from Wild Type and Mutants of Synechococcus sp. Strain PCC 7942.

    Science.gov (United States)

    Orus, M. I.; Rodriguez, M. L.; Martinez, F.; Marco, E.

    1995-04-01

    Immature inclusions representing three progressive steps of carboxysome biogenesis have been identified in Synechococcus during the period of adaptation to low-CO2 conditions: (a) ring-shaped structures, (b) electron-translucent inclusions with the shape of a carboxysome and the internal orderly arrangement of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) molecules, and (c) carboxysomes with an internal electron-translucent area, which seem to be the penultimate stage of carboxysome maturation. The ability to build up normal carboxysomes is impaired in three (M3, EK6, and D4) of four high-carbon-requiring mutants studied in this work. M3 and EK6 exhibit abundant immature electron-translucent carboxysomes but no mature ones. This finding supports the contention that an open reading frame located 7.5 kb upstream of the gene encoding the large subunit of Rubisco (altered in M3) is involved in the carboxysome composition and confirms the structural role of the small subunit of Rubisco (slightly modified in EK6) in the assembly of these structures. D4 shows few typical carboxysomes and frequent immature types, its genetic lesion affecting the apparently unrelated gene encoding a subunit of phosphoribosyl aminoamidazole carboxylase of the purine biosynthesis pathway. Revertants EK20 (EK6) and RK13 (D4) have normal carboxysomes, which means that the restoration of the ability to grow under low CO2 coincides with the proper assembling of these structures. N5, a transport mutant due to the alteration of the gene encoding subunit 2 of NADH dehydrogenase, shows an increase in the number and size of carboxysomes and frequent bar-shaped ones.

  10. Optimization of Process Parameters for Conversion of 3-cyanpyridine to Nicotinamide Using Resting Cells of Mutant 4D Strain of Rhodococcus rhodochrous PA-34

    Directory of Open Access Journals (Sweden)

    Amit Seth

    2011-11-01

    Full Text Available Mutant of Rhodococcus rhodochrous PA-34, named as 'mutant 4D' has been reported for the hyperconversion of 3-cyanopyridine to nicotinamide. This mutant 4D generated through chemical mutagenesis has much more hydration potential than its wild strain. The reaction conditions for prolonged reaction and process parameters for the conversion of 3-cyanopyridine to nicotinamide were optimized. Under the optimized reaction conditions the mutant 4D is stable at higher temperature (55°C, high ionic strength (0.3 M and at acidic pH conditions (5.5 and exhibited 8.0, 7.9 and 7.0 U/mg dcw NHase activity, respectively. In a batch reaction of (One litre, 7M 3-cyanopyridine was completely converted to nicotinamide in 3h at 55°C using 7g resting cells (dry cell mass of mutant 4D of R. rhodochrous PA-34.

  11. Protoplast fusion technology for improved production of coenzyme Q10 using Paracoccus denitrificans ATCC 19367 mutant strains

    Directory of Open Access Journals (Sweden)

    Pradipta Tokdar

    2014-01-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Induced mutants generated from Paracoccus denitrificans ATCC 19367 having antibiotic resistant markers, were used as parent strains to carry out protoplast fusion. The generated fusants were screened using standardized protocol for CoQ10 production. Among the generated fusants, one fusant namely PF-P1 showed 1.73 folds enhancements in specific CoQ10 content than wild type strain. Fusant PF-P1 was characterized by biochemical and molecular approaches where it showed differences than wild type strain. The fusant was further identified by 16S rRNA gene sequence analysis that showed eight nucleotide base pair mutation on conserved region and 99% homology with Paracoccus denitrificans strains. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  12. DNA polymorphism and total protein in mutants of Metarhizium anisopliae var. Anisopliae (Metsch.) Sorokin strain E9 Polimorfismo de DNA e proteína total em mutantes da linhagem E9 de Metarhizium anisopliae var. anisopliae (Metsch.) Sorokin

    OpenAIRE

    Laurineide Lopes de Carvalho Freire; Ana Bolena Lima da Costa; Larissa Brandão Góes; Neiva Tinti de Oliveira

    2001-01-01

    Five mutants (MaE10, MaE27, MaE24, MaE41 e MaE49) of Metarhizium anisopliae wild strain E9 were analysed for DNA profile through the RAPD technique and for changes in total protein content by spectrophotometry, polyacrylamide gel electrophoresis and densitometry. The pattern of RAPD markers showed genetic polymorphism among the strains: out of twenty primers seven were selected, producing 113 bands. Forty seven bands were present in all strains (41.6% of monomorphic bands) and 66 showed polym...

  13. Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits.

    Science.gov (United States)

    Lill, H; Burkovski, A; Altendorf, K; Junge, W; Engelbrecht, S

    1993-10-04

    The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.

  14. Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.

    Science.gov (United States)

    Lin, Xiaorong; Chacko, Nadia; Wang, Linqi; Pavuluri, Yashwant

    2015-04-01

    Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (∼10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.

  15. Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains

    Science.gov (United States)

    Li, Ming-Wen; Kinchen, Kristy L; Vallelunga, Jadine M; Young, Diana L; Wright, Kaleb D K; Gorano, Lisa N; Wasson, Katherine; Lloyd, K C Kent

    2013-01-01

    In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen–thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm–zona penetration defects. PMID:23315689

  16. Halorhodopsin and photosensory behaviour in Halobacterium halobium mutant strain L-33

    Energy Technology Data Exchange (ETDEWEB)

    Traulich, B.; Wagner, G. (Botanisches Institut l der Justus-Liebig-Universitat, Giessen (Germany, F.R.)); Hildebrand, E.; Schimz, A. (Kernforschungsanlage Juelich G.m.b.H. (Germany, F.R.). Inst. fuer Neurobiologie); Lanyi, J.K. (California Univ., Irvine (USA))

    1983-05-01

    Halobacterium halobium, strain L-33, which is deficient in bacteriorhodopsin (BR) but synthesizes increased amounts of halorhodopsin (HR), responds to changes in fluence rate with visible light or with UV light. The observations support an earlier report that BR is not essential for photosensing in H. halobium. In the UV-range, changes in light intensity elicit the maximal response at lambda = 370 nm. In the visible range, changes in light intensity show the maximal response at lambda = 565 nm and a secondary peak at lambda = 590 nm. The latter corresponds to the absorption maximum of HR (lambdasub(max) = 588 nm). This light-energy converting retinal pigment of H. halobium thus appears to contribute to photosensory behavior.

  17. 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.

  18. Monitoring the effect of pyrene on the germination and radial growth of the wild and mutant strains of Rhizopus arrhizus UCP402

    Directory of Open Access Journals (Sweden)

    Ricardo Kenji Shiosaki

    2008-06-01

    Full Text Available The physiological mutant of Rhizopus arrhizus was obtained in the pyrene resistance gradient test. Comparative studies were carried out about the behavior of the germination process and the radial growth of the mutant and wild strains of R. arrhizus UCP 402. Sabouraud Sucrose and Yeast Malt Broth cultures containing pyrene (10 mg/L induced the germination process of the sporangiospores of the wild and mutant strains of R. arrhizus. The radial growth of the strains was inversely proportional to the pyrene concentration in the culture medium. The results showed an adaptation of R. arrhizus UCP 402x (mutant in the pyrene (50mg/L and suggested a higher ability of application in the removal of pyrene from the contaminated areas.O mutante fisiológico de Rhizopus arrhizus foi obtido pelo teste do gradiente de resistência ao pireno. Estudos comparativos conduzidos sobre o comportamento do processo de germinação e o crescimento radial foram realizados entre as amostras selvagem e mutante de R. arrhizus UCP 402. Os meios Sabouraud Sacarose e Caldo de Levedura e Malte contendo pireno (10 mg/L induziram ao processo de germinação de esporangiosporos das amostras selvagem e mutante de R. arrhizus. O crescimento radial das amostras foi inversamente proporcional à concentração de pireno no meio de cultura. Os resultados demonstraram uma excelente adaptação da amostra mutante de R. arrhizus UCP 402x na concentração de pireno (50 mg/L, sugerindo uma alta habilidade e possibilidade de aplicação na remoção de pireno em áreas contaminadas.

  19. Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development.

    Science.gov (United States)

    Mott, Tiffany M; Vijayakumar, Sudhamathi; Sbrana, Elena; Endsley, Janice J; Torres, Alfredo G

    2015-01-01

    In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis. Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 10(4) CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.

  20. Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development.

    Directory of Open Access Journals (Sweden)

    Tiffany M Mott

    Full Text Available In this study, a Burkholderia mallei tonB mutant (TMM001 deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis.Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 10(4 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001.Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.

  1. An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

    Science.gov (United States)

    Mitobe, Jiro; Sinha, Ritam; Mitra, Soma; Nag, Dhrubajyoti; Saito, Noriko; Shimuta, Ken; Koizumi, Nobuo; Koley, Hemanta

    2017-07-01

    Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

  2. Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid : Growth profiles and catalase activities in relation to microbody proliferation

    NARCIS (Netherlands)

    Klei, Ida J. van der; Rytka, Joanna; Kunau, Wolf H.; Veenhuis, Marten

    1990-01-01

    The parental strain (A+T+) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A+T-), catalase A (A-T+) or both catalases (A-T-), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two olei

  3. Inactivation of ccmO in Synechococcus sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO(2).

    Science.gov (United States)

    Marco, E; Martinez, I; Ronen-Tarazi, M; Orus, M I; Kaplan, A

    1994-03-01

    Inactivation of ccmO in Synechococcus sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO(2) for growth.

  4. Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus

    Science.gov (United States)

    Gokce, Abdulmecit; Cakar, Zeynep Petek; Yucel, Meral; Ozcan, Orhan; Sencan, Sevde; Sertdemir, Ibrahim; Erguner, Bekir; Yuceturk, Betul; Sarac, Aydan; Yuksel, Bayram

    2016-01-01

    The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus. PMID:27284151

  5. A mutant lacking the glutamine synthetase gene (glnA) is impaired in the regulation of the nitrate assimilation system in the cyanobacterium Synechocystis sp. strain PCC 6803.

    Science.gov (United States)

    Reyes, J C; Florencio, F J

    1994-12-01

    The existence in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 of two genes (glnA and glnN) coding for glutamine synthetase (GS) has been recently reported (J.C. Reyes and F.J. Florencio, J. Bacteriol. 176:1260-1267, 1994). In the current work, the regulation of the nitrate assimilation system was studied with a glnA-disrupted Synechocystis mutant (strain SJCR3) in which the only GS activity is that corresponding to the glnN product. This mutant was unable to grow in ammonium-containing medium because of its very low levels of GS activity. In the SJCR3 strain, nitrate and nitrite reductases were not repressed by ammonium, and short-term ammonium-promoted inhibition of nitrate uptake was impaired. In Synechocystis sp. strain PCC 6803, nitrate seems to act as a true inducer of its assimilation system, in a way similar to that proposed for the dinitrogen-fixing cyanobacteria. A spontaneous derivative strain from SJCR3 (SJCR3.1), was able to grow in ammonium-containing medium and exhibited a fourfold-higher level of GS activity than but the same amount of glnN transcript as its parental strain (SJCR3). Taken together, these finding suggest that SJCR3.1 is a mutant affected in the posttranscriptional regulation of the GS encoded by glnN. This strain recovered regulation by ammonium of nitrate assimilation. SJCR3 cells were completely depleted of intracellular glutamine shortly after addition of ammonium to cells growing with nitrate, while SJCR3.1 cells maintained glutamine levels similar to that reached in the wild-type Synechocystis sp. strain PCC 6803. Our results indicate that metabolic signals that control the nitrate assimilation system in Synechocystis sp. strain PCC 6803 require ammonium metabolism through GS.

  6. Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Kim, Kiju; Park, Bo-Kyoung; Hahn, Tae-Wook

    2015-11-01

    Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

  7. Influence of pH on butyrate uptake and solvent fermentation by a mutant strain of Clostridium acetobutylicum

    Energy Technology Data Exchange (ETDEWEB)

    Soni, B.K. [Institute of Gas Technology, 1700 S. Mount Prospect Road, Des Plaines, IL 60018 (United States); Jain, M.K. [MBI International, P.O. Box 27609, 3900 Collins Road, Lansing, MI 48909 (United States)

    1997-11-01

    The effect of pH (between 4.4 and 6.6) on butyrate uptake by the mutant strain of Clostridium acetobutylicum was studied using the fermentation broth from fermentor-2 (solventogenic stage) of a two-fermentor continuous system. Low pH (< 4.6) adversely affected the overall metabolic activity as observed by low solvent production and carbohydrate consumption. Uptake of 4.0 {+-} 0.5 g l{sup -1} butyrate, under batch incubation at 30 C, was not inhibited at pH > 5.2, however, at pH < 5.2, a marked inhibition in butyrate uptake was noticed. A higher pH (e.g. pH 5.4) was required for the uptake of elevated concentration of externally added butyrate at 8.5 {+-} 1.0 g l{sup -1}. Batch incubation at relatively higher temperatures (35 and 37 C) indicated a similar trend i.e., a pH of >5.5 was required for uptake of >8 g l{sup -1} butyrate. Optimization studies for butyrate uptake by C. acetobutylicum suggested a direct correlation between minimum pH and butyrate concentration or temperature. The role of undissociated butyric acid appears to be critical in regulation of butyrate uptake. (orig.) With 5 figs., 6 tabs., 23 refs.

  8. Increase Hydrogen Production by Hydrogen-producing Mutant UV-d48 in Comparison with Wild Parent Strain Etanoligenens sp ZGX4

    Institute of Scientific and Technical Information of China (English)

    ZHENG Guo-xiang; REN Nan-qi; LIN Hai-long; LI Yong-feng

    2006-01-01

    Hydrogen production by hydrogen-producing mutant strain UV-d48 was one typical ethanol-type fermentative H2-producing mutant, which the main metabolic end production was ethanol and acetic acid. Compare to the wild type strain, the activity of hydrogenase of mutant UV-d48 was stronger in course of growth and fermentation. It can evolve hydrogen at stronger ability and a greater rate than that of its parent wild type, namely ZGX4. Resting cells of UVd48 and Ethanoligenens sp ZGX4 were set up in a batch mode in phosphate buffered saline (PBS) to decouple growth from hydrogen production at the expense of glucose of varying composition. Mutant UV-d48 evolved more hydrogen than ZGX4at glucose concentrations ranging from 2 mmol/L to 200 mmol/L. The difference in the amount of H2 evolved by both strains decreased as the concentration of glucose increased. The maximal H2 yield and H2-producing rate by strain UV-d48 was 3091.1 mL/L and 54 mL/(hODunitL) respectively, at a glucose concentration of 60 mmol/L. With strain ZGX4, the maximal H2 yield and H2-producing rate was 2 180.2 mL/L and 33 mL/(hODunitL) under these conditions, respectively.Experiments using wastewater with certain content molasses yielded similar results. In each case, strain UV-d48 evolved hydrogen at a faster rate than the wild type, showing a possible potential for commercial hydrogen production.

  9. Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ− mutant vaccine strain used for DIVA-strategies

    Science.gov (United States)

    Vilei, Edy M.; Frey, Joachim

    2010-01-01

    Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ− mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ− mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ− mutant vaccine strain. PMID:20381545

  10. DNA polymorphism and total protein in mutants of Metarhizium anisopliae var. Anisopliae (Metsch. Sorokin strain E9 Polimorfismo de DNA e proteína total em mutantes da linhagem E9 de Metarhizium anisopliae var. anisopliae (Metsch. Sorokin

    Directory of Open Access Journals (Sweden)

    Laurineide Lopes de Carvalho Freire

    2001-06-01

    Full Text Available Five mutants (MaE10, MaE27, MaE24, MaE41 e MaE49 of Metarhizium anisopliae wild strain E9 were analysed for DNA profile through the RAPD technique and for changes in total protein content by spectrophotometry, polyacrylamide gel electrophoresis and densitometry. The pattern of RAPD markers showed genetic polymorphism among the strains: out of twenty primers seven were selected, producing 113 bands. Forty seven bands were present in all strains (41.6% of monomorphic bands and 66 showed polymorphism (58.4%. The mean coefficient of similarity among all strains was 0.75 (75%. The total protein content varied, staining in the interval of 6.0-8.0 µg/µl. The electrophoresis analysis, through zymogram and protein fraction profiles by densitometry, allowed the observation of seven bands for the wild strain E9 and five bands for the mutants MaE10, MaE27, MaE34, MaE41 and MaE49, evidence of variations in µg% among protein fractions. The RAPD technique was very sensitive to detect genetic differences between the wild type and the mutants obtained through gamma radiation. The total protein analysis also showed changes in quantity and pattern of bands after electrophoresis in the mutants compared to the wild type.Foram analisados cinco mutantes MaE (MaE10, MaE27, MaE34, MaE41 e MaE49 da linhagem selvagem E9 de Metarhizium anisopliae var. anisopliae quanto ao perfil de DNA pela técnica de RAPD e também quanto ao conteúdo de proteína total por espectrometria e eletroforese em gel de poliacrilamida e densitometria. O padrão de marcadores de RAPD evidenciou polimorfismo nas amostras; dos 20 primers testados foram selecionados 7 que geraram 113 bandas. Deste total, 47 estavam presentes em todas as amostras (41.6% de bandas monomórficas e 66 mostraram polimorfismo (58.4%. O coeficiente médio de similaridade foi de 75%. O conteúdo de proteína total variou de 6 a 8 µg/µl. O zimograma e perfís das frações de proteínas obtidos por densitometria

  11. Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.

    Science.gov (United States)

    Vidal, O; Longin, R; Prigent-Combaret, C; Dorel, C; Hooreman, M; Lejeune, P

    1998-05-01

    Classical laboratory strains of Escherichia coli do not spontaneously colonize inert surfaces. However, when maintained in continuous culture for evolution studies or industrial processes, these strains usually generate adherent mutants which form a thick biofilm, visible with the naked eye, on the wall of the culture apparatus. Such a mutant was isolated to identify the genes and morphological structures involved in biofilm formation in the very well characterized E. coli K-12 context. This mutant acquired the ability to colonize hydrophilic (glass) and hydrophobic (polystyrene) surfaces and to form aggregation clumps. A single point mutation, resulting in the replacement of a leucine by an arginine residue at position 43 in the regulatory protein OmpR, was responsible for this phenotype. Observations by electron microscopy revealed the presence at the surfaces of the mutant bacteria of fibrillar structures looking like the particular fimbriae described by the Olsén group and designated curli (A. Olsén, A. Jonsson, and S. Normark, Nature 338:652-655, 1989). The production of curli (visualized by Congo red binding) and the expression of the csgA gene encoding curlin synthesis (monitored by coupling a reporter gene to its promoter) were significantly increased in the presence of the ompR allele described in this work. Transduction of knockout mutations in either csgA or ompR caused the loss of the adherence properties of several biofilm-forming E. coli strains, including all those which were isolated in this work from the wall of a continuous culture apparatus and two clinical strains isolated from patients with catheter-related infections. These results indicate that curli are morphological structures of major importance for inert surface colonization and biofilm formation and demonstrate that their synthesis is under the control of the EnvZ-OmpR two-component regulatory system.

  12. Growth phase effect on the dark anaerobic hydrogen production in the glucose tolerant mutant of unicellular cyanobacterium Synechocystis sp. strain PCC6803

    Directory of Open Access Journals (Sweden)

    Adipa Chongsuksantikul*

    2015-04-01

    Full Text Available This study has examined the effect of growth phase on hydrogenproduction from cells of a glucose tolerant mutant of Synechocystissp. strain PCC6803. The extracellular products including hydrogen,lactate and acetate from cyanobacteria cells in dark anaerobicnitrate-free solution, yielded different excretory profiles dependingon which growth phases were prepared from photosynthesis. Theamount of hydrogen generated cells prepared from stationary phasewas highest in HEPES buffer and nitrate-free solution, darkanaerobic condition.

  13. Comparative Evaluation of Agroindustrial Byproducts for the Production of Alkaline Protease by Wild and Mutant Strains of Bacillus subtilis in Submerged and Solid State Fermentation

    OpenAIRE

    Hamid Mukhtar; Ikramul Haq

    2013-01-01

    The present study describes the screening of different agroindustrial byproducts for enhanced production of alkaline protease by a wild and EMS induced mutant strain of Bacillus subtilis IH-72EMS8. During submerged fermentation, different agro-industrial byproducts were tested which include defatted seed meals of rape, guar, sunflower, gluten, cotton, soybean, and gram. In addition to these meals, rice bran, wheat bran, and wheat flour were also evaluated for protease production. Of all the b...

  14. Comparison of wild-type and UV-mutant beta-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications.

    Science.gov (United States)

    McCarthy, Tracey C; Lalor, Eoin; Hanniffy, Orla; Savage, Angela V; Tuohy, Maria G

    2005-04-01

    A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal beta-glucans. Activity against beta-(1, 3)(1, 4)-D: -glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less beta-glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley beta-glucan (13.0-16.9%), but were more active against crude beta-glucan from barley (16.0-24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash beta-glucan. Finally, TC2 and TC5 produce more efficient beta-glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications.

  15. A new osteopetrosis mutant mouse strain (ntl) with odontoma-like proliferations and lack of tooth roots.

    Science.gov (United States)

    Lu, Xincheng; Rios, Hector F; Jiang, Baichun; Xing, Lianping; Kadlcek, Renata; Greenfield, Edward M; Luo, Guangbin; Feng, Jian Q

    2009-12-01

    A new spontaneous mouse mutant (ntl) with autosomal-recessive osteopetrosis was characterized. These mice formed tartrate-resistant acid phosphate (TRAP)-positive osteoclasts but their osteoclasts had no ruffled border and did not resorb bone. These mice displayed no tooth eruption or tooth root formation. Adult mutant mice developed odontoma-like proliferations near the proximal ends of the incisors. Intraperitoneal injection of progenitor cells from the liver of 16.5 days postcoitum wild-type embryos into newborn mutants rescued the osteopetrosis phenotype, indicating that the defects were intrinsic to the osteoclasts. Our findings not only provide further support for a critical role of osteoclasts in tooth eruption and tooth root development, but also suggest that the perturbation of the homeostasis of the odontogenic precursors of the incisors is primarily responsible for the development of the odontoma-like proliferations in this osteopetrosis mutant. Genetic mapping has narrowed down the location of the mutant allele to a genetic interval of 3.2 cM on mouse chromosome 17.

  16. Technical Report on the Development of Mutant Paracoccus strain and Optimization of Medium Composition for the Mass Production of Astaxanthin

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jong Il; Lee, Ju Woon; Kim, Jae Hun; Song, Beom Seok

    2010-08-15

    Astaxanthin is used to role of provitamin A, and it is stronger antioxidant activity than vitamin E (100-500 times higher activity) and other carotenoids (10-fold). Furthermore, astaxanthin is also used as a nutraceutical and a medicinal ingredient against degenerative diseases such as cancer, heart disease, and skin related illness. The objective of this study was develop a carotenoid-hyperproducing mutant of Paracoccus N81106 using gamma irradiation and optimized medium composition. A mutant of Paracoccus having higher carotenoid content was isolated, and the production medium was optimized using response surface methodology. These results support that astaxanthin with strong antioxidant activity could be economically produced using the mutant and will be helpful for the related industry

  17. Computational studies on the resistance of penicillin-binding protein 2B (PBP2B) of wild-type and mutant strains of Streptococcus pneumoniae against β-lactam antibiotics.

    Science.gov (United States)

    Ramalingam, Jothi; Vennila, Jannet; Subbiah, Parthasarathy

    2013-09-01

    Mutations within transpeptidase domain of penicillin-binding protein 2B of the strains of Streptococcus pneumoniae leads to resistance against β-lactam antibiotics. To uncover the important residues responsible for sensitivity and resistance, the recently determined three dimensional structures of penicillin-binding protein 2B of both wild-type R6 (sensitive) and mutant 5204 (resistant) strains along with the predicted structures of other mutant strains G54, Hungary19A-6 and SP195 were considered for the interaction study with β-lactam antibiotics using induced-fit docking of Schrödinger. Associated binding energies of the complexes and their intermolecular interactions in the binding site clearly show that the wild-type R6 as sensitive, mutant strains 5204 and G54 as highly resistant, and the mutant strains Hungary19A-6 and SP195 as intermediate resistant. The study also reveals that the mutant strains Hungary19A-6 and SP195 exhibit intermediate resistant because of the existence of mutations till the intermediate 538th and 516th positions, respectively, and not till the end of the C-terminus. Furthermore, our investigations show that if the mutations are extended till the end of the C terminus, then the antibiotic resistance of induced-mutated strains increases from intermediate to high as in the strains 5204 and G54. The binding patterns obtained in the study are useful in designing potential inhibitors against multidrug resistant S. pneumoniae.

  18. Substrate specificity of glucose dehydrogenase and carbon source utilization pattern of pantoea dispersa strain P2 and its radiation induced mutants

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Keun; Murugesan, Senthilkumar [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-06-15

    Mineral phosphate solubilizing pantoea dispersa strain P2 produced 5.5 mM and 42.6 mM of gluconic acid on 24 h and 72 h incubation, respectively. Strain P2 exhibited glucose dehydrogenase (GDH) specific activity of 0.32 IU mg{sup -1} protein. We have studied the substrate specificity of GDH as well as carbon source utilization pattern of strain P2. GDH of strain P2 did not use ribose as substrate. Utilization of lactose with specific activity of 0.65 IU mg{sup -1} protein indicated that the enzyme belongs to GDH type B isozyme. Arabinose, galactose, ribose, sucrose and xylose did not induce the synthesis of GDH enzyme while mannose induced the synthesis of GDH with highest specific activity of 0.58 IU mg{sup -1} protein. Through radiation mutagenesis, the substrate specificity of GDH was modified in order to utilize side range of sugars available in root exudates. Ribose, originally not a substrate for GDH of strain P2 was utilized as substrate by mutants P2-M5 with specific activity of 0.44 and 0.57 IU mg{sup -1} protein, respectively. Specific activity of GDH on the media containing lactose and galactose was also improved to 1.2 and 0.52 IU mg{sup -1} protein in P2-M5 and P2-M6 respectively. Based on the carbon source availability in root exudate, the mutants can be selected and utilized as efficient biofertilizer under P-deficient soil conditions.

  19. Expression profiles of genes in wild-type DJ-1 and L10P mutant DJ-1 in monoclonal cell strains

    Directory of Open Access Journals (Sweden)

    LIU Zhen-hua

    2013-07-01

    Full Text Available Background DJ-1 gene is a causative gene which contributes to the onset of autosomal recessive early-onset parkinsonism (AREP. Many research suggest that DJ-1 protein may change expression of certain genes through regulate its transcriptional activity, which play a role in the pathogenesis of Parkinson's disease (PD. In our previous study, we found a new mutation of DJ-1 which we named as L10P. DJ-1 gene encodes the first frame 29 bp from the thymine (T→cytosine (C, so that the leucine on the 10th locus of DJ-1 protein was replaced by proline (L10P. To elucidate the effect of the L10P mutation, we identify genes for which expressions are abnormally regulated by L10P mutant DJ-1 protein using DNA microarray analysis. Methods Human embryonic kidney cell 293 (HEK293 monoclonal cell strains which can stably express pCMV-Tag2A-Flag, pCMV-Tag2A-Flag-DJ-1 and pCMV-Tag2A-Flag-DJ-1-L10P were selected by screening, and identified on the basis of DNA, RNA and protein levels to confirm whether the acquired HEK293 monoclonal cell strains can stably express empty vector, wild-type DJ-1 protein and L10P mutant DJ-1 protein. Gene chip technique was used to perform differential gene screening for different groups of HEK293 monoclonal cell strains. Results Compared with the expression in empty vector group, the expression of 14 genes was up-regulated and 28 genes was down-regulated in wild-type group; and the expression of 14 genes was up-regulated and 9 down-regulated in expressing L10P mutant group respectively. Comparison of the expression in wild-type group, expression of 59 genes was up-regulated and 27 genes down-regulated in L10P mutant group. These differential genes all took part in the biological processes including signal transduction, transcriptional regulation, cell cycle, apoptosis, oxidative stress and so on. Conclusion L10P mutant DJ-1 protein may directly or indirectly influence the singal transduction and play a role in the mechanism of PD.

  20. 2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases.

    Science.gov (United States)

    Curto, Miguel; Valledor, Luis; Navarrete, Clara; Gutiérrez, Dolores; Sychrova, Hana; Ramos, José; Jorrin, Jesús

    2010-11-10

    By using a 2-DE based workflow, the proteome of wild and potassium transport mutant trk1,2 under optimal growth potassium concentration (50mM) has been analyzed. At the exponential and stationary phases, both strains showed similar growth, morphology potassium content, and Vmax of rubidium transport, the only difference found being the Km values for this potassium analogue transport, higher for the mutant (20mM) than for the wild (3-6mM) cells. Proteins were buffer-extracted, precipitated, solubilized, quantified, and subjected to 2-DE analysis in the 5-8 pH range. More differences in protein content (37-64mgg(-1) cell dry weight) and number of resolved spots (178-307) were found between growth phases than between strains. In all, 164 spots showed no differences between samples and a total of 105 were considered to be differential after ANOVA test. 171 proteins, corresponding to 71 unique gene products have been identified, this set being dominated by cytosolic species and glycolitic enzymes. The ranking of the more abundant spots revealed no differences between samples and indicated fermentative metabolism, and active cell wall biosynthesis, redox homeostasis, biosynthesis of amino acids, coenzymes, nucleotides, and RNA, and protein turnover, apart from cell division and growth. PCA analysis allowed the separation of growth phases (PC1 and 2) and strains at the stationary phase (PC3 and 4), but not at the exponential one. These results are also supported by clustering analysis. As a general tendency, a number of spots newly appeared at the stationary phase in wild type, and to a lesser extent, in the mutant. These up-accumulated spots corresponded to glycolitic enzymes, indicating a more active glucose catabolism, accompanied by an accumulation of methylglyoxal detoxification, and redox-homeostasis enzymes. Also, more extensive proteolysis was observed at the stationary phase with this resulting in an accumulation of low Mr protein species.

  1. Construction and Genetic Analysis of Murine Hepatitis Virus Strain A59 Nsp16 Temperature Sensitive Mutant and the Revertant Virus

    Institute of Scientific and Technical Information of China (English)

    Guo-hui Chang; Bao-jun Luo; Pin Lu; Lei Lin; Xiao-yan Wu; Jing Li; Yi Hu; Qing-yu Zhu

    2011-01-01

    Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu"-ts18(cd) were indistinguishable from the reported ts mutant Wu"-ts 18. Then we cultured the ts mutant Wu"-ts 18(cd) at non-permissive temperature 39.5℃, which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu"-ts 18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

  2. Genome Sequences of the Oxytetracycline Production Strain Streptomyces rimosus R6-500 and Two Mutants with Chromosomal Rearrangements

    KAUST Repository

    Baranasic, Damir

    2014-07-17

    The genome sequence of Streptomyces rimosus R6-500, an industrially improved strain which produces high titers of the important antibiotic oxytetracycline, is reported, as well as the genome sequences of two derivatives arising due to the genetic instability of the strain.

  3. Production and Accumulation of Xylooligosaccharides with Long Chains by Growing Culture and Xylanase of a Mutant Strain of Bacillus pumilus X-6-19

    Institute of Scientific and Technical Information of China (English)

    Qingzhu Yuan; Tsuyoshi Adachi; Shinji Takenaka; Shuichiro Murakami; Machiko Tanaka; Kenji Aoki

    2008-01-01

    Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosacchatides with long chainsfrom xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increasein the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19.The addition of D-glucose to the culture of the mutant swain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but notxylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharideswith long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized.The hydrolyzates generated by the purified xylanase contained xylobiose, xylotrinse, xylotewaose, and xylopentaose, but not xylose.

  4. Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination.

    Directory of Open Access Journals (Sweden)

    Dedeke Rockx-Brouwer

    Full Text Available Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.

  5. Cloning, sequencing, and expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in E. coli.

    Science.gov (United States)

    Pratush, Amit; Seth, Amit; Bhalla, T C

    2012-10-01

    The NHase encoding gene of mutant 4D was isolated by PCR amplification. The NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli cells showed higher NHase activity (5.9 U/mg dcw) as compared to the wild (4.1 U/mg dcw) whereas it is less than the mutant strain (8.4 U/mg dcw). Addition of cobalt ion in Luria-Bertani medium is needed up to a very small concentration (0.4 mM) for NHase activity. The recombinant E. coli exhibited maximum NHase activity at 6 h of incubation and was purified with a yield of 56 % with specific activity of 37.1 U/mg protein.

  6. Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination.

    Science.gov (United States)

    Rockx-Brouwer, Dedeke; Chong, Audrey; Wehrly, Tara D; Child, Robert; Crane, Deborah D; Celli, Jean; Bosio, Catharine M

    2012-01-01

    Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.

  7. Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

    Science.gov (United States)

    Peters, S E; Paterson, G K; Bandularatne, E S D; Northen, H C; Pleasance, S; Willers, C; Wang, J; Foote, A K; Constantino-Casas, F; Scase, T J; Blacklaws, B A; Bryant, C E; Mastroeni, P; Charles, I G; Maskell, D J

    2010-01-01

    In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

  8. Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Strains of Synechocystis PCC 6803 for Maximal, 24-Hour Production of Hydrogen Gas

    Energy Technology Data Exchange (ETDEWEB)

    Ely, Roger L.; Chaplen, Frank W.R.

    2014-03-11

    enhanced H2 production profiles using selected culture conditions and inhibitors of specific pathways in WT cells and an NDH-1 mutant; 3. Create Synechocystis PCC 6803 mutant strains with modified hydrogenases exhibiting increased O2 tolerance and greater H2 production; and 4. Integrate enhanced hydrogenase mutants and culture and metabolic factor studies to maximize 24-hour H2 production.

  9. Screening of Saccharomyces Strains Highly Producing Glutathione and Breeding of Its Ethionine-resistant Mutants%高产谷胱甘肽酵母菌株的筛选及其抗乙硫氨酸诱变研究

    Institute of Scientific and Technical Information of China (English)

    冮洁; 单立峰; 吴耘红; 张鹭

    2008-01-01

    [Objective] The aim of this study was to screen Saccharomyces for glutathione over-production. [Method] Ethionine-resistant mutants were obtained through UV mutagenesis and rational screening. [Result] A high GSH-producing strain HSJB1 was isolated from soil, and the biomass for this strain by flask shaking fermentation was 3.87 g/L while the GSH yield was 91.87 mg/L. According to the morphological, physiological and biochemical characteristics of cells, this strain was primarily identified as Saccharomyces cerevisiae. An ethionine-resistant mutant YBS77 was obtained through UV mutagenesis of the original strain HSJB1, and the biomass for this strain by flask shaking fermentation was 7.60 g dry cell weight/L while the GSH yield was 211.96 mg/L. [Conclusion] The biomass of the mutant obtained by breeding is increased by 96.38% than that of the original strain, and the GSH yield of the mutant obtained by breeding is increased by 130.72% than that from the original strain, which indicates that the breeding method is feasible.

  10. Construction and characterization of a glycoprotein E deletion mutant of bovine herpesvirus type 1.2 strain isolated in Brazil

    NARCIS (Netherlands)

    Franco, A.C.; Rijsewijk, F.A.M.; Flores, E.F.; Weiblen, R.; Roehe, P.M.

    2002-01-01

    This paper describes the construction and characterization of a Brazilian strain of bovine herpesvirus type 1.2a (BoHV-1.2a) with a deletion of the glycoprotein E (gE) gene. The deletion was introduced by co-transfection of a deletion fragment containing the 5´and 3´gE flanking regions and genomic D

  11. The Use of NF1 and NF2 Mutant Mouse Strains in the Investigation of Gene Function and Disease Development

    Science.gov (United States)

    1998-10-01

    the major symptoms of the disease are schwannomas affecting the eighth cranial nerves, meningiomas and ependymomas. NF2 patients also frequently exhibit...not develop the schwannomas or other CNS tumors that occur in human NF2 (28,29). The generation and initial characterization of these strains was...animal’s back and tongue , respectively. (Note the multinodular nature of the tongue lesion(s)). Only cells associated with the central normal nerve (N

  12. [Characteristics of the structural organization of the DIP1 gene in Drosophila melanogaster strains mutant for the flamenco gene].

    Science.gov (United States)

    Nefedova, L N; Romanova, N I; Kim, A I

    2007-01-01

    Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flamSS (SS) and flamMS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flampy + (P) carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco+. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flamSS, flamMS, flampy +(P), and flamenco+ considerably differ from one another in the structure of DIP1. Strains flamss and flamMS have no Dral restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The Dral gene of strainsflamSS andflamMS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tcl/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIPI and, probably, neighboring genes. In strains flamenco+ and flampy + (P), the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either

  13. Identificação do potencial amilolítico de linhagens mutantes do fungo filamentoso Aspergillus nidulans Identification of the amylolytic potential of mutant strains of the filamentous fungi Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Izabel Aparecida Soares

    2010-09-01

    Full Text Available As amilases estão entre as mais importantes enzimas industriais, apresentando grande importância biotecnológica, principalmente na indústria alimentícia. Com o avanço no conhecimento das enzimas, a utilização dos fungos como fonte de enzimas vem adquirindo um status de destaque nas mais variadas áreas industriais e comerciais. Diante disso, o presente estudo procurou identificar a presença de atividade amilolítica em quatro linhagens do fungo filamentoso Aspergillus nidulans, selvagem, PAT, biA1methG1 e CLB3, utilizando dois meios distintos de cultura, BDA e Meio Completo a 2% amido, variando os tratamentos com adição ou não de glicose. Foram determinados o diâmetro médio da colônia, o diâmetro médio do halo e o Índice Enzimático. Como resultados, todas as linhagens testadas foram capazes de degradar o amido quando na ausência de glicose, porém o tratamento que obteve estatisticamente melhor crescimento e maior degradação do amido foi o MC sem glicose a 2% amido e a linhagem que se demonstrou potencialmente degradadora de amido foi o mutante CLB3. Conclui-se, portanto, que Aspergillus nidulans pode ser considerado como um produtor de amilases.The amylases are among the most important industrial enzymes showing great biotechnological importance, mainly in the food industry. Due to the advance knowledge in enzyme research, the use of fungi as source of enzymes has acquired a prominent status in most varied industrial and commercial areas. Thus, the present study tried to identify the presence of the amylolytic activity in four stains of filamentous fungi Aspergillus nidulans, selvagem, PAT, biA1methG1 and CLB3 by using two different culture means, PDA and Complete mean with 2% starch, varying the treatments with and without the addition of glucose. The colony average diameter, the halo average diameter, and the enzymatic index were determined. The results show that all strains tested were capable of degrading starch in the

  14. Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Mezard, C.; Nicolas, A. [Universite Paris-Sud, Orsay (France)

    1994-02-01

    Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions; (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product. 67 refs., 4 figs., 5 tabs.

  15. Study on the molecular basis of glucoamylase overproduc-tion of a mutant strain Aspergillus niger T21

    Institute of Scientific and Technical Information of China (English)

    FAN; Xiaochun

    2001-01-01

    [1]Qiao, D., Tang, G., Zhong, L. et al., The overall analysis and comparison of the expression of glucoamylase in the over-production strain A. niger T21 and its original strain A. niger 3.795, Acta Microbiologica Sinica (in Chinese), 1997, 37(5): 349.[2]Zhong, L, Qiao, D, Tang, G. et al., Cloning, sequencing and comparison of the 5′-flanking regions of glaA gene from high and low glucoamylase producing strains of Aspergillus niger, Acta Microbiologica Sinica (in Chinese), 1996, 36: 181.[3]Qiao, D., Zhong, L., Tang, G. et al., Cloning and sequencing of glucoamylase gene from Aspergillus niger 3.795, Chinese J. Biochem. and Mol. Biology (in Chinese), 1998, 14: 254.[4]Zhong, L., Fan, X., Tang, G., Cotransformation of glucoamylase overproducing strain Aspergillus niger T21 with niaD and uidA gene, Mycosystema (in Chinese), 1999, 18: 172.[5]Roberts, I. N., Oliver, R. P., Punt, P. J. et al., Expression of the Escherichia coli b-glucuronidase gene in industrial and phytopathogenic filamentous fungi, Curr. Genet., 1989, 15: 177.[6]Lowry, O. H., Rosebrough, N. J., Farr, A. L. et al., Protein measurement with the folin phenol reagent, J. Biochem., 1951, 193: 268.[7]Zhu, H., Qu, F., Zhu, L., Isolation of genomic DNAs from fungi using benzyl chloride, Acta Mycologica Sinica (in Chi-nese), 1994, 13: 34.[8]Verwoerd, T. C., Dekker, B. M. M., Hoekema, A., A small-scale procedure for the rapid isolation of plant RNAs, Nucleic Acids Research, 1989, 17: 2362.[9]Campbell, E. I., Unkles, S. E., Marco, J. A. et al., Improved transformation efficiency of Aspergillus niger using the ho-mologous niaD gene for nitrate reductase, Curr. Genet., 1989, 16: 53.[10]Fowler, T., Berka, R. M., Ward, M., Regulation of the glaA gene of Aspergillus niger, Curr. Genet., 1990, 18: 537.[11]Verdoes, J. C., Punt, P. J., Stouthamer, A. H. et al., The effect of multiple copies of the upstream region on expression of the Aspergillus niger glucoamylase

  16. Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

    Science.gov (United States)

    Galindo-Leva, Luz Ángela; Hughes, Stephen R; López-Núñez, Juan Carlos; Jarodsky, Joshua M; Erickson, Adam; Lindquist, Mitchell R; Cox, Elby J; Bischoff, Kenneth M; Hoecker, Eric C; Liu, Siqing; Qureshi, Nasib; Jones, Marjorie A

    2016-07-01

    Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.

  17. Nif- Hup- mutants of Rhizobium japonicum.

    OpenAIRE

    Moshiri, F; Stults, L; Novak, P.; Maier, R J

    1983-01-01

    Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains...

  18. Hydrogen production by hup(-) mutant and wild-type strains of Rhodobacter capsulatus from dark fermentation effluent of sugar beet thick juice in batch and continuous photobioreactors.

    Science.gov (United States)

    Uyar, Basar; Gürgan, Muazzez; Özgür, Ebru; Gündüz, Ufuk; Yücel, Meral; Eroglu, Inci

    2015-10-01

    Photofermentative production of hydrogen is a promising and sustainable process; however, it should be coupled to dark fermentation to become cost effective. In order to integrate dark fermentation and photofermentation, the suitability of dark fermenter effluents for the photofermentative hydrogen production must be demonstrated. In this study, thermophilic dark fermenter effluent (DFE) of sugar beet thick juice was used as a substrate in photofermentation process to compare wild-type and uptake hydrogenase-deficient (hup (-)) mutant strains of Rhodobacter capsulatus by means of hydrogen production and biomass growth. The tests were conducted in small-scale (50 mL) batch and large-scale (4 L) continuous photobioreactors in indoor conditions under continuous illumination. In small scale batch conditions, maximum cell concentrations were 0.92 gdcw/L c and 1.50 gdcw/L c, hydrogen yields were 34 % and 31 %, hydrogen productivities were 0.49 mmol/(L c·h) and 0.26 mmol/(Lc·h), for hup (-) and wild-type cells, respectively. In large-scale continuous conditions, maximum cell concentrations were 1.44 gdcw/L c and 1.87 gdcw/L c, hydrogen yields were 48 and 46 %, and hydrogen productivities were 1.01 mmol/(L c·h) and 1.05 mmol/(L c·h), for hup (-) and wild-type cells, respectively. Our results showed that Rhodobacter capsulatus hup (-) cells reached to a lower maximum cell concentration but their hydrogen yield and productivity were in the same range or superior compared to the wild-type cells in both batch and continuous operating modes. The maximum biomass concentration, yield and productivity of hydrogen were higher in continuous mode compared to the batch mode with both bacterial strains.

  19. Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

    Directory of Open Access Journals (Sweden)

    Deepali Bisht

    2013-01-01

    Full Text Available Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100. The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L-1; starch, 15.0 g.L-1; triton-X-100, 0.93 mL.L-1; incubation temperature, 34.12 ºC and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL-1. The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R² value (0.9987. The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.

  20. High-yield production of aryl alcohol oxidase under limited growth conditions in small-scale systems using a mutant Aspergillus nidulans strain.

    Science.gov (United States)

    Pardo-Planas, Oscar; Prade, Rolf A; Wilkins, Mark R

    2017-02-01

    Aryl alcohol oxidase (MtGloA) is an enzyme that belongs to the ligninolytic consortium and can play an important role in the bioenergy industry. This study investigated production of an MtGloA client enzyme by a mutant strain of Aspergillus nidulans unable to synthesize its own pyridoxine. Pyridoxine limitation can be used to control cell growth, diverting substrate to protein production. In agitated culture, enzyme production was similar when using media with 1 mg/L and without pyridoxine (26.64 ± 6.14 U/mg mycelia and 26.14 ± 8.39 U/mg mycelia using media with and without pyridoxine, respectively). However, the treatment lacking pyridoxine had to be supplemented with pyridoxine after 156 h of fermentation to sustain continued enzyme production. Use of extremely diluted pyridoxine levels allowed reduced fungal growth while maintaining steady enzyme production. Concentrations of 9 and 13.5 µg/L pyridoxine allowed MtGloA production with a growth rate of only 5% of that observed when using the standard 1 mg/L pyridoxine media.

  1. Purification and Characterization of a New Heme-Binding Protein (HBP59) from the Mutant Strain DJ35 of Azotobacter vinelandii

    Institute of Scientific and Technical Information of China (English)

    Shao-Min Bian; Huang-Ping Wang; Hui-Na Zhou; Ying Zhao; Jian-Feng Zhao; Ju-Fu Huang

    2007-01-01

    A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandli Lipmann. After analysis by matrix-assisted laser desorptlon ionization time-offlight mass spectrometry, the protein was identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra (ABS) in the reduced state exhibited three peaks at 421,517, and 556nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichroism (CD) spectrum of HBP59 in the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the Δε (CD extinction coefficient) values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully interacted with heme, its maximal ABS value and Soret CD intensity were increased by approximately 10-fold compared with values before interaction. Therefore, it seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with iow spin and may be involved in heme utilization or adhesion.

  2. A Salmonella Enteritidis hilAssrAfliG deletion mutant is a safe live vaccine strain that confers protection against colonization by Salmonella Enteritidis in broilers.

    Science.gov (United States)

    De Cort, W; Geeraerts, S; Balan, V; Elroy, M; Haesebrouck, F; Ducatelle, R; Van Immerseel, F

    2013-10-17

    Consumption of contaminated poultry meat is an important cause of Salmonella infections in humans. Therefore, there is a need for control methods that protect broilers from day-of-hatch until slaughter age against infection with Salmonella. Colonization-inhibition, a concept in which a live Salmonella strain is orally administered to day-old chickens and protects against subsequent challenge, can potentially be used as control method. In this study, the safety and efficacy of a Salmonella Enteritidis ΔhilAssrAfliG strain as a colonization-inhibition strain for protection of broilers against Salmonella Enteritidis was evaluated. After administration of the Salmonella Enteritidis ΔhilAssrAfliG strain to day-old chickens, this strain could not be isolated from the gut, internal organs or faeces after 21 days of age. In addition, administration of this strain to one-day-old broiler chickens decreased faecal shedding and caecal and internal organ colonization of a Salmonella Enteritidis challenge strain administered one day later using a seeder bird model. To our knowledge, this is the first report of an attenuated Salmonella strain for which both the safety and efficacy has been shown in long-term experiments (until slaughter age) in broiler strain can potentially be used as a live colonization-inhibition strain for controlling Salmonella Enteritidis infections in broilers.

  3. Observation on biological characters of Streptococcus mutans luxS gene knockout mutant strain%变异链球菌luxS基因缺陷突变株生物学性状的初步观察

    Institute of Scientific and Technical Information of China (English)

    黄正蔚; 刘正; 马瑞; 唐子圣; 朱彩莲

    2008-01-01

    目的 为研究口腔主要致龋菌变异链球菌的种属间密度感应(quorum sensing)相关基因luxS对口腔生态的影响,构建此菌的luxS基因缺失突变株.方法 采用同源重组的方法,利用红霉素耐药基因erm置换变异链球菌基因组中的luxS基因,并在含抗性标记的选择性培养基上筛选出阳性克隆.初步研究该基因的缺失突变对变异链球菌在生长与生物被膜成熟分化中的作用.结果经PCR鉴定,luxS基因的置换突变位置正确,并能在体外稳定传代,突变株与野生株相比在达静止期后总菌数量上有明显差别,但在生物被膜的形成与分化上并未发现显著差异.结论 通过此研究建立了可稳定传代的变异链球菌luxS基因的缺失突变株,生长表型和生物被膜的形成与野生株无显著差异,为进一步研究此基因功能与调控机制提供了技术平台.%Objective To investigate the construction of Streptococcus mutans luxS gene knockout mutant which can act as the technical platform for following researches on luxS quorum sensing function in oral ecosystem.Methods Erythromycin resistance gene was inserted between two 1 kb fragments containing regions of DNA immediately upstream and downstream of the luxS translational start and stop codons.The resuhing construct was linearized and electro-transformed into Streptococcus mutans cells.After allelic exchange,the luxS gene knockout mutant strains were selected on 10μg/ml erythromycin plates,and compared the growth and biofilms formation of luxS knockout mutant with wild type strains.Results The luxSknockout mutant was confirmed by PCR,and it was also confirmed that this gene mutant could be stably passed through in vitro.The growth mode of luxS knockout mutant showed obvious difierences against that of wild type at stationary phase,the knockout mutant gained more bacteria cells growth.Conclusion Streptococcus mutans luxS gene has been successfully disrupted with allelic

  4. Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations.

    Science.gov (United States)

    Suchland, Robert J; Jeffrey, Brendan M; Xia, Minsheng; Bhatia, Ajay; Chu, Hencelyn G; Rockey, Daniel D; Stamm, Walter E

    2008-12-01

    Clinical isolates of Chlamydia trachomatis that lack IncA on their inclusion membrane form nonfusogenic inclusions and have been associated with milder, subclinical infections in patients. The molecular events associated with the generation of IncA-negative strains and their roles in chlamydial sexually transmitted infections are not clear. We explored the biology of the IncA-negative strains by analyzing their genomic structure, transcription, and growth characteristics in vitro and in vivo in comparison with IncA-positive C. trachomatis strains. Three clinical samples were identified that contained a mixture of IncA-positive and -negative same-serovar C. trachomatis populations, and two more such pairs were found in serial isolates from persistently infected individuals. Genomic sequence analysis of individual strains from each of two serovar-matched pairs showed that these pairs were very similar genetically. In contrast, the genome sequence of an unmatched IncA-negative strain contained over 5,000 nucleotide polymorphisms relative to the genome sequence of a serovar-matched but otherwise unlinked strain. Transcriptional analysis, in vitro culture kinetics, and animal modeling demonstrated that IncA-negative strains isolated in the presence of a serovar-matched wild-type strain are phenotypically more similar to the wild-type strain than are IncA-negative strains isolated in the absence of a serovar-matched wild-type strain. These studies support a model suggesting that a change from an IncA-positive strain to the previously described IncA-negative phenotype may involve multiple steps, the first of which involves a translational inactivation of incA, associated with subsequent unidentified steps that lead to the observed decrease in transcript level, differences in growth rate, and differences in mouse infectivity.

  5. A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity.

    Directory of Open Access Journals (Sweden)

    Vignesh M P Babu

    Full Text Available The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V, catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.

  6. A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity.

    Science.gov (United States)

    Babu, Vignesh M P; Sutton, Mark D

    2014-01-01

    The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.

  7. Attenuated Actinobacillus pleuropneumoniae double-deletion mutant S-8∆clpP/apxIIC confers protection against homologous or heterologous strain challenge.

    Science.gov (United States)

    Xie, Fang; Li, Gang; Zhou, Long; Zhang, Yanhe; Cui, Ning; Liu, Siguo; Wang, Chunlai

    2017-01-06

    Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which leads to large economic losses to the swine industry worldwide. In this study, S-8△clpP△apxIIC, a double-deletion mutant of A. pleuropneumoniae was constructed, and its safety and protective efficacy were evaluated in pigs. The S-8△clpP△apxIIC mutant exhibited attenuated virulence in a murine (BALB/c) model, and caused no detrimental effects on pigs even at a dose of up to 1.0 × 10(9) CFU. Furthermore, the S-8△clpP△apxIIC mutant was able to induce a strong immune response in pigs, which included high levels of IgG1 and IgG2, stimulated gamma interferon (IFN-γ), interleukin 12 (IL-12), and interleukin 4 (IL-4) production, and conferred effective protection against the lethal challenge with A. pleuropneumoniae serovars 7 or 5a. The pigs in the S-8△clpP△apxIIC immunized groups have no lesions and reduced bacterial loads in the lung tissue after challenge. The data obtained in this study suggest that the S-8△clpP△apxIIC mutant can serve as a highly immunogenic and potential live attenuated vaccine candidate against A. pleuropneumoniae infection.

  8. The isolation and characterization of a mutant strain of Saccharomyces cerevisiae that requires a long chain base for growth and for synthesis of phosphosphingolipids.

    Science.gov (United States)

    Wells, G B; Lester, R L

    1983-09-10

    A mutant of Saccharomyces cerevisiae has been obtained that shows an absolute growth requirement for long chain bases found in sphingolipids. In the absence of a long chain base, the cells are unable to synthesize the phosphoinositol-containing sphingolipids characteristic of yeast. These results suggest that one or more of the yeast sphingolipids plays a vital biological role.

  9. 突变株CTM2降解苯酚和4-氯酚的生物降解特性%Biodegradation of Phenol and 4-Chlorophenol by the Mutant Strain CTM 2

    Institute of Scientific and Technical Information of China (English)

    姜岩; 任南琪; 蔡徇; 吴迪; 乔丽艳; 林森

    2008-01-01

    The biodegradations of phenol and 4-chlorophenol(4-cp)were studied using the mutant strain CTM 2 obtained by the He-Ne laser irradiation on wild-type Candida tropicalis.The results showed that the capacity of the CTM 2 to biodegrade 4-cp was increased up to 400 mg.L-1 within 59.5 h.In the dual.substrate biodegradation,both 2 to degrade phenol.In addition,the kinetic behaviors were described using the kinetic model proposed in this lab.

  10. The effects of modeled microgravity on growth kinetics, antibiotic susceptibility, cold growth, and the virulence potential of a Yersinia pestis ymoA-deficient mutant and its isogenic parental strain.

    Science.gov (United States)

    Lawal, Abidat; Kirtley, Michelle L; van Lier, Christina J; Erova, Tatiana E; Kozlova, Elena V; Sha, Jian; Chopra, Ashok K; Rosenzweig, Jason A

    2013-09-01

    Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent.

  11. Nif- Hup- mutants of Rhizobium japonicum.

    Science.gov (United States)

    Moshiri, F; Stults, L; Novak, P; Maier, R J

    1983-01-01

    Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity. Images PMID:6874648

  12. Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17.

    Science.gov (United States)

    Marsden, H S; Crombie, I K; Subak-Sharpe, J H

    1976-06-01

    The polypeptides induced in cells infected with a Glasgow isolate of HSV-I (17 syn+) have been characterized by SDS polyacrylamide gel electrophoresis. Study of the kinetics of synthesis in three cell lines has detected a total of 52 polypeptides, 33 of which can be identified in polypeptide profiles of purified virions. These include six low mol. wt. polypeptides that have not been previously reported. Several polypeptides were labelled with glucosamine in infected BHK cells. The different polypeptide patterns obtained at permissive (31 degrees C) and nonpermissive (38 degrees C) temperature in cells infected with 16 temperature-sensitive (ts) mutants are reported. The effect of multiplicity of infection (m.o.i.) on the polypeptide profile has been examined for two of the DNA -ve mutants: below ten, the profile varied with the m.o.i. whereas above ten it was constant. All mutants were therefore examined at an m.o.i. of approx. 20. Mutants from the same complementation group showed very similar profiles. A number of general conclusions concerning control of protein synthesis in HSV infected cells can be made: (I) As most of the 16 ts mutants affected the synthesis of several or many polypeptides it follows that a large proportion of genome specifies controlling functions. (2) The high frequency with which some polypeptides were affected suggests they are at or near the terminus of biosynthetic pathways which are under multiple control. (3) Conversely, some polypeptides were affected with a low frequency suggesting that their synthesis is not dependent on the expression of many virus functions. (4) Several individual ts mutations lead to the synthesis of increased amounts of different large polypeptides. (5) Analysis of every band detectably affected by at least one ts mutation has disclosed nine classes of dependence relationship between polypeptide synthesis and the DNA phenotype of the mutants, illustrating that this relationship is complex and different for

  13. An organic acid-tolerant HAA1-overexpression mutant of an industrial bioethanol strain of Saccharomyces cerevisiae and its application to the production of bioethanol from sugarcane molasses.

    Science.gov (United States)

    Inaba, Takuya; Watanabe, Daisuke; Yoshiyama, Yoko; Tanaka, Koichi; Ogawa, Jun; Takagi, Hiroshi; Shimoi, Hitoshi; Shima, Jun

    2013-12-30

    Bacterial contamination is known as a major cause of the reduction in ethanol yield during bioethanol production by Saccharomyces cerevisiae. Acetate is an effective agent for the prevention of bacterial contamination, but it negatively affects the fermentation ability of S. cerevisiae. We have proposed that the combined use of organic acids including acetate and lactate and yeast strains tolerant to organic acids may be effective for the elimination of principally lactic acid bacterial (LAB) contamination. In a previous study employing laboratory S. cerevisiae strains, we showed that overexpression of the HAA1 gene, which encodes a transcriptional activator, could be a useful molecular breeding method for acetate-tolerant yeast strains. In the present study, we constructed a HAA1-overexpressing diploid strain (MATa/α, named ER HAA1-OP) derived from the industrial bioethanol strain Ethanol Red (ER). ER HAA1-OP showed tolerance not only to acetate but also to lactate, and this tolerance was dependent on the increased expression of HAA1 gene. The ethanol production ability of ER HAA1-OP was almost equivalent to that of the parent strain during the bioethanol production process from sugarcane molasses in the absence of acetate. The addition of acetate at 0.5% (w/v, pH 4.5) inhibited the fermentation ability of the parent strain, but such an inhibition was not observed in the ethanol production process using ER HAA1-OP.

  14. 耐甲氧西林表皮葡萄球菌 psm-mec 缺失突变株的构建%Construction of mutant strains of methicillin resistant Staphylococcus epidermidis with psm-mec gene deletion

    Institute of Scientific and Technical Information of China (English)

    杨永长; 胡洪华; 陈亮; 刘华; 喻华; 黄文芳

    2015-01-01

    Objective To construct mutant strains of methicillin resistant Staphylococcus epidermi-dis (MRSE) with psm-mec gene deletion and to investigate the function of psm-mec gene.Methods The drug sensitivity test and DNA sequence analysis were performed to screen out the tetracycline and chloram -phenicol sensitive clinical strains of MRSE , whose upstream and downstream sequences of psm-mec gene were identical to those of the Staphylococcus epidermidis reference strain RP62A.The recombinant plasmid pBT2-Δpsm-mec was constructed by using the fusion PCR and a temperature sensitive shuttle plasmid .After being identified , the plasmid was transformed into the Staphylococcus aureus RN4220 strain by electropora-tion, and then transformed into the selected clinical isolates of MRSE .The mutant strains of MRSE with psm-mec deletion were screened out and identified after homologous recombination .The differences in biofilm formation between the mutant and wild-type strains were analyzed for further elucidation the relationships be-tween the psm-mec gene and biofilm formation in MRSE strains .Results Three clinical MRSE isolates for the construction of mutant strains with psm-mec gene deletion were screened out and identified by using drug sensitivity test and sequence alignment analysis .The mutants constructed via homogenous recombination were screened out and identified .Compared with the corresponding wild-type strains, the three mutants with psm-mec gene deletion showed significantly decreased ability of biofilm formation , demonstrating that the psm-mec genes strains induced the biofilm formation of MRSE .Conclusion The Δpsm-mec mutant strains were successfully constructed .The psm-mec gene played an important role in the biofilm formation of Staphy-lococcus epidermdis.%目的:构建耐甲氧西林表皮葡萄球菌( MRSE)的psm-mec缺失突变株,并对psm-mec的功能进行初步研究。方法运用药敏试验、DNA序列分析技术筛选psm-mec上下游序

  15. Growth, physicochemical properties, and morphogenesis of Chinese wild-type PRV Fa and its gene-deleted mutant strain PRV SA215

    Directory of Open Access Journals (Sweden)

    Tang Shanhu

    2011-06-01

    Full Text Available Abstract Background PRV Fa is common in China and causes most of the pseudorabies in the pig industry. A PRV SA215 strain with deleted gE, gI, and TK genes was constructed to develop a commercial attenuated live vaccine. However, the physicochemical properties, growth pattern, penetration kinetics, and morphogenesis of the PRV SA215 and its parental PRV Fa strain are unclear. Results A series of experiments were conducted to characterize both strains and provide more information. PRV Fa and PRV SA215 were found to have similar penetration patterns, with about 5 min half-time of penetration. The SA215 strain exhibited a slight delay in entry compared with PRV Fa. In the one-step growth test, the titers of the SA215 strain were first detected at 8 h, rapidly increased, and peaked at 12 h. A plateau was formed between 12-36 h of culturing. PRV SA215 showed delayed replication and approximately 10-30-fold lower titers during 0-16 h of culturing compared with the PRV-Fa strain. After 16 h, the PRV Fa titers dramatically decreased, whereas those of PRV SA215 were prolonged to 36 h and reached a titer value equal to that of PRV Fa and then decreased. Both strains were sensitive to both heat and acid-alkali treatments; however, PRV Fa was relatively more stable to heat treatment than PRV SA215. Both strains could propagate in the cultures with pH values from 5.0 to 9.0. Cultures with pH below 3.0 or above 11.0 were fatal to both strains. Both strains had considerable resistance to freeze-thawing treatments. Morphogenetic investigations showed that typical phases in the maturation pathway were observed in the PRV Fa-infected PK15 cells, whereas secondary envelopment was not observed in the PRV SA215 strain. Instead, capsid aggregations with concomitants of electrodense materials were observed. Conclusions These results suggest that PRV SA215 is a promising strain for vaccine development

  16. Properties of S-adenosyl-L-methionine:macrocin O-methyltransferase in extracts of Streptomyces fradiae strains which produce normal or elevated levels of tylosin and in mutants blocked in specific O-methylations.

    Science.gov (United States)

    Seno, E T; Baltz, R H

    1981-01-01

    An efficient assay for S-adenosyl-L-methionine:macrocin O-methyltransferase, the enzyme which carries out the terminal step in tylosin biosynthesis, is described. Macrocin O-methyltransferase requires Mg2+ and S-adenosyl-L-methionine for activity, has a temperature optimum of about 31 degrees C, and has a pH optimum of 7.5 to 8.2. Macrocin O-methyltransferase specifically converts macrocin to tylosin by O-methylation of the 3" ' position of macrocin. In vitro methylation studies with extracts from a tylosin-producing Streptomyces fradiae strain and from mutant strains blocked in 2" '- or 3" '-O-methylations indicated that: (i) the 2" '- and 3" '-O-methylations occur after 6-deoxy-D-allose is attached to the macrolide ring; (ii) the 2" '- and 3" '-O-methylations are carried out by separate enzymes; and (iii) the 2" '-O-methylation precedes the 3" '-O-methylation. Macrocin O-methyltransferase was inhibited by high levels of its substrate, macrocin, by its product, tylosin, and by other tylosin analogs which contained mycinose or demethyl analogs of mycinose. Macrocin O-methyltransferase was produced early in the tylosin fermentation cycle by S. fradiae and preceded the onset of rapid tylosin biosynthesis by about 24 h. The enzyme specific activity reached maximum at about 72 h and then slowly declined. A mutant strain of S. fradiae selected for increased tylosin production synthesized macrocin O-methyltransferase more rapidly and accumulated a higher enzyme specific activity than a wild-type strain. PMID:7305323

  17. Expression and Immunogenicity of a Mutant Diphtheria Toxin Molecule, CRM197, and Its Fragments in Salmonella typhi Vaccine Strain CVD 908-htrA

    Science.gov (United States)

    Orr, Nadav; Galen, James E.; Levine, Myron M.

    1999-01-01

    Mutant diphtheria toxin molecule CRM197 and fragments thereof were expressed in attenuated Salmonella typhi CVD 908-htrA, and the constructs were tested for their ability to induce serum antitoxin. Initially, expressed proteins were insoluble, and the constructs failed to induce neutralizing antitoxin. Soluble CRM197 was expressed at low levels by utilizing the hemolysin A secretion system from Escherichia coli. PMID:10417208

  18. Locating a modifier gene of Ovum mutant through crosses between DDK and C57BL/6J inbred strains in mice

    Indian Academy of Sciences (India)

    JING TAN; GEN DI SONG; JIA SHENG SONG; SHI HAO REN; CHUN LI LI; ZHEN YU ZHENG; WEI DONG ZHAO

    2016-06-01

    A striking infertile phenotype has been discovered in the DDK strain of mouse. The DDK females are usually infertile when crossed with males of other inbred strains, whereas DDK males exhibit normal fertility in reciprocal crosses. This phenomenon is caused by mutation in the ovum (Om) locus on chromosome 11 and known as the DDK syndrome. Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/+) females and males of other strains. This embryonic mortality exhibited an aggravated trend with increasing background genes of other strains. These results indicated that some modifier genes of Om were present in other strains. In the present study, a population of N 2(Om/+) females from the backcrosses between C57BL/6J (B6) and F1(B6♀×DDK♂) was used to map potential modifier genes of Om. Quantitative trait locus showed that a major locus, namely Amom1 (aggravate modifier gene of Om 1), was located at the middle part of chromosome 9 in mice. The Amom1 could increase the expressivity of Om gene, thereby aggravating embryonic lethality when heterozygous (Om/+)females mated with males of B6 strain. Further, the 1.5 LOD-drop analysis indicated that the confidence interval was between 37.54 and 44.46 cM,∼6.92 cM. Amom1 is the first modifier gene of Om in the B6 background.

  19. Construction and Characterization of Campylobacter jejuni flhA Mutant Strain%空肠弯曲菌flhA突变株的构建及其功能研究

    Institute of Scientific and Technical Information of China (English)

    惠星星; 李烨; 王小元

    2011-01-01

    flhA, encoded by the gene flhA, is an important component of the flagellar export apparatus of Campylobacter jejuni. Here we constructed an flhA mutant strain HXW001.Compared with that of the wild type, the flhA mutant cell could grow well but lack flagella, the motility of the mutant cells was lost, and the ability of the mutant cells for the formation of biofilm and autoagglutination also decreased. In addition, it was found that the expression of the flagellin gene flaA was affected by flhA. These findings indicated that flhA was the molecular basis of flagellar synthesis and motility of Campylobacter jejuni and that flhA was closely related to pathogenicity of Carnpylobacter jejuni.%flhA是空肠弯曲茵鞭毛输出装置的关键基因,通过构建Campylobacterjejuni flhA突变株HXW001,进行毒力相关表型研究和分析.结果显示HXW001生长性能稳定,但其鞭毛和运动能力丧失,生物膜形成和自身凝集现象明显下降.RT-PCR实验证明flhA对鞭毛丝主要结构蛋白基因flaA的表达有一定的调控功能.研究表明flhA是空肠弯曲茵鞭毛生成和运动能力重要的分子基础,与空肠弯曲菌致病性密切有关.

  20. Construction and characteristic of srtA gene mutant strain of Streptococcus suis 2%猪链球菌2型srtA基因缺失菌株的构建及生物学特性

    Institute of Scientific and Technical Information of China (English)

    吴涛; 常海涛; 谭臣; 付婷; 司有辉; 贝为成; 陈焕春

    2009-01-01

    In the present study,srtA gene mutant strain was constructed by using the thermo-sensitive suicide vector pSET4s and confirmed by PCR and Southern blotting.The results showed that the construction of the mutant strain was successful,named SC211.In addition complementation mutant strain was constructed by using the E.coli/Streptococcus shuttle vector pAT18,named SC212.The characteristic of SC211 and SC212 was further researched.The virulence of different strains was determined by animal infection,which demonstrated that the virulence was decreased clearly when the srtA gene knocked out.The virulence of the mutant was restored when strain mediated by plasmid-containing the srtA gene.However,it could not reach the level of the wild type.The hemolytic activity of the srtA gene mutant strain and the complementation strain had no difference when compared with the wild type strain.The adherence to the Hep2 cell of the srtA gene mutant strain degraded obviously,and the adherence to the Hep2 cell of the complementation strain had no obvious difference compared with the wild type.%扩增了srtA基因的上、下游同源臂P1和P2及其全长序列,利用温度敏感型"自杀性"质粒pSET4s构建了重组质粒pSET4s-P1-P2,并将该质粒电转化入野生菌株SS2(SC21)中,通过抗生素和温度双重筛选,得到srtA基因缺失菌株,命名SC211.同时,将srtA基因定向插入穿梭质粒pAT18,构建穿梭质粒pAT18-srtA,并将该质粒电转入srtA基因缺失菌株SC211中,通过抗生素筛选,得到质粒介导的srtA基因互补菌株SC212.通过PCR、South-ern blotting等对缺失突变菌株和互补菌株进行了鉴定.对基因缺失突变菌株和回复菌株传代培养遗传稳定性试验结果显示,缺失突变菌株和互补菌株能够稳定遗传.比较了基因缺失突变菌株、互补菌株及野生菌株的生长特性、溶血活性、细胞粘附特性,结果表明,3个菌株的生长速度、溶血活性没有

  1. Degradation of n-Haloalkanes and α,ω-Dihaloalkanes by Wild-Type and Mutants of Acinetobacter sp. Strain GJ70

    NARCIS (Netherlands)

    Janssen, Dick B.; Jager, Dick; Witholt, Bernard

    1987-01-01

    A 1,6-dichlorohexane-degrading strain of Acinetobacter sp. was isolated from activated sludge. The organism could grow with and quantitatively release halide from 1,6-dichlorohexane, 1,9-dichlorononane, 1-chloropentane, 1-chlorobutane, 1-bromopentane, ethylbromide, and 1-iodopropane. Crude extracts

  2. Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase and cysteine protease, and the transcriptional regulator Rgg

    Directory of Open Access Journals (Sweden)

    Finne Jukka

    2006-02-01

    Full Text Available Abstract Background Flow cytometry based adherence assay is a potentially powerful but little used method in the study of bacterial binding to host structures. We have previously characterized a glycoprotein-binding activity in Streptococcus pyogenes called 'strepadhesin' binding to thyroglobulin, submaxillar mucin, fetuin and asialofetuin. We have identified surface-associated pullulanase (PulA and cysteine protease (SpeB as carriers of strepadhesin activity. In the present paper, we investigated the use of flow cytometry as a method to study the binding of Rgg, SpeB and PulA knock-out strains to cultured human epithelial cells. Results Streptococcal mutants were readily labelled with CFDA-SE and their binding to epithelial cells could be effectively studied by flow cytometry. A strain deficient in Rgg expression showed increased binding to the analyzed epithelial cell lines of various origin. Inactivation of SpeB had no effect on the adhesion, while PulA knock-out strains displayed decreased binding to the cell lines. Conclusion These results suggest that the flow cytometric assay is a valuable tool in the analysis of S. pyogenes adherence to host cells. It appears to be an efficient and sensitive tool for the characterization of interactions between the bacteria and the host at the molecular level. The results also suggest a role for Rgg regulated surface molecules, like PulA, in the adhesion of S. pyogenes to host cells.

  3. Experimental infection of commercial layers with wild or attenuated Salmonella Gallinarum mutant strains: anatomic pathology, total blood cell count and serum protein levels

    Directory of Open Access Journals (Sweden)

    KO Garcia

    2013-06-01

    Full Text Available The aim of the present study was to comparatively evaluate hemogram, blood serum components and anatomopathologic alterations in commercial layers experimentally challenged with an attenuated vaccine candidate strain (SG∆cobS∆cbiA and other two pathogenic strains (SGDcobS and SGNalr of Gallinarum (SG. In total, 280 commercial layers were randomly divided into 4 groups (G1, G2, G3 and G4. At five days of age, birds from groups G1 received approximately 107 colony forming units (CFU of SGDcobS; meanwhile birds from group G2 and G3 received the same dose of SGNalr and SG∆cobS∆cbiA, respectively. Birds from G4 were not infected. At 24 hours before (DBI and 24 hours after (1 DAI, and three (3 DAI, five (5 DAI, seven (7 DAI ten (10 DAI, and fifteen (15 DAI days after the infection, 10 birds of each group were humanely killed and blood samples collected to hematological and serum tests. Samples of liver, spleen, thymus, bursa of Fabricius, kidney and heart were also collected for the histological examination. Birds inoculated with SGDcobS and SGNalr showed similar alterations in hemogram, blood serum components and anatomopathologic exams. On the other hand, the exams of birds inoculated with SG∆cobS∆cbiA strain were similar to those of the uninfected birds. However, changes could be noticed in levels of uric acid and cholesterol during the course of the infection of birds from G3. Decrease in levels of light IgG 3 DAI was also observed in birds from this group. Pyknosis in kidney cells was a microscopic alteration found in birds from G3. Further studies must be done to verify if these alterations will not interfere in the performance of the vaccinate birds with SG∆cobS∆cbiA strain.

  4. LuxS基因缺失的变形链球菌突变株的构建及鉴定%Construction and identification of a LuxS-deleted mutant strain of S.mutans

    Institute of Scientific and Technical Information of China (English)

    于丹妮; 韩福胜; 韩玉植; 陈杰

    2008-01-01

    目的 通过同源重组法构建LuxS基因缺失的变形链球菌(Streptococcus mutans)突变株.方法 运用基因同源重组方法将红霉素抗性基因(Eymr)连接到PCR扩增LuxS基因两端区域产生的2个基因片段之间,并共同插入到pUCl9载体的多克隆位点中,构建出带红霉素抗性标志的缺失突变载体pUCluxKO.将突变载体转化到含完整LuxS基因的突变受体变形链球菌标准株中,红霉素筛选出LuxS基因缺失的变形链球菌突变株,并经PCR、生物荧光检测及DNA测序鉴定.结果 构建的突变载体经限制性内切核酸酶酶切分析显示,产生的条带与设计结果完全一致.PCR方法扩增突变株LuxS和Eymr基因显示,LuxS基因已被完整敲除掉,经生物荧光检测,突变株不能诱导哈氏弧菌(Vibrio harveyi)BBl70的生物发光,说明不能产生信号分子AI-2(autoinducer-2).DNA测序证实筛选得到了LuxS基因缺失的变形链球菌突变株.连续传代培养后证实,变形链球菌LuxS基因突变株具有良好的稳定性.结论 成功构建出LuxS基因缺失的变形链球菌突变株,为研究LuxS基因对变形链球菌致龋毒力的影响奠定了基础.%Objective To knock out the entire LuxS gene of Streptococcus mutans UA159 strain via homologous recombination and construct a LuxS-deleted mutant strain of S.mutans.Methods The erythromycin resistance gene(Eymr)was inserted between the two DNA fragments located in the upper and downstream of LuxS gene that had been amplified by PCR.Then the two DNA fragments along with the inserted Eymr were engineered into pUCl9 plasmid to construct the recombination plasmid pUCluxKO.Electrotransformation of S. mutans cells with pUCluxKO-mutant resulted in the isolation of erythromycin resistant S.mutans,transformants,which was then subjected to polymerase chain reaction,Vibrio harveyi BBl70 luminescence bioassay and sequencing analysis.Results Restriction endonuclease analysis showed that pUCluxKOmutant vector

  5. Cellulase production by two mutant strain of Trichoderma longibranchiatum QM9414 and Rut C30; Produccion de celulasas a partir de dos cepas hiperproductoras de trichoderma longibranchiatum Qm9-41 4 y Rut C30

    Energy Technology Data Exchange (ETDEWEB)

    Blanco, M. J.

    1991-07-01

    Native or pretreated biomass from Onopordum nervosum Boiss, has been examined as candidate feedstock for cellulase production by two mutant strain of Trichoderma Ionqibrachiatum QM9414 and Rut C30. Batch cultivation methods were evaluated and compared with previous experiments using ball-milled, crystalline cellulose (Solka floc). Batch cultivation of T. Ionqibrachiatum Rut C30 on 5% (w/v) acid pretreated O. nervosum biomass yielded enzyme productivities and activities comparable to those obtained on Solka floc. However, the overall enzyme production performance was lower than on Solka floc at comparable cellulose concentrations. This fact may be due to the accumulation of pretreated by products and lignin in the ferment. (Author) 40 refs.

  6. Factors affecting growth and antibiotic susceptibility of Helicobacter pylori: effect of pH and urea on the survival of a wild-type strain and a urease-deficient mutant.

    Science.gov (United States)

    Sjöström, J E; Larsson, H

    1996-06-01

    This study investigated how pH and the presence of urea affect the survival and growth of Helicobacter pylori and whether these factors affect susceptibility to antibiotics in vitro. The viability of a wild-type strain and a urease-deficient mutant of H. pylori was studied after incubation for 1 h in buffers at different pH values at 37 degrees C under microaerophilic conditions. Viable counts were not affected at pH 5 and pH 7. In buffer at pH 3, there were no viable organisms, but urea (6.25 mM) protected the wild-type strain, which survived well. At pH 9, urea further reduced the viability of H. pylori and flurofamide almost abolished the effect of urea on the wild-type strain. Neither urea nor flurofamide affected the viability of the urease-deficient mutant under the same conditions. Growth was also pH dependent and was enhanced in shake-cultures. At pH 5, urea supported growth of the wild-type strain, but at pH 7 a toxic effect on the bacteria was observed. Growth of H. pylori at pH 5.9 was poor, and susceptibility to amoxycillin, erythromycin and clarithromycin was markedly less than at pH 7.2 and 7.9. The bactericidal activities of metronidazole and tetracycline were similar at the different pH values studied. At neutral pH the killing rates of amoxycillin and clarithromycin were growth rate dependent. Susceptibility to metronidazole was enhanced in stationary cultures. The interaction obtained between the proton pump inhibitor, omeprazole, and amoxycillin at pH 7 was of additive type. These results suggest that pH and growth conditions may be important in the antibacterial efficacy of different antibiotics in vivo and also provide a possible explanation for the potentiating effect of omeprazole with antibiotics in the treatment of H. pylori infections.

  7. Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains: Evidence of a New Pathway of d-Alanine Biosynthesis.

    Science.gov (United States)

    Marshall, Darrell D; Halouska, Steven; Zinniel, Denise K; Fenton, Robert J; Kenealy, Katie; Chahal, Harpreet K; Rathnaiah, Govardhan; Barletta, Raúl G; Powers, Robert

    2017-03-03

    In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc(2)155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc(2)155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.

  8. Isolation of a novel mutant from Bacillus subtilis natto.

    Science.gov (United States)

    Yoshida, Kazuo

    2006-01-01

    For the construction of strains with full probiotics function in intestines, deoxycholate resistant mutants were isolated from Bacillus subtilis natto. The partial characterization of the mutants was carried out and described.

  9. Construction and Identification of omp31-Deleted Mutant of Brucella Standard Strain 16M%布鲁氏菌16M△omp31基因缺失株的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    王慧; 张亚丽; 王远志; 陈创夫; 任晓丽

    2013-01-01

    To construct the omp31 deletion mutant of Brucella melitensis 16M,the upstream and downstream of the omp31 gene and SacB gene were amplified by PCR from Brucella melitensis 16M and Bacillus subtilis. After constructing omp31-SacB re-combinant fragment into plasmid 18-T simple vector, the suicide plasmid pGEM-7zf+-△omp31-SacB was further obtained and transformed into Brucella melitensis 16M by electroporation. The Aomp31 mutant strain was screened out by homologous recombination and its stability was detected by continuous bacteria culture. The results showed that Brucella melitensis 16M △omp31 mutant strain was successfully generated and reversion was not observed in 15 generations. This research lays a foundation of further study on the anti-apoptosis mechanism and construction of new types of vaccines of Brucella.%为了构建布鲁氏菌16M△omp31基因缺失株,采用PCR方法分别从亲本株16 M上扩增omp31基因的侧翼看序列及枯草芽孢杆菌SacB基因,并将所得片段与pMD18-T载体相连并测序,利用双酶切的方法分别将其连入自杀载体pGEM-7zf+,获得亚克隆pGEM-7zf+-△omp31-SacB.将所构建好的自杀载体通过电转化入布鲁氏菌16M感受态细胞中,经2次同源重组后筛选出16M△omp31基因缺失株,并对获得缺失株进行遗传稳定性检测.结果显示:成功获得布鲁氏菌16 M△omp31基因缺失株,该缺失株在15代内未发生回复性突变.本研究为今后研究布鲁氏菌抗凋亡机制奠定基础.

  10. Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli.

    Science.gov (United States)

    Lou, Hubing; Chen, Min; Black, Susan S; Bushell, Simon R; Ceccarelli, Matteo; Mach, Tivadar; Beis, Konstantinos; Low, Alison S; Bamford, Victoria A; Booth, Ian R; Bayley, Hagan; Naismith, James H

    2011-01-01

    Antibiotic-resistant bacteria, particularly gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.

  11. Altered Antibiotic Transport in OmpC Mutants Isolated from a Series of Clinical Strains of Multi-Drug Resistant E. coli

    Science.gov (United States)

    Ceccarelli, Matteo; Mach, Tivadar; Beis, Konstantinos; Low, Alison S.; Bamford, Victoria A.; Booth, Ian R.; Bayley, Hagan; Naismith, James H.

    2011-01-01

    Antibiotic-resistant bacteria, particularly Gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane. PMID:22053181

  12. Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli.

    Directory of Open Access Journals (Sweden)

    Hubing Lou

    Full Text Available Antibiotic-resistant bacteria, particularly gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate. We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.

  13. 念珠菌类细菌样变异株生物学性状及遗传学特征研究%Study on Biological Characters and Genetic Characteristics of Oidiomycetes Mutant Strains Like Bacterial Morphology

    Institute of Scientific and Technical Information of China (English)

    王华; 苍金荣; 任健康; 苏宝凤; 归巧娣; 张利侠; 刘文康; 闫福堂; 刘英

    2015-01-01

    Objective To investigate the changes of the morphology,structure and biological characters of mutated Candida and through its genetic characteristics,research and reveal the mechanism of the variation at the molecular level.Methods Used different nutritional conditions,different growth conditions and different azole antifungal agents to induce mutation of the standard strains of Candida albicans.In clinical study,Candida mutant strains was isolated from vaginal secretions,pleu-ral effusion and gastric juice samples in patients of poor effect with Antifungal therapy,and studied on the morphological characteristics,and the nuclear structure,the biochemical reaction,the drug resistance,the bacterial composition and the ge-netic characteristics of above variants,etc.Results Mycelial?morphology:Candida were prone to mutation like bacteria, mutant bacteria could show G+ Aureus shape,G+ Bacillus,G+ long filamentous,G- Aureus shape,G- Bacillus and G- long filamentous;Nuclear structure:Candida mutant strains changed like prokaryotes under the electron microscope because it lost the original structure of eukaryotic cells.Biochemical reaction:there were 5 different items in 20 biochemical test ob-served.Drug sensitivity test:Candida mutated to antifungal drugs being originally sensitive was completely resistant,sensi-tive and resistant originally was completely sensitive,and the same as ordinary bacteria resistant.The cell component chan-ges:there was significantly different in Candida variant strain and the atavism of variant strain identified by mass spectrome-try.The most conservative fungalgene expression:Candida mutated had conservative gene expression of eukaryotes.It could be demonstrated that oidiomycetes mutant strains like bacterial morphology with prokaryotic cell biological characteristics was derived from Candida with eukaryotic cells.Conclusion Candida was prone to variation like bacterial morphology.The biological characteristics of mutant resembled prokaryote

  14. The live attenuated Actinobacillus pleuropneumoniae triple-deletion mutant ΔapxIC ΔapxIIC ΔapxIV-ORF1 strain, SLW05, Immunizes pigs against lethal challenge with Haemophilus parasuis.

    Science.gov (United States)

    Fu, Shulin; Ou, Jiwen; Zhang, Minmin; Xu, Juan; Liu, Huazhen; Liu, Jinlin; Yuan, Fangyan; Chen, Huanchun; Bei, Weicheng

    2013-02-01

    Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (ΔapxIC ΔapxIIC ΔapxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-γ) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine.

  15. A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain.

    Science.gov (United States)

    Gutgsell, N S; Del Campo, M; Raychaudhuri, S; Ofengand, J

    2001-07-01

    This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.

  16. 单核细胞增生性李斯特菌prfA基因缺失株的构建及其生物学特性%Construction and characterization of Listeria monocytogenes ΔprfA mutant strains

    Institute of Scientific and Technical Information of China (English)

    白春光; 殷月兰; 贾艳艳; 付红; 高云飞; 焦新安

    2011-01-01

    [目的]单核细胞增生性李斯特菌(Lm)是人兽共患李斯特菌病的病原菌,其致病性与调控因子PrfA蛋白作用下毒力基因的表达有着密切关系,本文初步探讨了PrfA蛋白对细菌毒力因子的调控作用.[方法]利用同源重组技术对血清型分别为1/2a和4b的LM4、F4636进行prfA基因的敲除,并构建其回复突变株,对获得的突变株LM4ΔprfA、F4636ΔprfA进行生物学特性研究.[结果]实验结果表明:两株缺失株的溶血活性丧失、回复突变株的溶血活性得到恢复,突变株还丧失磷脂酶活性,黏附和侵袭特性显著下降(P<0.05),对BALB/c小鼠的半数致死剂量提高了105个数量级.[结论]由此表明,PrfA蛋白对hly、plcB、inl家族基因的表达及细菌毒力具有重要的调控作用.prfA基因缺失株的构建为进一步研究PrfA蛋白的调控功能提供了材料,为研究其在Lm致病性中的作用奠定了基础.%[Objective] Listeria monocytogenes ( Lm) is an important pathogen that can cause serious listeriosis in humans and animals. The pathogenicity of Lm has a close relationship with the PrfA protein regulating the expression of virulence genes. Therefore, we studied the regulation functions of PrfA and its role on Lm's virulence. [Methods] The prfA genes of LM4, serotype l/2a, and F4636, serotype 4b, were deleted by homologous recombination technology, and the biological characteristics of the mutants were further studied. [ Results ] The prfA gene deleted strains LM4△prfA and F4636△prfA and their back mutation strains were successfully constructed. The results show that the hemolysis activity was lost in prfA deleted strains and was recovered in the reverse mutant strains. The prfA deleted strains lost phospholipase activity; their adhesion and invasion ability significantly decreased. Furthermore, their 50% lethal doses ( LD50) were 5 logs higher comparing with wild type strains. [Conclusion] PrfA regulates hly, plcB and inl gene

  17. Construction and characterization of double mutant strain hif-1(ia04);glb-13(tm2825)in C.elegans%秀丽隐杆线虫双突变株系hif-1(ia04);glb-13(tm2825)的制备与鉴定

    Institute of Scientific and Technical Information of China (English)

    石锦平; 任长虹; 李媛; 张成岗

    2011-01-01

    Objective To investigate the physiological function of the glb-13 gene, to backcross glb-13 (tm2825) and to construct double mutant strain hif-1 (ia04) ;g lb-13 (tm2825) in Caenorhabditis elegans. Methods The glb-13 (tm2825) mutant male strain was obtained by mating the glb-13 (tm2825) mutant hermaphrodite with the male N2 strain, and then with the N2 hermaphrodite. It was then backcrossed twice, and the homozygote was identified by single C. elegans genome PCR. The male hif-1(ia04) mutant strain, which was obtained by mating the hif-l(ia04) mutant hermaphrodite with wildtype N2 strain, was further mated with the glb-13(tm2825) mutant hermaphrodite. The homozygote was also identified by single C. elegans genome PCR. Results The backcrossed mutant strain glb-13 ( tm2825 ) and the homozygotes hif-1 ( ia04 ) ;glb-13(tm2825) double mutant were obtained. Conclusion Backcross can maintain the mutant's genetic stability while mutant mating can construct a double mutant. This finding will facilitate further functional studies of the glb-13 gene.%目的 以秀丽隐杆线虫为模式生物研究珠蛋白(globin)13(lgb-13)的生理作用,以回交2次的glb-13(tm2825)突变株系和hif-1(ia04)构建线虫双突变株系hif-1(ia04);glb-13(tm2825).方法 glb-13(tm2825)与野生型N2株系交配获得glb-13(tm2825)的雄虫,再与N2的雌雄同体线虫进行交配,回交2次后,通过单只线虫基因组PCR法鉴定出交配后的纯合体.使用hif-1(ia04)和野生型线虫N2进行交配得到hif-1(ia04)的雄虫,然后再与glb-13(tm2825)雌雄同体线虫交配,通过单只线虫基因组PCR法鉴定出交配后的纯合体.结果 获得hif-1(ia04);glb-13(tm2S25)双突变株系.结论 通过线虫交配回交可以使突变株系线虫保持稳定性状,而突变株系之间的交配可以制备出双突变株系,为深入开展glb-13的功能研究奠定了物质基础.

  18. Methods of producing protoporphyrin IX and bacterial mutants therefor

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  19. Construction and characterization of the cAMP receptor protein gene deletion mutant of Salmonella typhimurium SL1344 strain%鼠伤寒沙门菌SL1344株cAMP受体蛋白基因缺失株的构建及其生物学特性

    Institute of Scientific and Technical Information of China (English)

    廖成水; 程相朝; 赵战勤; 张春杰; 李银聚; 吴庭才; 郁川; 王晓利; 胡阿勇

    2011-01-01

    The cAMP receptor protein gene(crp) deletion mutant of Salmonella typhimurium SL1344 strain was constructed by the allelic exchange introduced by the transduction of suicide plasmid.In addition,the biological characteristics of the mutant were determined.Firstly,the upstream and downstream fragments of crp gene were amplified from SL1344 strain genome.The two fragments were successively cloned into the suicide pRE112 vector to construct the recombinant suicide vector pREΔcrp harboring the 321 bp-deleted crp fragment.The recombination suicide vector was conjugated with SL1344 and the unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR.The serotype of the mutant was 1,4,5,12:i:1,2,identical to the parent SL1344.The mutant was stable with the recombinant Δcrp gene in vitro.However,the carbohydrate fermentation or utilization assays of the mutant were differed from the parent SL1344 strain,obviously.The growth velocity of the mutant was more slowly compared with SL1344.The chicken lethal test showed that the virulence of the SL1344 Δcrp mutant strain with LD50 of 7.40×109 CFU was 32 456 times lower than the parent SL1344 strain with LD50 of 2.28×105 CFU.These results showed that the SL1344 Δcrp mutant was constructed successfully.It is likely that this Δcrp mutant could be adapted to develop attenuated Salmonella vaccine.%通过自杀性质粒介导的细菌同源重组技术,构建鼠伤寒沙门菌SL1344株的crp基因缺失疫苗候选菌株,并对其生物学特性进行初步研究。首先构建含缺失321bp crp基因的重组自杀性质粒pREΔcrp,然后利用重组自杀性质粒介导的等位基因交换技术,两步法筛选SL1344的Δcrp缺失株,用PCR鉴定结果表明该缺失株构建成功。生物学特性研究发现,缺失株ΔcrpSL1344保留了亲本菌株SL1344的血清型1,4,5,12:i:1,2,且能够稳定遗传缺失321bp的crp

  20. Behavioral characterization of system xc- mutant mice.

    Science.gov (United States)

    McCullagh, Elizabeth A; Featherstone, David E

    2014-05-15

    The slc7a11 gene encodes xCT, an essential component of 'system xc-', a plasma membrane exchanger that imports cystine and exports glutamate. Slc7a11 is expressed primarily in the brain, but its role there is not clear. We performed behavioral tests on two different strains of homozygous slc7a11 mutant mice ('sut' and 'xCT'), as well as heteroallelic offspring of these two strains ('xCT/sut') and their associated genetic backgrounds. Homozygous sut mutant males showed reduced spontaneous alternation in spontaneous alternation tasks as well as reduced movement in an open field maze, but xCT and xCT/sut strains did not show significant changes in these tasks compared to appropriate controls. Neither xCT nor sut mutants showed differences from controls in rotarod tests. Female behavioral phenotypes were independent of estrus cycle stage. To ensure that homozygous xCT, sut, and xCT/sut strains all represent protein null alleles, we measured whole brain xCT protein levels using immunoblots. xCT, sut and xCT/sut strains showed no detectable xCT protein expression, confirming them as null alleles. Previously published microdialysis experiments showed reduced striatal glutamate in xCT mutants. Using the same methods, we measured reduced interstitial glutamate levels in the striatum but not cerebellum of sut mutants. However, we detected no glutamate change in the striatum or cerebellum of sut/xCT mice. We detected no changes in whole brain EAAT-1, -2, or -3 expression. We conclude that the behavioral and chemical differences exist between slc7a11 mutant strains, but we were unable to definitively attribute any of these differences to loss of system xc-.

  1. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.;

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosine......-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5...... phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5...

  2. Selection and characterization of L-ethionine resistant mutants of Trichosporon cutaneum.

    Science.gov (United States)

    Georgieva, Nelly; Alexieva, Zlatka

    2005-01-01

    Trichosporon cutaneum R57 and its L-ethionine resistant mutant NZ94 strain were investigated. The amino acid analyses of cell content of both strains were carried out. The pool of free methionine in the mutant strain is enhanced 16.5 times. The total amount of sulphur-containing amino acids in the mutant cells was significantly increased from 36.8 in the wild strain to 113.4 mg/g protein in the mutant strain. In the process of mutant strain cultivation there was found a high excretion of free methionine (259 microg/ml) in the medium. It was shown that the amino acid content of both wild and mutant strains would be helpful for formulating of new improved animal nutritional diets.

  3. Characterization of Glutamine-Requiring Mutants of Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Janssen, Dick B.; Joosten, Han M.L.J.; Herst, Patricia M.; Drift, Chris van der

    1982-01-01

    Revertants were isolated from a glutamine-requiring mutant of Pseudomonas aeruginosa PAO. One strain showed thermosensitive glutamine requirement and formed thermolabile glutamine synthetase, suggesting the presence of a mutation in the structural gene for glutamine synthetase. The mutation conferri

  4. Study on culturing Trichodema mutants

    Institute of Scientific and Technical Information of China (English)

    CHEN Jian-ai; WANG Wei-ming

    2004-01-01

    @@ Trichodema mutants strains T5, T0803, T1010, T1003were cultured in different conditions and media, also in the presence of fungicides at 40 mg/kg (CK or procymidone + chlorothalonil, or maneb or phosethyl-Al) . The pH values of media were 5, 6, 7 and 8 and hyphae were grown at temperatures of 15, 20, 25 and 30 ℃. After being cultured for 3, 4, 5, or 6 days, the strains were transferred at a lower temperature to sporulate (20℃) Obtained data were analyzed statistically, with the orthogonal array and ranges (R) differing dependes on the treatments (R = 40.0,42.4, 48.0, 62.8,107.0). The results indicated that the most important factor was the nature of the strain (R =107.0), while the change in temperature and time of cultivation produced the lowest effect (R =40.0). Each factor variance was significant and A3B4C2D1E3 was the optimum combined condition, in which strain T1010 grew more quickly and sporulated most.

  5. Comparison of the X-gal- and P-gal-based systems for screening of mutant λlacZ phages originating from the transgenic mouse strain 40.6

    NARCIS (Netherlands)

    Mientjes, E.J.; Steenwinkel, M.J.S.T.; Delft, J.H.M. van; Lohman, P.H.M.; Baan, R.A.

    1996-01-01

    The recent introduction of the phenyl-β-D-galactopyranoside (P-gal)-based positive-selection system for screening of λlacZ phages originating from the λlacZ transgenic mouse (Muta Mouse) has made the determination of mutant frequencies (MF) a much simpler task. Previously, MF data from these mice ha

  6. Repair effects of laser on mutants of filamentous fungi

    Science.gov (United States)

    Zhao, Yansheng; Xiao, Canpeng; Qian, Hailun; Su, Baoliang; Hu, Yujun; Deng, Jianhui

    1999-09-01

    The paper reports that penicillin-producing strains and lovastatin-producing strains were irradiated by UV and subsequently by laser (632.8 nm), and the reparation rate reached 297% and 264%. High-yield mutant was selected with improved potency of 24.5% and 30%, respectively; Gibberellin producing strains were treated with chemical agent LiCl, and then irradiated with 632.8 nm laser. One mutant with 189.6% increased potency was obtained. The experimental results indicated that using laser irradiation after UV or chemical agent mutation was a new useful method in breeding high-yield strains.

  7. Phosphoribosylpyrophosphate (PRPP)-less mutants of Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1989-01-01

    R and prs-4::KanR were obtained. These strains were fully viable, but required guanosine, uridine, histidine, tryptophan and nicotinamide mononucleotide. There was no phosphoribosylpyrophosphate synthetase activity or phosphoribosylpyrophosphate pool in the mutant strains. These results show...

  8. Construction of a mutant strain of Streptococcus mutans with clpC-deletion to study the role of clpC ;gene in genetic competence%clpC 基因对变异链球菌感受态形成的影响

    Institute of Scientific and Technical Information of China (English)

    徐巧丽; 饶慧华; 马晓波; 黄朝阳; 郑港森; 张加勤; 宋秀宇

    2015-01-01

    目的:构建变异链球菌clpC缺陷突变株,检测该基因对变异链球菌感受态形成的影响。方法分别以变异链球菌UA159基因组和pIB107质粒为模板,PCR扩增clpC基因片段和卡那霉素基因盒(lox71-KMR-lox66);将clpC基因片段插入pMD-19T simple载体,经ClaⅠ/EcoRⅠ酶切、补平后连入卡那霉素基因盒,构建clpC缺陷突变同源重组载体pCKX2;SalⅠ线性化pCKX2并转化变异链球菌,卡那霉素筛选阳性菌落;质粒pCrePA转化阳性菌株,30℃培养剔除卡那霉素基因盒;37℃培养去除pCrePA,获得clpC缺陷突变株,PCR和测序鉴定;提取细菌总RNA并逆转录成cDNA,用RT-PCR法扩增clpC缺失序列的核苷酸片段并进行产物的电泳分析;pDL276分别转化变异链球菌和clpC缺陷突变株,观察感受态细胞形成变化。结果 PCR和测序结果证实成功构建同源重组载体pCKX2及变异链球菌clpC缺陷突变株;RT-PCR结果显示,△clpC缺失的核苷酸片段PCR产物电泳结果并无相应的条带出现;clpC缺陷突变株的感受态形成期延迟并延长维持期。结论 clpC基因具有负调控变异链球菌晚期感受态细胞形成的作用。%Objective To construct a mutant strain of Streptococcus mutans ( S.mutans ) with clpC-deletion and to investigate the role of clpC gene in genetic competence.Methods The fragment of clpC gene and the kanamycin resistant cassette flanked by two loxP sites were amplified by PCR.The purified fragment of clpC gene was cloned into pMD-19T simple vector to construct pCKX1.The pCKX1 vector was digested with ClaⅠ/EcoRⅠ, then blunted and introduced into lox71-KMR-lox66 to obtain pCKX2 vector via homologous recombination.The pCKX2 vector was linearized with SalⅠ and transformed into S.mutans UA159 strain.The positive strains constructed via homologous recombination were screened with kanamycin and transformed with the thermosensitive plasmid pCrePA.The KMR

  9. New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA

    DEFF Research Database (Denmark)

    Asklund, Marlene; Atlung, Tove

    2004-01-01

    an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection...

  10. Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity

    Energy Technology Data Exchange (ETDEWEB)

    Upchurch, R.G.; Walker, D.C.; Rollins, J.A.; Ehrenshaft, M.; Daub, M.E. (North Carolina State Univ., Raleigh (United States))

    1991-10-01

    The authors have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

  11. Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity.

    Science.gov (United States)

    Upchurch, R G; Walker, D C; Rollins, J A; Ehrenshaft, M; Daub, M E

    1991-10-01

    We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

  12. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    From an Escherichia coli purine auxotroph a mutant defective in phosphoribosylpyrophosphate (PRib-PP) synthetase has been isolated and partially characterized. In contrast to the parental strain, the mutant was able to grow on nucleosides as purine source, whereas growth on purine bases was reduced......, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

  13. Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization.

    Science.gov (United States)

    Bellemann, P; Geider, K

    1992-05-01

    Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

  14. Spontaneous Nif- mutants of Rhodopseudomonas capsulata.

    OpenAIRE

    Wall, J D; Love, J.; Quinn, S P

    1984-01-01

    Revertible, spontaneous Nif- mutants of Rhodopseudomonas capsulata have been shown to accumulate in cultures growing photosynthetically with an amino acid as the nitrogen source such that H2 is maximally produced. The majority of such strains carry mutations which are clustered in a short region of the chromosome, probably representing one or two genes. Because this cluster includes temperature-sensitive mutations, it is also likely that it identifies the structural gene of a polypeptide. The...

  15. Comparative production of cellulases by mutants of Trichoderma parceramosume PTCC5140

    Directory of Open Access Journals (Sweden)

    Hoda Nouri

    2017-06-01

    Discussion and conclusion: Evaluation of cellulase production in mutant strains of Trichoderma parceramosume PTCC 5140 showed that use of chemical mutagenesis with 2 to 11 fold increasing in enzyme activity is a potent method to improve cellulase complex activity. In the current study, obtained mutant strains could be introduced as a potent cellulase producer for further studies in bioconversion processes.

  16. Phanerochaete mutants with enhanced ligninolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  17. Isolation and characterization of Streptococcus mutans mutants defective in adherence and aggregation.

    OpenAIRE

    Murchison, H; Larrimore, S; Curtiss, R

    1981-01-01

    A method was developed which enriched for mutants of Streptococcus mutans that exhibit defects in adherence to glass, aggregation, or both. Mutants were isolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) after mutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survival after mutagenesis was kept above 1 to 2% to enhance the probability that mutants resulted from single mutational events. A total of 117 mutants were isolated; they also displayed non...

  18. Selection of a recombinant Marek's disease virus in vivo through expression of the Marek's EcoRI-Q (Meq)-encoded oncoprotein: characterization of an rMd5-based mutant expressing the Meq of strain RB-1B.

    Science.gov (United States)

    Kumar, Pankaj; Dong, Huimin; Lenihan, Dawn; Gaddamanugu, Syamsundar; Katneni, Upendra; Shaikh, Shireen; Tavlarides-Hontz, Phaedra; Reddy, Sanjay M; Peters, Wachen; Parcells, Mark S

    2012-06-01

    Marek's disease (MD) is a highly contagious viral disease of chickens (Gallus gallus domesticus) caused by MD virus (MDV), characterized by paralysis, neurologic signs, and the rapid onset of T-cell lymphomas. MDV-induced T-cell transformation requires a basic leucine zipper protein called Marek's EcoRI-Q-encoded protein (Meq). We have identified mutations in the coding sequence of Meq that correlated with virus pathotype (virulent, very virulent, and very virulent plus). The aim of this study was to determine whether recombinant viruses could be isolated based on Meq expression through in vivo selection. Chicken embryo fibroblasts (CEFs) were cotransfected with an rMd5 strain-based Meq deletion virus (rMd5deltaMeq) and meq loci from strains representing different pathotypes of MDV. Transfected CEFs were inoculated into chickens in two independent studies. We were able to isolate a single recombinant virus, rMDV-1137, in a contact-exposed chicken. rMDV-1137 had recombined two copies of the meq gene of RB-1B and was found to have pathogenicity similar to both RB-1B and rMd5 parental strains. We found the RB-1B- and rMd5-induced lymphomas showed differences in composition and that rMDV-1137-induced lymphomas were intermediate in their composition. We were able to establish cell lines from both RB-1B- (MDCC-UD35, -UD37) and rMDV-1137 (MDCC-UD36, -UD38)-induced, but not rMd5-induced, lymphomas. To date, no rMd5- or parent Md5-transformed T-cell lines have been reported. Our results suggest that 1) a recombinant MDV can be selected on the basis of oncogenicity; 2) changes in Meq sequence seem to affect tumor composition and the ability to establish cell lines; and 3) in addition to meq, other genomic loci affect MDV pathogenicity and oncogenicity.

  19. 炭疽芽孢杆菌A16R株lysA基因缺失突变株的构建%Construction of lysA deletion mutant of Bacillus anthracis vaccine strain A16R

    Institute of Scientific and Technical Information of China (English)

    高飞; 王东澍; 冯尔玲; 朱力; 王恒樑; 廖祥儒; 刘先凯

    2013-01-01

    Objective To construct the lysA site-deleted mutagenesis of Bacillus anthracis vaccine strain A16R in order to provide scientific reference for subsequent study on quantitative proteomics. Methods Using lysA Site-deleted mutagenesis as the target gene, software was used to design primers of upstream and downstream of lysA and antibiotic resistance genes. The recombinant plasmid was constructed by inserting three fragments into the vector and electroporated into competence A16R cells. Finally, A16R mutagenesis strain was screened and verified. Growth curves of the mutagenesis strain and wild strain were drawn, and physiological and biochemical characteristics were analyzed. Result and Conclusion lysA Site-deleted mutagenesis is obtained, contributing to quantitative proteomics research and establishing a good technical platform for functional genomics research of B. anthracis.%目的 构建炭疽芽孢杆菌(Bacillus anthracis)A16R株lysA基因缺失突变株,为后续的定量蛋白质组学研究奠定基础.方法 以炭疽杆菌活疫苗A16R株lysA基因为目的缺失基因,利用软件设计上下游同源臂以及抗性基因的引物,用同源重组酶将3个片段连入质粒中,构建重组质粒,并将重组质粒导入炭疽杆菌A16R感受态细胞中,筛选炭疽杆菌A16R株lysA基因缺失突变株,对其进行验证.最后绘制缺失突变株和野生株生长曲线并进行生理生化分析.结果 成功构建了重组质粒,经同源重组后获得lysA基因缺失突变株.鉴定表明目的基因已经丢失.结论 成功获得炭疽杆菌A16R株lysA基因缺失突变株,为定量蛋白质组学研究奠定了基础,也为炭疽杆菌重要基因功能的研究建立了良好的技术平台.

  20. Phanerochaete mutants with enhanced ligninolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Kakar, S.N.; Perez, A.; Gonzales, J. (Argonne National Lab., IL (United States))

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Most of the organic compounds are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated, or are hyperproducers or supersectors of key enzymes under enriched conditions. Through UV-light and [gamma]-ray mutagenesis, the authors have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants, 76UV, produced 272 U of lignin peroxidases enzyme activity/L after 9 d under high nitrogen (although the parent strain does not produce this enzyme under these conditions). The mutant and the parent strains produced up to 54 and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments, the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 d. 17 refs., 1 fig., 3 tabs.

  1. Construction and characterization of invasion protein B gene deleted mutant of Salmonella typhimurium SL1344 strain%鼠伤寒沙门菌SL1344株侵袭性蛋白B缺失株的构建及生物学特性

    Institute of Scientific and Technical Information of China (English)

    陈松彪; 陈桂华; 赵战勤; 李静; 郁川; 何雷; 张春杰; 程相朝; 李银聚; 颜云飞; 金修哲

    2015-01-01

    Objective:In order to develop a safer vaccine strain exploit Salmonella typhimurium vaccine strain .A ΔsipB mutant of Salmonella typhimurium SL 1344 strain was constructed.Methods: Firstly, the recombinant suicide plasmid containing the missing 585 bp sipB ( PREΔsipB ) was built by homologous recombination , and screenned by two-step method.Results: PCR and sequencing results showed that SL 1344ΔsipB was successfully constructed.It was no significant changes compared with SL 1344.But compared with the parent strains SL 1344 , the mutant strain had obvious change in its virulence , oral challenge of bacteria in mice revealed that LD50 of the mutant strain was 1.70 ×108 CFU,the toxicity reduced about 1.4%.The protection rate induced by the sipB mutant was 50%,and the serum antibody peaked 14 d post-immunization.Conclusion:The SL1344ΔsipB mutant was constructed suc-cessfully,and genetic stability ,significantly reduced virulence.The study provides a new approach for further study of the relationship between the gene and pathogenicity of Salmonella typhimurium.It is likely that the ΔsipB mutant could be adapted to develope attenuated Salmonella vaccine.%目的:为了研制更加安全的鼠伤寒沙门菌弱毒株,本研究构建了鼠伤寒沙门菌SL1344ΔsipB基因缺失突变株。方法:首先构建含缺失585 bp sipB基因的重组自杀性质粒PREΔsipB,利用重组自杀性质粒介导的等位基因交换技术,两步法筛选出SL1344的sipB缺失株。结果:PCR及测序结果表明SL1344ΔsipB构建成功。进一步生物学特性研究表明,缺失株SL1344ΔsipB保留了亲本菌株SL1344的血清型1,4,5,12:i:1,2,且能够稳定遗传缺失585 bp的sipB基因,生长速度没有发生明显改变;但是,与亲本株SL1344相比,其毒力发生明显改变,缺失株SL1344ΔsipB口服感染6周龄BALB/c小鼠的LD50为1.70×108 CFU,毒力较亲本株SL1344降低至1.4%,免疫

  2. Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene.

    Directory of Open Access Journals (Sweden)

    Cristina W Cunha

    Full Text Available Herpes simplex virus 1 (HSV-1 ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture.

  3. Molecular analysis of mutants of the Neurospora adenylosuccinate synthetase locus

    Indian Academy of Sciences (India)

    A. Wiest; A. J. McCarthy; R. Schnittker; K. McCluskey

    2012-08-01

    The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5′ end of the gene and no mutants were identified in the 3′ untranslated region of this gene. We additionally analysed ad-8+ alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.

  4. Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

    Science.gov (United States)

    Gottschalk, Leda Maria Fortes; de Sousa Paredes, Raquel; Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant’Ana; da Silva Bon, Elba Pinto

    2013-01-01

    The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 °C and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture. PMID:24294256

  5. Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

    Directory of Open Access Journals (Sweden)

    Leda Maria Fortes Gottschalk

    2013-01-01

    Full Text Available The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 ºC and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L, which was not detected in the T. reesei culture.

  6. Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1.

    Science.gov (United States)

    Gottschalk, Leda Maria Fortes; de Sousa Paredes, Raquel; Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; da Silva Bon, Elba Pinto

    2013-01-01

    The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 °C and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture.

  7. [Eremothecium ashbyii mutants resistant to 2,6-diaminopurine].

    Science.gov (United States)

    Stepanov, A I; Beburov, M Iu; Zhdanov, V G

    1975-01-01

    3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.

  8. Mutant prevention concentration and mutant selection window for 10 antimicrobial agents against Rhodococcus equi.

    Science.gov (United States)

    Berghaus, Londa J; Giguère, Steeve; Guldbech, Kristen

    2013-10-25

    The objectives of this study were to determine the mutant prevention concentration (MPC), time above the MPC and mutant selection window for 10 antimicrobial agents against Rhodococcus equi and to determine if the combination of a macrolide with rifampin would decrease emergence of resistant mutants. Antimicrobial agents investigated (erythromycin, clarithromycin, azithromycin, rifampin, amikacin, gentamicin, enrofloxacin, vancomycin, imipenem, and doxycycline) were selected based on in vitro activity and frequency of use in foals or people infected with R. equi. Each antimicrobial agent or combination of agents was evaluated against four virulent strains of R. equi. MPC were determined using an agar plate assay. Pharmacodynamic parameters were calculated using published plasma and pulmonary pharmacokinetic variables. There was a significant (Pequi. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Connexin mutants and cataracts

    Directory of Open Access Journals (Sweden)

    Eric C Beyer

    2013-04-01

    Full Text Available The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8 have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death and formation of cytoplasmic accumulations (that may act as light scattering particles. These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.

  10. Cytokinin production by plant growth promoting rhizobacteria and selected mutants.

    Science.gov (United States)

    García de Salamone, I E; Hynes, R K; Nelson, L M

    2001-05-01

    One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G20-18, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G20-18 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G20-18 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 10(-5) M adenine increased cytokinin production in 96- and 168-h cultures of strain G20-18 by approximately 67%. G20-18 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR.

  11. Isolation and characterization of Rhizobium meliloti mutants affected in exopolysaccharide production.

    Science.gov (United States)

    Rodríguez-Navarro, D N; Palomares, A J; Casadesús, J

    1991-06-01

    Rhizobium meliloti mutants affected in the production of exopolysaccharide (EPS) were isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutants were classified into three phenotypic classes: (I) Exo-, rough mutants lacking exopolysaccharide; (II) Exos (for "small") which form tiny, compact colonies and synthesize reduced amounts of EPS; and (III) Exoc (for "constitutive"), hypermucoid mutants which overproduce EPS. Hypermucoid strains showed increased resistance to desiccation. All the mutants were able to nodulate, although a significant decrease in infectivity degree and/or competitiveness was found in rough and compact strains. Two mutants proved to be deficient in nitrogen fixation. Complementation analysis with cloned R. meliloti exo genes could not be applied to the study of these Fix- mutants because introduction of plasmids derived from cosmid vector pLAFR1 caused loss of nodulating ability. However, complementation of calcofluor staining and EPS production was observed. Complementation with certain exo genes also caused a marked increase in motility.

  12. [Isolation of a high hydrogen-producing mutant TB34 generated by transposon insertion and analysis of hydrogen production].

    Science.gov (United States)

    Liu, Hong-Yan; Wang, Guang-Ce; Shi, Liu-Yang; Zhu, Da-Ling

    2012-07-01

    To increase the hydrogen-producing capacity of Pantoea agglomerans BH18, isolated from mangrove sludge, we constructed a stable transposon mutagenesis library of this strain. A Tn7-based transposon was randomly inserted into the genomic DNA. Mutants were screened by kanamycin resistance and identified by amplification of the inserted transposon sequences. A mutant strain TB34 was isolated, whose hydrogen production capacity was significantly improved compared to the wild type strain. In seawater-containing medium supplemented with 10 g x L(-1) glucose and had an initial pH of 7.0, the hydrogen yield (H2/glucose) of the mutant strain was (2.04 +/- 0.04) mol x mol(-1), which was 43% higher than that of the wild type strain. The mutant TB34 showed steady hydrogen production capacity for five consecutive passages. Different carbon sources were tested in the hydrogen production by the mutant TB34 and the results showed that both the mutant strain TB34 and the wild type strain BH18 were able to produce hydrogen on sucrose, glucose and fructose. However, different from the wild type strain, the mutant strain TB34 was also able to produce hydrogen using xylose as substrate, with a hydrogen yield (H2/xylose) of (1.34 +/- 0.09) mol x mol(-1), indicating a broader substrate spectrum in the mutant.

  13. Characterization of a Mutant of Alteromonas aurantia A18 and Its Application in Mariculture

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Mutant J61321 with enhanced siderophore production of Alteromonas aurantia A18 was obtained after a series of chemical-physical mutageneses. It was found that the antibacterial activity against Vibrio anguillarum W-1 and siderophore production of the mutant were higher than those by the original strain A18 which had been used in mariculture. The results of the specific J61321 and the original strain A18, respectively, while the siderophore with catechol group was yielded by strain W-1 (Aibrio anguillarum). Meanwhile, the siderophore yield, antibacterial activity and anti-chelator activity of strain J61321 were higher than those of its parent strain A18.

  14. Ethanol fermentation on glucose/xylose mixture by co-cultivation of restricted glucose catabolite repressed mutants of Pichia stipitis with respiratory deficient mutants of Saccharomyces cerevisiae.

    Science.gov (United States)

    Kordowska-Wiater, Monika; Targoński, Zdzisław

    2002-01-01

    Restricted glucose catabolite repressed mutants of P. stipiti CCY 39501 were selected using UV irradiation. Four mutants were obtained which assimilated glucose slower than the native strain of P. stipitis and the degree of glucose repression was about 2-fold lower for P5-90-133 and P5-200-16 mutants and about 10-fold lower for P5-80-7 and P5-80-35 mutants. P5-80-7 and P5-80-35 produced very small amounts of ethanol from glucose and xylose, whereas P5-90-133 and P5-200-16 fermented sugars at the wild-type level. These two mutants were selected for co-fermentation process with native strain of S. cerevisiae V30 or Ja(a), as well as with their respiratory deficient mutants. During co-culture process of P. stipitis mutants with native strains of S. cerevisiae the ethanol yields obtained ranged from 0.38 to 0.45 g/g, and this alcohol was produced mainly from glucose. But, when also xylose, besides glucose was fermented to ethanol during co-fermentation of both mutant strains, lower yields of ethanol (0.28-0.40 g/g) were obtained.

  15. Secretos de Mutantes

    OpenAIRE

    Marín, Martha; Muñoz, Germán; Serrano, Rafael

    2017-01-01

    Apartándose de enfoques que consideran las culturas juveniles como ‘desviaciones sociales', ‘tribus urbanas' o ‘nuevos movimientos políticos', Secretos de mutantes bucea en culturas juveniles urbanas como la Skinhead, el Punk, el Metal, el Hardcore, el Grunge y el Hip Hop, explorándolas desde un punto de vista inédito: su dimensión de creación, para percibir los cruciales y casi desconocidos procesos que sus miembros llevan a cabo en estos vastos universos de experimentación. Esta obra se nut...

  16. 人egf基因在突变枯草芽孢杆菌WYBS2001中的转化、分泌表达及其产物功能研究%Transforming and Secreting Expression of Human egf in Mutant Strain WYBS2001 of Bacillas and Its Functions

    Institute of Scientific and Technical Information of China (English)

    王关林; 易庆; 方宏筠

    2003-01-01

    通过紫外线突变诱导获得了对感染致病菌具有显著抑菌作用的枯草芽孢杆菌突变株WYBS2001.采用PCR技术人工合成了175 bp的人表皮生长因子(hEGF)的基因片段,并引入Pst Ⅰ、HindⅢ酶切位点、起始密码子及pUS186载体信号肽序列CTTAGA.经DNA测序分析,合成的片段与人egf基因序列完全一致.然后将其克隆至枯草杆菌分泌型质粒载体pUS186上构建成重组质粒pUSE,并转化枯草杆菌突变菌株WYBS2001,获得转人egf基因的枯草杆菌生态工程菌WYBS2001T.RIA检测结果表明,WYBS2001T阳性工程菌培养的上清液中可检测到hEGF,含量为7.6 ng/ml.如果培养液中添加蛋白酶抑制剂可提高hEGF检测量.通过多代的培养仍然能够稳定地分泌表达hEGF.生物学功能实验表明,分泌的hEGF对人K562体外培养细胞的增殖和生长具有明显生物学活性.对烧伤动物模型的功能性实验观察到,WYBS2001T工程菌制剂对动物的烧伤有明显的治疗作用.该研究表明,微生态基因工程菌有很好的应用前景.%Mutant strain WYBS2001 of B. subtilis with strong anti-pathgenic activity was obtained by mutagenic ultraviolet rays. The gene fragment of Human Epidermal Growth Factor(hegf) of 175 bp was synthesized by PCR and the restriction sites Pst Ⅰ and Hind Ⅲ , original code and the signal sequence CTTAGA of secreting vector pUS186 were induced in the fragment. The DNA sequencing result revealed that the synthesized fragment was identical with that of human egf. Then the biological engineering strain WYBS2001T with human egf was obtained by transforming pUSE which was constructed by cloning egf into the secreting plasmid pUS186, into mutant strain WYBS2001. The result of RIA showed that hEGF can be found in the supernatant of the cultures and its content was 7.6 ng/ml. And the content can be increased if the proteinase inhibitor was added into the medium. After several generations' culturing, WYBS2001T positive

  17. Eficiência e competitividade de variantes espontâneos isolados de estirpes de Bradyrhizobium spp recomendadas para a cultura da soja (Glycine max Effectiveness and competitiveness of spontaneous mutants isolated from Bradyrhizobium spp strains recommended for soybean crop (Glycine max

    Directory of Open Access Journals (Sweden)

    Fabíola Gomes de Carvalho

    2005-12-01

    Full Text Available O cultivo sucessivo de soja inoculada numa mesma área proporcionou a adaptação de uma população de rizóbios, que podem não ser tão eficientes quanto à capacidade de fixação de N2, mas apresentam alta competitividade, dificultando a introdução de novas estirpes mais eficientes. Com a finalidade de avaliar o desempenho simbiótico (eficiência e competitividade de variantes espontâneos isolados de estirpes de B. japonicum (SEMIA 5079 e SEMIA 5080 e B. elkanii (SEMIA 587 e SEMIA 5019, realizou-se um experimento em casa de vegetação onde os variantes foram inoculados isoladamente e em diferentes combinações entre os variantes e uma estirpe comprovadamente mais competitiva (SEMIA 587 ou SEMIA 5019 a partir da adição de inóculos mistos (1/1; v/v no cultivar de soja BR-16. Por meio da avaliação das variáveis analisadas (nodulação, produção de matéria de seca da parte aérea, N total acumulado na parte aérea e ocupação nodular, foi possível constatar que o determinante da maior eficiência em tratamentos co-inoculados não foi a ocupação nodular de determinada estirpe ou variante presente no inóculo, mas, sim, o tipo de interação (sinérgica ou antagônica predominante no tratamento co-inoculado e que é possível selecionar variantes eficientes e competitivos para a cultura da soja a partir de estirpes parentais que já apresentam características desejáveis para utilização em inoculantes comerciais.The continuous cultivation of inoculated soybean in the same area can determine the soil colonization with a rhizobia population presenting low nitrogen fixation effectiveness. This fact can be a problem for the establishment of a more effective population. A greenhouse experiment was carried out to evaluate the symbiotic effectiveness and competitiveness of spontaneous mutants isolated from B. japonicum (SEMIA 5079 and SEMIA 5080 and B. elkanii (SEMIA 587 and SEMIA 5019 strains. The soybean biovar BR 16 was

  18. ECB deacylase mutants

    Science.gov (United States)

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  19. Phenotypic characterization and virulence of a sae- agr- mutant of Staphylococcus aureus.

    Science.gov (United States)

    Giraudo, A T; Rampone, H; Calzolari, A; Nagel, R

    1996-02-01

    A sae::Tn551 agr::tetM double mutant was constructed and characterized. The production of several exoproteins (e.g., beta-hemolysin, DNase, and proteases) by this mutant was determined and found to be lower than the already diminished production of either isogenic single mutant sae- or agr-. The double mutant also showed, like the agr- mutant, null production of alpha- and delta-hemolysins and diminished levels of lipase. The reduced levels of many exoproteins in the double mutant as compared with their already diminished levels in either single mutant suggest that there is an additive or synergistic interaction between the two mutations involved, sae- and agr-. However, inactivation of both loci, sae and agr, had a different effect on the two exoproteins that are up regulated in the agr- mutant; thus, coagulase dropped to levels close to the null levels of the sae- parental strain, while extracellular protein A displayed the high levels characteristic of the agr- single mutant. The virulence of the sae- agr- double mutant, determined by intraperitoneal injection in mice, was found to be significantly diminished as compared with that of the sae+ agr+ parental strain or the sae- agr+ single mutant.

  20. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib....... Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate...

  1. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    . Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate......, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

  2. C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

    Science.gov (United States)

    Kanteti, Rajani; Dhanasingh, Immanuel; El-Hashani, Essam; Riehm, Jacob J; Stricker, Thomas; Nagy, Stanislav; Zaborin, Alexander; Zaborina, Olga; Biron, David; Alverdy, John C; Im, Hae Kyung; Siddiqui, Shahid; Padilla, Pamela A; Salgia, Ravi

    2016-01-01

    We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

  3. Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

    OpenAIRE

    Biel, S W; Biel, A J

    1990-01-01

    A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between...

  4. Investigation into the resistance of lactoperoxidase tolerant Escherichia coli mutants to different forms of oxidative stress.

    Science.gov (United States)

    De Spiegeleer, Philipp; Vanoirbeek, Kristof; Lietaert, Annelies; Sermon, Jan; Aertsen, Abram; Michiels, Chris W

    2005-11-15

    Six lactoperoxidase tolerant Escherichia coli transposon mutants isolated and characterized in an earlier study, and some newly constructed double mutants, were subjected to peroxide, superoxide and hypochlorite stress, and their inactivation was compared to that of the wild type strain MG1655. Knock out mutants of waaQ and waaO, which owed their lactoperoxidase tolerance to an impaired outer membrane permeability due to a reduced porin content, also exhibited higher resistance to hypochlorite, as did a knock-out strain of lrp, encoding a regulatory protein affecting a wide range of cellular functions. Unlike the outer membrane mutants however, the lrp strain was also more resistant to t-butyl hydroperoxide, but more susceptible to the superoxide generating compound plumbagin. Finally, a lactoperoxidase tolerant knock-out strain of ulaA, involved in ascorbic acid uptake, did not show resistance to any of the other oxidants. The possible modes of action of these different oxidants are discussed.

  5. Isolation of Rhizobium phaseoli Tn5-induced mutants with altered expression of cytochrome terminal oxidases o and aa3.

    Science.gov (United States)

    Soberón, M; Membrillo-Hernández, J; Aguilar, G R; Sánchez, F

    1990-01-01

    Two Rhizobium phaseoli mutants affected in cytochrome expression were obtained by Tn5-mob mutagenesis of the wild-type strain (CE3). Mutant strain CFN031 expressed sevenfold less cytochrome o in culture, expressed cytochrome aa3 under microaerophilic culture conditions, in contrast to strain CE3, and was affected in its vegetative growth properties and proliferation inside plant host cells. Mutant CFN037 expressed cytochrome aa3 under microaerophilic culture conditions, while bacteroid development and nitrogen fixation occurred earlier than in strain CE3. Images FIG. 2 PMID:2155209

  6. Direct Selection of Clostridium acetobutylicum Fermentation Mutants by a Proton Suicide Method

    OpenAIRE

    Cueto, Pablo H.; Méndez, Beatriz S.

    1990-01-01

    Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain.

  7. Construction and characterization of a glycoprotein E deletion mutant of bovine herpesvirus type 1.2 strain isolated in Brazil Construção e caracterização de uma amostra de BoHV-1.2 isolada no Brasil com uma deleção no gene da glicoproteína E

    Directory of Open Access Journals (Sweden)

    Ana C. Franco

    2002-09-01

    Full Text Available This paper describes the construction and characterization of a Brazilian strain of bovine herpesvirus type 1.2a (BoHV-1.2a with a deletion of the glycoprotein E (gE gene. The deletion was introduced by co-transfection of a deletion fragment containing the 5´and 3´gE flanking regions and genomic DNA of wild type BoHV-1 into bovine cells. Isolation of gE deletion mutant was performed by immunoperoxidase staining with an anti-gE monoclonal antibody. Viral clones were plaque purified and further examined by restriction endonuclesase digestion and Southern blot hybridization. This gE deletion mutant will be evaluated as a vaccinal virus, in order to determine its potential use for a differential vaccine.Este artigo descreve a construção e caracterização de uma amostra de um herpesvírus bovino tipo 1.2a (BoHV-1.2a que apresenta uma deleção na região genômica que codifica a glicoproteína E (gE. A deleção gênica foi induzida através da co-transfecção de um fragmento de deleção, contendo as regiões 5´e 3´flanqueadoras da gE, com o DNA viral intacto de uma amostra viral isolada de um animal que apresentava doença respiratória. O isolamento do vírus gE negativo (gE- foi realizado com auxílio da técnica de imunoperoxidase em que foi utilizado como anticorpo primário um anticorpo monoclonal anti-gE. O vírus gE- foi purificado e o DNA isolado desta amostra foi examinado através das técnicas de análise por enzimas de restrição e "Southern blot". Esta amostra gE- será avaliada como candidata para compor uma vacina diferencial contra a rinotraqueíte infecciosa dos bovinos.

  8. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    Science.gov (United States)

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

  9. Survival of Brucella abortus aqpX mutant in fresh and ripened cheeses.

    Science.gov (United States)

    Santiago-Rodríguez, María Del Rosario; Díaz-Aparicio, Efrén; Arellano-Reynoso, Beatriz; García-Lobo, Juan M; Gimeno, Miquel; Palomares-Reséndiz, Erika G; Hernández-Castro, Rigoberto

    2015-02-01

    The objective of this work was to evaluate the survival of a Brucella abortus aqpX mutant during the elaboration and conservation of fresh and ripened cheeses at 4 °C and 24 °C. The pH values and water activity were monitored for each type of cheese. The fresh cheese was elaborated with raw milk inoculated with 6×10⁸ colony-forming units (CFU)/mL each of parental and mutant strain. Ripening cheeses were elaborated with both raw and pasteurized milk and inoculated with 12×10⁸ CFU/mL each of parental and mutant strains. In fresh cheese, survival was observed during elaboration and conservation for 7 days at 4 °C in mutant and parental strains. The number of survivors of the mutant strain was 10 times lower compared with the parental strain at pH 5 and a(w) of 0.930. In the cheese elaborated with raw milk and ripened at 24 °C, both strains survived until day 17 at pH 4.0 and a(w) of 0.89. However, when the cheese was elaborated with pasteurized milk, the parental strain survived until day 31 of ripening, and the mutant strain survived 24 days at pH 4 and a(w) of 0.886. The survival of the mutant strain showed a diminution of one logarithm during elaboration and ripening of cheese as compared with the parental strain. When the cheese was elaborated with raw milk and ripened at 4 °C, survival of the parental strain was 24 days, whereas the mutant strain survived only 17 days (pH 5 and a(w) 0.90). Regarding the cheese elaborated with pasteurized milk and maturated at 4 °C, both strains survived 31 days (pH 5 and a(w) 0.90), with the same survival diminution during elaboration and ripening. Our results show that in both types of cheese, the mutated aqpX strain survived 10 times less than the parental strain, which shows that the aqpX gene can be related to the survival of Brucella abortus in this type of cheese.

  10. Isolation and characterization of Escherichia coli mutants lacking inducible cyanase.

    Science.gov (United States)

    Guilloton, M; Karst, F

    1987-03-01

    To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

  11. 雄甾-4-烯-3,17-二酮11α羟化突变株Metarhizium anisopliae M28-203的代谢调控%Metabolic regulation of androst-4-ene-3,17-dione 11α-hydroxylation mutant strain Metarhizium anisopliae M28-203

    Institute of Scientific and Technical Information of China (English)

    叶丽; 周珮; 冯美卿

    2011-01-01

    目的 从营养代谢调控和转化条件控制两个方面入手提高雄甾-4-烯-3,17-二酮(androst-4-ene-3,17-dione,AD)11α羟化突变株Metarhizium anisopliae M28-203转化效率.方法 通过正交试验确定发酵培养基配方,对影响转化的一系列因素如:pH、发酵时间、通气量、底物投料量及溶解性、细胞通透性进行控制,考察以上因素对羟化反应的影响,确定最佳转化条件.结果 该突变株培养基最佳配方为葡萄糖30 g/L、玉米浆20 g/L、蚕蛹粉5 g/L、硫酸铵2.5 g/L、磷酸氢二钾1 g/L、硫酸镁0.5 g/L和硫酸亚铁0.02 g/L.在pH 6.0~6.5、菌龄48~60 h、250 mL摇瓶装液量为30和40 mL、转化时间72 h时能取得最佳转化效果.在底物投料量为2 mg/mL时,采用4%无水乙醇溶解底物或加入0.75 mg/mL Tween 80均有利于羟化反应,在以上优化条件下转化率分别达到62.5%和66.8%.结论 突变株Metarhizium anisopliae M28-203能有效地在AD上引入11α羟基,为工业生产依普利酮及其他甾体药物提供关键中间体.%Objective To improve the transformation efficiency of androst-4-ene-3, 17-dione (AD) llα-hydroxylation mutant strain Metarhizium anisopliae M28-203 through regulations of nutrition metabolism and biotransformation conditions. Methods The optimal fermentation culture was determined by orthogonal test. The effects of pH, fermentation time, air flow, substrate addition method and substrate concentration on hydroxylation were investigated and the optimal transformation condition was constructed. Results The optimal fermentation culture contained glucose 30 g/L, corn steep 20 g/L, silkworm chrysalis powder 5 g/L, (NH4)2SO4 2.5 g/L, K2HPO4 1 g/L, MgSO4 0.5 g/L and FeSO4 0.02 g/L. The optimal transformation condition was pH 6.0 - 6.5, 48 - 60 h mycelium age, 30 and 40 mL medium volume in 250 mL shake flask and 72 hour transformation time. Both 4% ethanol and 0.75 mg/mL Tween 80 were helpful for substrate solubility, and then

  12. Comparative study of the mutant prevention concentrations of moxifloxacin, levofloxacin, and gemifloxacin against pneumococci.

    Science.gov (United States)

    Credito, Kim; Kosowska-Shick, Klaudia; McGhee, Pamela; Pankuch, Glenn A; Appelbaum, Peter C

    2010-02-01

    We tested the propensity of three quinolones to select for resistant Streptococcus pneumoniae mutants by determining the mutant prevention concentration (MPC) against 100 clinical strains, some of which harbored mutations in type II topoisomerases. Compared with levofloxacin and gemifloxacin, moxifloxacin had the lowest number of strains with MPCs above the susceptibility breakpoint (P<0.001), thus representing a lower selective pressure for proliferation of resistant mutants. Only moxifloxacin gave a 50% MPC (MPC50) value (1 microg/ml) within the susceptible range.

  13. Comparative Study of the Mutant Prevention Concentrations of Moxifloxacin, Levofloxacin, and Gemifloxacin against Pneumococci▿ †

    Science.gov (United States)

    Credito, Kim; Kosowska-Shick, Klaudia; McGhee, Pamela; Pankuch, Glenn A.; Appelbaum, Peter C.

    2010-01-01

    We tested the propensity of three quinolones to select for resistant Streptococcus pneumoniae mutants by determining the mutant prevention concentration (MPC) against 100 clinical strains, some of which harbored mutations in type II topoisomerases. Compared with levofloxacin and gemifloxacin, moxifloxacin had the lowest number of strains with MPCs above the susceptibility breakpoint (P < 0.001), thus representing a lower selective pressure for proliferation of resistant mutants. Only moxifloxacin gave a 50% MPC (MPC50) value (1 μg/ml) within the susceptible range. PMID:20008781

  14. Isolation and characterization of stable mutants of Streptomyces peucetius defective in daunorubicin biosynthesis

    Indian Academy of Sciences (India)

    K. S. Vetrivel; K. Dharmalingam

    2001-04-01

    Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, -rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed by dnrE (mutants SPAK and SPMAG), dnrS (SPFS and SPRHO) and doxA (SPDHC) gene products.

  15. The isolation of Staphylococcus aureus tea tree oil-reduced susceptibility mutants.

    Science.gov (United States)

    Cuaron, Jesus A; Dulal, Santosh; Cooke, Peter H; Torres, Nathanial J; Gustafson, John E

    2014-08-01

    Tea tree oil (TTO)-reduced susceptibility (TTORS) mutants of two Staphylococcus aureus laboratory strains were isolated utilizing TTO gradient plates. Attempts to isolate TTORS mutants employing agar plates containing single TTO concentrations failed. All TTORS mutants demonstrated a small colony variant (SCV) phenotype and produced cells with a smaller diameter, as determined by scanning electron microscopy. The addition of SCV auxotrophic supplements to media did not lead to an increase in TTORS mutant colony size. Revertants were also isolated from the TTORS mutants following growth in drug-free media, and all revertant strains demonstrated phenotypes similar to their respective parent strains. Transmission electron microscopy revealed that an SH1000 TTORS mutant demonstrated a thinner cell wall and novel septal invaginations compared with parent strain SH1000. In addition, comparative genomic sequencing did not reveal any mutations in an SH1000 TTORS mutant previously linked to well-characterized SCV genotypes. This study demonstrates that TTO can select for a unique SCV phenotype.

  16. Isolation and characterization of selenate resistant mutants of Acremonium chrysogenum

    Directory of Open Access Journals (Sweden)

    Airton Vialta

    1999-01-01

    Full Text Available Mutants unable to convert exogenous sulfate to sulfite were isolated using the toxic analogue selenate. Three of twenty-eight isolated mutants were chromate sensitive. They showed a possible lesion in the gene that codes the ATP sulfurylase. The others were chromate resistant, and probably had a lesion in one or both of the genes that code the sulfate permease. Methionine increased the resistance levels to selenate. In addition, the frequency of spontaneous mutants obtained in a medium containing methionine was higher (between 2.4 x 10-6 and 18.0 x 10-6 than that obtained using a medium without any intentional source of sulfur (between 0.7 x 10-6 and 5.0 x 10-6. The original strain, as well as the mutants, were able to grow in a sulfur-free liquid medium even after 4 consecutive inoculation procedures. These results indicated the existence of sulfur traces in the medium and/or an efficient intracellular storage system. There was no significant difference between cephalosporin C production in mutants and the original strain.Mutantes incapazes de converter o sulfato extracelular em sulfito foram isolados utilizando o análogo tóxico selenato. De 28 mutantes isolados, apenas 3 foram sensíveis ao cromato, provavelmente apresentando lesão no gene que codifica a ATP sulfurilase. Os demais foram resistentes ao cromato e devem conter lesão no gene sB ou também no gene sC. A metionina elevou os níveis de resistência ao selenato e a freqüência de mutantes espontâneos obtida em meio contendo este aminoácido foi maior (entre 2,42 x 10-6 e 18,04 x 10-6 do que a obtida no meio sem a adição de qualquer fonte intencional de enxofre (entre 0,71 x 10-6 e 5,0 x 10-6. A linhagem original e os mutantes foram capazes de crescer, mesmo depois de quatro etapas de inóculo, fato que pode ser explicado pela existência de traços do referido elemento no meio e/ou a presença de um sistema eficiente de estocagem intracelular. A produção de cefalosporina C

  17. Construction of acetoin high-producing Bacillus subtilis strain

    Directory of Open Access Journals (Sweden)

    Yanjun Tian

    2016-07-01

    Full Text Available This paper describes the construction and selection of a high-producing mutant, Bacillus subtilis HB-32, with enhanced acetoin yield and productivity. The mutant was obtained by the protoplast fusion of a Bacillus subtilis mutant TH-49 (Val− producing acetoin and Bacillus licheniformis AD-30 producing α-acetolactate decarboxylase, with the fusogen polyethylene glycol and after the regeneration and selection, etc. of the fusant. The acetoin production reached 49.64 g/L, which is an increase of 61.8% compared to that of B. subtilis strain TH-49. Random amplified polymorphic DNA analysis was performed to determine the mutagenic and protoplast fusion effects and the genomic changes in the acetoin high-producing strain compared to the parent strains at the molecular level. The constructed strain was shown to be promising for large-scale acetoin production. Future studies should focus on the application of the mutant strain in practice.

  18. Production of astaxanthin from cellulosic biomass sugars by mutants of the yeast Phaffia rhodozyma

    Science.gov (United States)

    Astaxanthin is a carotenoid of high value to the aquaculture, nutraceutical, and pharmaceutical industries. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were test...

  19. Repressor mutant forms of the Azospirillum brasilense NtrC protein.

    Science.gov (United States)

    Huergo, Luciano F; Assumpção, Marcelo C; Souza, Emanuel M; Steffens, M Berenice R; Yates, M Geoffrey; Chubatsu, Leda S; Pedrosa, Fábio O

    2004-10-01

    The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source. Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site. The phenotypes of these strains are discussed in relation to their genotypes.

  20. HBV genotypes prevalence, precore and basal core mutants in Morocco.

    Science.gov (United States)

    Baha, Warda; Ennaji, My Mustapha; Lazar, Fatiha; Melloul, Marouane; El Fahime, Elmostafa; El Malki, Abdelouahad; Bennani, Abdelouaheb

    2012-08-01

    The study of hepatitis B virus (HBV) genomic heterogeneity has become a major issue in investigations aimed at understanding the relationship between HBV mutants and the wide spectrum of clinical and pathological conditions associated with HBV infection. The objective of the current study was to find out the pattern of HBV genotypes circulating in Morocco and to investigate the precore (PC) and basal core promoter (BCP) mutants' status in Moroccan chronic hepatitis B patients. Viral genotypes were determined in 221 chronic carriers using INNO-LiPA HBV assay and hemi-nested PCR. Phylogenetic analysis was performed in 70 samples, and multiplex PCR method was used to confirm some genotyping results. PC and CP mutants were determined using Inno-Lipa. All isolates were successfully genotyped. The genotype distribution was D in 90.45% of cases, A (5.9%), E (1 case), and mixed genotypes (5 A/D and 2 D/F) in 3.17% patients. HBV carried in the HBV/D samples could be assigned to D7 (63.3%), D1 (32.7%) and 2% of strains to each D4 and D5, all HBV/A belonged to A2 subgenotype and HBV/E strain could not be sub-genotyped. In 70 studied strains, HBV mutants were detected in 88.6% of cases; PC mutants were detected in (40%) of patients and 21.5% present a mixture of wild type and G1896A mutation. BCP mutants were observed in 65.7% of cases, 22.9% were found to have the T1762/1764A double mutation, 18.6% had A1762/1764T mutation and 22.9% of patients showed the A1762T/G1764A double mutation with either A1762T/G1764T mutation. Co-infection by PC and BCP mutants was detected in 52.9% of cases. Movement from place to place most likely shapes the observed genotype distribution and consequent prevalence of genotypes other than A2 or D7 in this population. High circulation of PC and BCP mutants is common in chronic hepatitis B infection in Morocco.

  1. Strain improvement of Gluconacetobacter xylinus NCIM 2526 for ...

    African Journals Online (AJOL)

    TUOYO

    2010-08-09

    Aug 9, 2010 ... wild strain. The mutant GHUV4 gave cellulose yield of 3.92 g/l which was 30% more than the wild strain ... One of the. BC application problems in industry is its low productivity. One approach to combat BC productivity is strain.

  2. Characterization and E protein expression of mutant strains during persistent infection of KN73 cells with Japanese encephalitis virus%KN73细胞持续感染乙脑病毒的模型中变异株性状及E蛋白表达

    Institute of Scientific and Technical Information of China (English)

    冯国和; 竹上勉; 赵桂珍

    2002-01-01

    目的研究乙脑病毒变异的特性以及变异株性状与E蛋白表达的关系.方法通过应用乙脑病毒的野生株以及人肝癌细胞KN73建立持续感染模型.采用胰蛋白酶消化技术每周进行细胞传代.经反复冻融收集持续感染细胞内病毒.采用BHK细胞空斑实验方法进行病毒滴定.采用间接免疫荧光方法检测E和NS3蛋白抗原.采用Western印迹杂交检测E和NS3蛋白的表达.结果感染早期(24小时-36小时)野生株感染的KN73细胞的培养液中病毒滴度为106 PFU/ml.在感染后期(3年)滴度为103-4 PFU/ml.病毒重叠感染实验发现在急性重叠感染野生株的持续感染KN73细胞中培养液的病毒滴度比在同一时相急性感染的正常KN73细胞培养液的滴度要低得多.间接免疫荧光检测表明,在持续感染的KN73细胞中病毒抗原的存在低于急性感染的KN73细胞.Western印迹杂交检测表明,E、NS3蛋白的分子量分别为53KD和73KD.在持续感染的KN73细胞中NS3蛋白的表达很稳定,但E蛋白的表达却明显受抑.结论从持续感染的KN73细胞中获得的病毒具有缺陷干扰病毒的一些特性且参与持续感染,其毒力和增殖均低于亲本病毒.这些变异可能与E蛋白表达减少有关.%Objective To study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.Methods Persistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Wester*# n blot analysis was used to test

  3. Mutant prevention concentrations of four carbapenems against gram-negative rods.

    Science.gov (United States)

    Credito, Kim; Kosowska-Shick, Klaudia; Appelbaum, Peter C

    2010-06-01

    We tested the propensities of four carbapenems to select for resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii mutants by determining the mutant prevention concentrations (MPCs) for 100 clinical strains with various ss-lactam phenotypes. Among the members of the Enterobacteriaceae family and A. baumannii strains, the MPC/MIC ratios were mostly 2 to 4. In contrast, for P. aeruginosa the MPC/MIC ratios were 4 to > or =16. The MPC/MIC ratios for beta-lactamase-positive K. pneumoniae and E. coli isolates were much higher (range, 4 to >16 microg/ml) than those for ss-lactamase-negative strains.

  4. Mutant Prevention Concentrations of Four Carbapenems against Gram-Negative Rods▿ †

    Science.gov (United States)

    Credito, Kim; Kosowska-Shick, Klaudia; Appelbaum, Peter C.

    2010-01-01

    We tested the propensities of four carbapenems to select for resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii mutants by determining the mutant prevention concentrations (MPCs) for 100 clinical strains with various ß-lactam phenotypes. Among the members of the Enterobacteriaceae family and A. baumannii strains, the MPC/MIC ratios were mostly 2 to 4. In contrast, for P. aeruginosa the MPC/MIC ratios were 4 to ≥16. The MPC/MIC ratios for β-lactamase-positive K. pneumoniae and E. coli isolates were much higher (range, 4 to >16 μg/ml) than those for ß-lactamase-negative strains. PMID:20308376

  5. Cariogenicity of a lactate dehydrogenase-deficient mutant of Streptococcus mutans serotype c in gnotobiotic rats.

    OpenAIRE

    FitzGerald, R.J.; Adams, B. O.; Sandham, H. J.; Abhyankar, S

    1989-01-01

    A lactate dehydrogenase-deficient (Ldh-) mutant of a human isolate of Streptococcus mutans serotype c was tested in a gnotobiotic rat caries model. Compared with the wild-type Ldh-positive (Ldh+) strains, it was significantly (alpha less than or equal to 0.005) less cariogenic in experiments with two different sublines of Sprague-Dawley rats. The Ldh- mutant strain 044 colonized the oral cavity of the test animals to the same extent as its parent strain 041, although its initial implantation ...

  6. Repair of ultraviolet-irradiated transforming DNA in A recA mutant of Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Stuy, J.H.; Walter, R.B. (Florida State Univ., Tallahassee (USA). Dept. of Biological Science)

    1983-04-01

    Ultraviolet-irradiated transforming DNA was assayed on a wild-type strain of Haemophilus influenzae strain Rd, on an excision repair-deficient (uvr-2) mutant, on a recombination repair-deficient (recA4) mutant, and on a strain carrying both mutations. The donor DNA had a point mutation genetic marker (strAl) and a long nonhomologous plasmid-derived DNA segment inserted in the HPl prophage. The shape of the inactivation curves suggested that only recombination was responsible for the inverse square root kinetics observed with excision repair-proficient recipients.

  7. [Production of carotene and lycopene by mutants of Streptomyces globisporus 1912 cultivated on mealy media].

    Science.gov (United States)

    Holembiovs'ka, S L; Matseliukh, B P

    2008-01-01

    Carotenoids produced by strains 4Crt and 4Lcp, spontaneous mutants oflandomycin E producer Streptomyces globisporus 1912 with activated cryptic genes of carotenogenesis, were identified, and their quantitative output was studied after growth of Streptomycete cultures on different mealy media in shake flasks. On the basis of chromatographic mobility and maxima of absorption spectra of purified pigments they were related to lycopene, beta- and phi-carotene (isorenieratene). It was shown that strain 4Crt synthesizes both carotenoids, while its spontaneous mutant 4Lcp--only lycopene. The greatest output of lycopene (27.24 mg/l) was observed after cultivation of 4Lcp strain on soy-corn medium.

  8. Ultra-violet-resistant mutants of Bacillus thuringiensis

    Energy Technology Data Exchange (ETDEWEB)

    Jones, D.R.; Karunakaran, V. (Polytechnic of Central London (UK). Faculty of Engineering and Science, School of Biological and Health Sciences); Burges, H.D. (Institute of Horticultural Research, Littlehampton (UK)); Hacking, A.J. (Reading Univ. (UK). Dextra Labs.Ltd.)

    1991-06-01

    One of the main disadvantages of using Bacillus thuringiensis as an insecticide is that the spore and crystal preparations applied to foliage are readily washed away by rain and are inactivated by sunlight. Spores from some strains of B. thuringiensis have been shown to be highly sensitive to u.v. light. This study has demonstrated how mutants with increased resistance to u.v., isolated by successive rounds of u.v. irradiation, and additionally with increased specific pathogenicity can be isolated. These techniques should be applied to strains that are frequently used in the industrial production of B.thuringiensis toxin. (author).

  9. Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

    Institute of Scientific and Technical Information of China (English)

    WANG Hua; ZHANG Jian; YANG Fan; WANG Kai; SHEN Si-Le; LIU Bing-Bing; ZOU Bo; ZOU Guang-Tian

    2011-01-01

    The mutant strains of aspergillus oryzae (HP300a) are screened under 300 Mpa for 20min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

  10. Azotobacter vinelandii mutS: nucleotide sequence and mutant analysis.

    OpenAIRE

    Le, O; Shen, B.; Iismaa, S E; Burgess, B K

    1993-01-01

    An Azotobacter vinelandii homolog to the Salmonella typhimurium mutS gene was discovered upstream of the fdxA gene. The product of this gene is much more similar to S. typhimurium MutS than either is to the HexA protein of Streptococcus pneumoniae. An A. vinelandii delta mutS mutant strain was shown to have a spontaneous mutation frequency 65-fold greater than that of the wild type.

  11. Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

    OpenAIRE

    de Montaigu Amaury; Magneschi Leonardo; Catalanotti Claudia; Yang Wenqiang; Mus Florence; Pootakham Wirulda; Gonzalez-Ballester David; Higuera Jose J; Prior Matthew; Galván Aurora; Fernandez Emilio; Grossman Arthur R

    2011-01-01

    Abstract A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment...

  12. Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

    Science.gov (United States)

    Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

    2016-01-10

    To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph).

  13. Construction and immunogenicity of a ∆apxIC/ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis

    Directory of Open Access Journals (Sweden)

    Qiong Liu

    2013-03-01

    Full Text Available The apxIC genes of the Actinobacillus pleuropneumoniae serovar 5 (SC-1, encoding the ApxIactivating proteins, was deleted by a method involving sucrose counter-selection. In this study, a mutant strain of A. pleuropneumoniae (SC-1 was constructed and named DapxIC/ ompP2. The mutant strain contained foreign DNA in the deletion site of ompP2 gene of Haemophilus parasuis. It showed no haemolytic activity and lower virulence of cytotoxicity in mice compared with the parent strain, and its safety and immunogenicity were also evaluated in mice. The LD50 data shown that the mutant strain was attenuated 30-fold, compared with the parent strain (LD50 of the mutant strain and parent strain in mice were determined to be 1.0 × 107 CFU and 3.5 × 105 CFU respectively. The mutant strain that was attenuated could secrete inactivated ApxIA RTX toxins with complete antigenicity and could be used as a candidate live vaccine strain against infections of A. pleuropneumoniae and H. parasuis.

  14. Construction and immunogenicity of a ∆apxIC/ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis.

    Science.gov (United States)

    Liu, Qiong; Gong, Yuheng; Cao, Yuqin; Wen, Xintian; Huang, Xiaobo; Yan, Qigui; Huang, Yong; Cao, Sanjie

    2013-03-06

    The apxIC genes of the Actinobacillus pleuropneumoniae serovar 5 (SC-1), encoding the ApxIactivating proteins, was deleted by a method involving sucrose counter-selection. In this study, a mutant strain of A. pleuropneumoniae (SC-1) was constructed and named DapxIC/ ompP2. The mutant strain contained foreign DNA in the deletion site of ompP2 gene of Haemophilus parasuis. It showed no haemolytic activity and lower virulence of cytotoxicity in mice compared with the parent strain, and its safety and immunogenicity were also evaluated in mice. The LD50 data shown that the mutant strain was attenuated 30-fold, compared with the parent strain (LD50 of the mutant strain and parent strain in mice were determined to be 1.0 × 10(7) CFU and 3.5 × 10(5) CFU respectively). The mutant strain that was attenuated could secrete inactivated ApxIA RTX toxins with complete antigenicity and could be used as a candidate live vaccine strain against infections of A. pleuropneumoniae and H. parasuis.

  15. EMMA—mouse mutant resources for the international scientific community

    Science.gov (United States)

    Wilkinson, Phil; Sengerova, Jitka; Matteoni, Raffaele; Chen, Chao-Kung; Soulat, Gaetan; Ureta-Vidal, Abel; Fessele, Sabine; Hagn, Michael; Massimi, Marzia; Pickford, Karen; Butler, Richard H.; Marschall, Susan; Mallon, Ann-Marie; Pickard, Amanda; Raspa, Marcello; Scavizzi, Ferdinando; Fray, Martin; Larrigaldie, Vanessa; Leyritz, Johan; Birney, Ewan; Tocchini-Valentini, Glauco P.; Brown, Steve; Herault, Yann; Montoliu, Lluis; de Angelis, Martin Hrabé; Smedley, Damian

    2010-01-01

    The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org. PMID:19783817

  16. Hepatitis B escape mutants in Scottish blood donors.

    Science.gov (United States)

    Larralde, Osmany; Dow, Brian; Jarvis, Lisa; Davidson, Fiona; Petrik, Juraj

    2013-06-01

    Hepatitis B virus (HBV) remains as the viral infection with the highest risk of transmission by transfusion. This risk is associated with window period donations, occult HBV infection (OBI) and the emergence of escape mutants, which render blood donations false negative for hepatitis B surface antigen (HBsAg) serological testing. A retrospective study was conducted to gain insights into the molecular epidemiology of HBV escape mutants in Scottish blood donors. The criterion for selection was HBV positivity either by serology or nucleic acid testing (NAT). HBsAg detection was compared across several commercial immunoassays. The full length S gene from plasma samples was PCR amplified, cloned and expressed in HepG2 cells. Eight samples showed HBsAg discordant results, while 5 OBI samples were found. Four escape mutants, containing missense mutations in the S gene, are described here. These mutations impaired HBsAg detection both from HBV infected plasma samples and from recombinant proteins derived from its infected donors. Phylogenetic analysis showed that most of the mutants were clustered in the genotype D and were closely related to strains from Asia and the Middle East. We report here a proline substitution, outside the major hydrophilic region, that impaired HBsAg detection in vivo and in vitro, warning about the risk for the emergence of vaccine escape mutants with mutations outside the major neutralisation site.

  17. Identification of a mutant PfCRT-mediated chloroquine tolerance phenotype in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Stephanie G Valderramos

    2010-05-01

    Full Text Available Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ, the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1 suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2 implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3 uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt.

  18. Screening of Toxin Mutant of Dickeya zeae and Its Biological Characters

    Institute of Scientific and Technical Information of China (English)

    Jingyi ZHANG; Yutao WANG; Yanchang LI; Qiongguang LIU

    2013-01-01

    [Objective] This study aimed to screen toxin mutant of Dickeya zeae (Er-winia chrysanthemi pv. zeae) and investigate its biological characters. [Method] We obtained a toxin mutant strain D. zeae Ech7-3-42 by using acridine orange as a mutagenic agent and compared their biological characteristics and virulence between the toxin mutant and wild strain. [Result] There was no significant difference in pectin lyase, protease, cellulase and the production of extracellular polysaccharide and lipopolysaccharide, but significant difference in toxin biological activities and vir-ulence. Ech7-3-42 mutant did not produce toxin, as wel as the loss of virulence on rice and HR on tobacco, but did not lose the ability to soft rot on potato. Mutant strain Ech7-3-42 can infect rice root and then enriched in the root neck and stalk, but it could not cause rice foot rot. Dickeya zeae (wild and mutant strain) could be detected by PCR in the root neck and below the 1-2 cm long stem area, but could not be detected in the leaves. [Conclusion] We believed that toxin may be one of the important factors for D. zeae virulence on rice.

  19. Survival, growth, and localization of epiphytic fitness mutants of pseudomonas syringae on leaves

    Energy Technology Data Exchange (ETDEWEB)

    Beattie, G.A.; Lindow, S.E. (Univ. of California, Berkeley, CA (United States))

    1994-10-01

    Among 82 epiphytic fitness mutants of a Pseudomonas syringae pv. syringae strain that were characterized in a previous study, 4 mutants were particularly intolerant of the stresses associated with dry leaf surfaces. These four mutants each exhibited distinctive behaviors when inoculated into and into plant leaves. For example, while non showed measurable growth on dry potato leaf surfaces, they grew to different population sizes in the intercellular space of bean leaves and on dry bean leaf surfaces, and one mutant appeared incapable of growth in both environments although it grew well on moist bean leaves. The presence of the parental strain did not influence the survival of the mutants immediately following exposure of leaves to dry, high-light incubation conditions, suggesting that the reduced survival of the mutants did not result from an inability to produce extracellular factors in planta. On moist bean leaves that were colonized by either a mutant or the wild type, the proportion of the total epiphytic population that was located in sizes protected from a surface sterilant was smaller for the mutants than for the wild type, indicating that the mutants were reduced in their ability to locate, multiply in, and/or survive in such protected sites. This reduced ability was only one of possible several factors contributing to the reduced epiphytic fitness of each mutant. Their reduced fitness was not specific to the host plant bean, since they also exhibited reduced fitness on the nonhost plant potato; the functions altered in these strains are thus of interest for their contribution to the general fitness of bacterial epiphytes. 52 refs., 6 figs., 1 tab.

  20. Isolation and characterization of Streptococcus mutans mutants defective in adherence and aggregation.

    Science.gov (United States)

    Murchison, H; Larrimore, S; Curtiss, R

    1981-12-01

    A method was developed which enriched for mutants of Streptococcus mutans that exhibit defects in adherence to glass, aggregation, or both. Mutants were isolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) after mutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survival after mutagenesis was kept above 1 to 2% to enhance the probability that mutants resulted from single mutational events. A total of 117 mutants were isolated; they also displayed non-wild-type colony morphology on mitis salivarius agar. These mutants were examined for (i) adherence and aggregation after overnight growth in sucrose-containing medium, (ii) aggregation of nongrowing cells in the presence of 200 microgram of sucrose per ml or 20 microgram of dextran per ml, and (iii) dextranase production on blue dextran agar plates. Although we isolated mutants which exhibited a variation from the parent strain in only one of the traits tested, the majority of mutants exhibited defects in two or more characteristics. Thirty-eight stable mutants of independent origin were categorized into 13 separate phenotypic groups.

  1. Selection of Pseudomonas sp. strain HBP1 Prp for metabolism of 2-propylphenol and elucidation of the degradative pathway

    NARCIS (Netherlands)

    Kohler, Hans-Peter E.; Maarel, Marc J.E.C. van der; Kohler-Staub, Doris

    1993-01-01

    A mutant of Pseudomonas sp. strain HBP1, originally isolated on 2-hydroxybiphenyl, was selected for the ability to grow on 2-propylphenol as the sole carbon and energy source. In the mutant strain, which was designated as Pseudomonas sp. strain HBP1 Prp, the cellular induction mechanism involved in

  2. Isolation and characterization of radioresistant mutants in Bacillus subtilis and Bacillus thuringiensis

    Energy Technology Data Exchange (ETDEWEB)

    Kalinin, V.L.; Petrov, V.N.; Petrova, T.M. (AN SSSR, Leningrad. Inst. Yadernoj Fiziki)

    Vegetative cells of Bac. thuringiensis var. galleriae (the wild-type strain 351) are much more sensitive to lethal effects of UV light and /sup 60/Co-..gamma..-rays than those of Bac. subtilis (the wild-type strain 168). This difference is less pronounced for spores of these strains. By means of repeated ..gamma..-irradiation-regrowth cycles radioresistant mutants of Bac. thuringiensis Gamsup(r) 14 and Bac. subtilis Gamsup(r) 9 were selected. The vegetative cells of these mutants are correspondingly 19 times and 3.9 times more resistant to lethal effects of ..gamma..-radiation than the cells of the parental strains. The resistance of the Gamsup(r) mutant cells to lethal effects of UV light and H/sub 2/O/sub 2/ is also increased. The spores of the Gamsup(r) 14 mutant are 1.5-1.7 times more resistant to ..gamma..-radiation and UV light than the wild-type spores. The radioresistant mutants and the parental strains do not vary in their capacity for host-cell reactivation of UV- or ..gamma..-irradiated phages Tg13 and 105.

  3. Extract from a mutant Rhodobacter sphaeroides as an enriched carotenoid source

    Directory of Open Access Journals (Sweden)

    Chih-Chiang Wang

    2016-03-01

    Full Text Available Background: The extract Lycogen™ from the phototrophic bacterium Rhodobacter sphaeroides (WL-APD911 has attracted significant attention because of its promising potential as a bioactive mixture, attributed in part to its anti-inflammatory properties and anti-oxidative activity. Objective: This study aims to investigate the components of Lycogen™ and its anti-inflammatory properties and anti-oxidative activity. Design and results: The mutant strain R. sphaeroides (WL-APD911 whose carotenoid 1,2-hydratase gene has been altered by chemical mutagenesis was used for the production of a new carotenoid. The strain was grown at 30°C on Luria–Bertani (LB agar plates. After a 4-day culture period, the mutant strain displayed a 3.5-fold increase in carotenoid content, relative to the wild type. In the DPPH test, Lycogen™ showed more potent anti-oxidative activity than lycopene from the wild-type strain. Primary skin irritation test with hamsters showed no irritation response in hamster skins after 30 days of treatment with 0.2% Lycogen™. Chemical investigations of Lycogen™ using nuclear magnetic resonance (NMR 1H, 13C, and COSY/DQCOSY spectra have identified spheroidenone and methoxyneurosporene. Quantitative analysis of these identified compounds based on spectral intensities indicates that spheroidenone and methoxyneurosporene are major components (approximately 1:1; very small quantities of other derivatives are also present in the sample. Conclusions: In this study, we identified the major carotenoid compounds contained in Lycogen™, including spheroidenone and methoxyneurosporene by high-resolution NMR spectroscopy analysis. The carotenoid content of this mutant strain of R. sphaeroides was 3.5-fold higher than that in normal strain. Furthermore, Lycogen™ from the mutant strain is more potent than lycopene from the wild-type strain and does not cause irritation in hamster skins. These findings suggest that this mutant strain has the

  4. Wild Accessions and Mutant Resources

    DEFF Research Database (Denmark)

    Kawaguchi, Masayoshi; Sandal, Niels Nørgaard

    2014-01-01

    Lotus japonicus, Lotus burttii, and Lotus filicaulis are species of Lotus genus that are utilized for molecular genetic analysis such as the construction of a linkage map and QTL analysis. Among them, a number of mutants have been isolated from two wild accessions: L. japonicus Gifu B-129...

  5. Study on Several Characteristics of Agaricus blazei Murril Strain J3 Irradiated by 60Co

    Institute of Scientific and Technical Information of China (English)

    WENG Bo-qi; JIANG Zhi-he; HUANG Ting-jun; CHEN Jian; ZHENG Wei-wen

    2003-01-01

    A new mutant strain J3 from Agaricus blazei Murril was obtained by 60Co irradiation. The yields of successive generations were increased more than 70% to compare with their original strain. The component analysis on amino acids and fatty acid illuminated that the nutrient value of strain J3 fruiting body was better than the original strain. The apparent nodule structure was found in the hyphea of J3 strain through the observation by scanning electron microscope. RAPD analysis showed the great difference of PCR fingerprints between J3 and its original strain. It is a promising mutant strain for further commercial development in the future.

  6. Nitrogen Control in Pseudomonas aeruginosa : Mutants Affected in the Synthesis of Glutamine Synthetase, Urease, and NADP-Dependent Glutamate Dehydrogenase

    NARCIS (Netherlands)

    Janssen, Dick B.; Habets, Winand J.A.; Marugg, Joey T.; Drift, Chris van der

    1982-01-01

    Mutants were isolated from Pseudomonas aeruginosa that were impaired in the utilization of a number of nitrogen sources. In contrast to the wild-type strain, these mutants appeared to be unable to derepress the formation of glutamine synthetase and urease under nitrogen-limited growth conditions, wh

  7. Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase

    NARCIS (Netherlands)

    CREMERS, HCJC; Batley, M; Redmond, J W; Eijdems, Elisabeth; BREEDVELD, MW; ZEVENHUIZEN, LPTM; Pees, Elisabeth; Wijffelman, C A; Lugtenberg, Ben J.J.

    1990-01-01

    Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the

  8. Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Hassan Abdel-Latif A. Mohamed

    2006-06-01

    Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

  9. Isolation and characterization of ultraviolet light-sensitive mutants of the blue-green alga Anacystis nidulans.

    Science.gov (United States)

    Asato, Y.

    1972-01-01

    Three independently isolated ultraviolet light sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 showed the highest sensitivity to UV by its greatly reduced photoreactivation capacity following irradiation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, thus indicating the presence of excision enzymes involved in dark repair. Under 'black' and 'white' illumination, strain uvs-1 shows photorecovery properties comparable with wild-type cultures. Results indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.

  10. Construction and Verification of LuxS-negative Mutants of Streptococcus Mutans and the Effect of the Absence of LuxS Gene on the Acid Tolerance

    Institute of Scientific and Technical Information of China (English)

    YU Dan-ni; CHEN Jie; ZHANG Yao-chao; HAN Yu-zhi

    2009-01-01

    Objective: To knock out the entire Luxs gene of Streptococcus mutans(S.mutans) UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. Mutans. To study the difference between the acid resistance of S. Mutans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Two DNA fragments locating in the upper and downstream of Luxs gene were amplified and a erythromycin resistance gene of PJT10 between them were engineered into PUC19 plasmid for constructing the recombination plasmid pUCluxKO. Electrotransformation of S.mutans cells with pUCluxKO-mutant resulted in isolation of erythromycin resistant S. Mutans transformants, which was identified by polymerase chain reaction, V.harveyi BB170 luminescence bioassay and sequencing analysis. Solutions of S. Mutans standard strain and LuxS mutant strain with same density were made and cultured at pH 3.5 to 7.0 BHI liquid for the same period.Terminal growth situation was compared.Firstly acidized in pH 5.5 BHI liquid,the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared.Results:Restriction endonuclease analyses showed that pUCluxKO-mutant vector had been successfully recombined. The Luxs-deleted status of S.mutans mutants was confirmed by PCR with primers which were specific for the genes of Luxs and Erythromycin resistance. S.mutans mutant can not induce bioluminescence, indiating the mutant had been successfully recombined. After twenty generations of culture, the constructed Chinese S.mutans mutants were confirmed to be stable. Significant difference of aciduricity was observed between S.mutans standard strain and LuxS mutant strain.The acid resistance of standard strain was stronger than that of LuxS mutant strain.The two strains both displayed the capability of acid tolerance responses. Conclusion:The S.mutans gene allelic exchange plasmid is constructed correctively and a Luxs

  11. Temperature-sensitive mutants of fowl plague virus: isolation and genetic characterization. [UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Almond, J.W.; McGeoch, D.; Barry, R.D.

    1979-01-30

    Forty-nine temperature-sensitive mutants of fowl plague virus (FPV) strain Rostock and four ts mutants of FPV-strain Dobson were isolated by utilizing two methods of plaque screening, after either spontaneous or chemically induced mutagenesis. Twenty-nine of the FPV-Rostock mutants were further characterized by genetic recombination studies and were found to fall into six high frequency recombination groups. The genome segment carrying the ts mutation in each group was identified by analyzing the gene composition of ts/sup +/ recombinants generated from crosses between representatives of each group and ts mutants of FPV-Dobson. It was concluded that the six groups correspond to mutations in six different genome segments, namely, those coding for the P/sub 1/, P/sub 2/, P/sub 3/, HA, NP, and NS proteins.

  12. Isolation of mutants deficient in acetyl-CoA synthetase and a possible regulator of acetate induction in Aspergillus niger.

    Science.gov (United States)

    Sealy-Lewis, H M; Fairhurst, V

    1998-07-01

    Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.

  13. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

    Directory of Open Access Journals (Sweden)

    Eliton da Silva Vasconcelos

    2013-12-01

    Full Text Available Clavulanic acid (CA is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064. The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  14. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

    Science.gov (United States)

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-12-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  15. Prevention of GABA reduction during dough fermentation using a baker's yeast dal81 mutant.

    Science.gov (United States)

    Ando, Akira; Nakamura, Toshihide

    2016-10-01

    γ-Aminobutyric acid (GABA) is consumed by yeasts during fermentation. To prevent GABA reduction in bread dough, a baker's yeast mutant AY77 deficient in GABA assimilation was characterized and utilized for wheat dough fermentation. An amber mutation in the DAL81 gene, which codes for a positive regulator of multiple nitrogen degradation pathways, was found in the AY77 strain. The qPCR analyses of genes involved in nitrogen utilization showed that transcriptional levels of the UGA1 and DUR3 genes encoding GABA transaminase and urea transporter, respectively, are severely decreased in the AY77 cells. The AY77 strain cultivated by fed-batch culture using cane molasses exhibited inferior gas production during dough fermentation compared to that of wild-type strain AY13. However, when fed with molasses containing 0.5% ammonium sulfate, the mutant strain exhibited gas production comparable to that of the AY13 strain. In contrast to the AY13 strain, which completely consumed GABA in dough within 5 h, the AY77 strain consumed no GABA under either culture condition. Dough fermentation with the dal81 mutant strain should be useful for suppression of GABA reduction in breads.

  16. Molecular characterization of Tn5-induced symbiotic (Fix-) mutants of Rhizobium meliloti.

    OpenAIRE

    Zimmerman, J L; Szeto, W W; Ausubel, F M

    1983-01-01

    To investigate the expression of specific symbiotic genes during the development of nitrogen-fixing root nodules, we conducted a systematic analysis of nodule-specific proteins and RNAs produced after the inoculation of alfalfa roots with a series of Rhizobium meliloti mutants generated by site-directed transposon Tn5 mutagenesis. The mutagenized region of the Rhizobium genome covered approximately 10 kilobases and included the region encoding the nitrogenase polypeptides. All mutant strains ...

  17. Herpes simplex virus type 1 ribonucleotide reductase null mutants induce lesions in guinea pigs.

    Science.gov (United States)

    Turk, S R; Kik, N A; Birch, G M; Chiego, D J; Shipman, C

    1989-12-01

    Two herpes simplex virus type 1 ribonucleotide reductase null mutants, hrR3 and ICP6 delta, produced cutaneous lesions in guinea pigs as severe as those of wild-type strains. The lesions induced by hrR3 resulted from in vivo replication of the mutant virus, suggesting that this virus-encoded enzyme is nonessential for virus replication in guinea pigs.

  18. Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae

    OpenAIRE

    Hironori eKawakami; Eiji eOhashi; Toshiki eTsurimoto; Tsutomu eKatayama

    2016-01-01

    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as ...

  19. Imipenem- and meropenem-resistant mutants of Enterobacter cloacae and Proteus rettgeri lack porins.

    Science.gov (United States)

    Raimondi, A; Traverso, A; Nikaido, H

    1991-01-01

    Carbapenems such as imipenem and meropenem are not rapidly hydrolyzed by commonly occurring beta-lactamases. Nevertheless, it was possible, by mutagenesis and selection, to isolate mutant strains of Enterobacter cloacae and Proteus rettgeri that are highly resistant to meropenem and imipenem. Two alterations were noted in the E. cloacae mutants. First, the mutant strains appeared to be strongly derepressed in the production of beta-lactamases, which reached a very high level when the strains were grown in the presence of imipenem. Second, these mutants were deficient in the production of nonspecific porins, as judged by the pattern of outer membrane proteins as well as by reconstitution assays of permeability. As with most porin-deficient mutants, their cultures were unstable, and their cultivation in the absence of carbapenems rapidly led to an overgrowth of porin-producing revertants. Analysis of the data suggests that the synergism between the lowered outer membrane permeability and the slow but significant hydrolysis of carbapenems by the overproduced enzymes can explain the resistance phenotypes quantitatively, although the possibility of alteration of the target cannot be excluded at present. With P. rettgeri mutants, there was no indication of further derepression of beta-lactamase, but the enzyme hydrolyzed imipenem much more efficiently than the E. cloacae enzyme did. In addition, the major porin was absent in one mutant strain. These results suggest that a major factor for the carbapenem resistance of these enteric bacteria is the porin deficiency, and this conclusion forms a contrast to the situation in Pseudomonas aeruginosa, in which the most prevalent class of imipenem-resistant mutants appears to lack the specific channel protein D2 yet retains the major nonspecific porin F. Images PMID:1656855

  20. Isolation of a Defective Prion Mutant from Natural Scrapie

    Science.gov (United States)

    Migliore, Sergio; Cosseddu, Gian Mario; Pirisinu, Laura; Riccardi, Geraldina; Nonno, Romolo

    2016-01-01

    It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of

  1. Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

    Science.gov (United States)

    Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

  2. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP and generation of a mutant library with diverse phenotypes.

    Directory of Open Access Journals (Sweden)

    Mingyue Fang

    Full Text Available In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.

  3. Phenotypic characterization of glucose repression mutants of Saccharomyce cerevisiae usinge experiments with C-13-labelled glucose

    DEFF Research Database (Denmark)

    Vijayendran, Raghevendran; Gombert, A.K.; Christensen, B.

    2004-01-01

    glucose. Through GC-MS analysis of the C-13 incorporated into the amino acids of cellular proteins, it was possible to obtain quantitative information on the function of the central carbon metabolism in the different mutants. Traditionally, such labelling data have been used to quantify metabolic fluxes...... through the use of a suitable mathematical model, but here we show that the raw labelling data may also be used directly for phenotypic characterization of different mutant strains. Different glucose derepressed strains investigated employed are the disruption mutants reg1, hxk2, grr1, mig1 and mig1mig2...... and the reference strain CEN.PK113-7D. Principal components analysis of the summed fractional labelling data show that deleting the genes HXK2 and GRR1 results in similar phenotype at the fluxome level, with a partial alleviation of glucose repression on the respiratory metabolism. Furthermore, deletion...

  4. [Biofilm Formation by the Nonflagellated flhB1 Mutant of Azospirillum brasilense Sp245].

    Science.gov (United States)

    Shelud'ko, A V; Filip'echeva, Yu A; Shumiliva, E M; Khlebtsov, B N; Burov, A M; Petrova, L P; Katsy, E I

    2015-01-01

    Azospirillum brasilense Sp245 with mixed flagellation are able to form biofilms on various surfaces. A nonflagellated mutant of this strain with inactivated chromosomal copy of the flhB gene (flhB1) was shown to exhibit specific traits at the later stages of biofilm formation on a hydrophilic (glass) surface. Mature biofilms of the flhB1::Omegon-Km mutant Sp245.1063 were considerably thinner than those of the parent strain Sp245. The biofilms of the mutant were more susceptible to the forces of hydrodynamic shear. A. brasilense Sp245 cells in biofilms were not found to possess lateral flagella. Cells with polar flagella were, however, revealed by atomic force microscopy of mature native biofilms of strain Sp245. Preservation of a polar flagellum (probably nonmotile) on the cells of A. brasilense Sp245 may enhance the biofilm stability.

  5. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    Science.gov (United States)

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A

    1989-12-01

    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

  6. [Phenotype analysis and mutant gene location of ventral yellow mouse (VY(Slac))].

    Science.gov (United States)

    Shi, Mei-Lian; Xu, Ping; Yin, Xiao-Shu; Yang, Wei-Wei; Gu, Mei-Er; Yu, Li-Ping; Liu, Gui-Jie; Wu, Bao-Jin

    2012-06-01

    The ventri-yellow pigmentation mouse (temporarily named VY(Slac)) arose spontaneously in the C57BL/6J inbred mouse strain, found and bred by Shanghai SLAC Laboratory Animal Co., Ltd. VY(Slac) presented a special phenotype marked by yellow coat on the ventral surface of neck and trunk that was without melanin deposition but maintained a normal structure. The number of melanocytes in epidermis and melanin in hair follicle of the abdominal skin of the mutant mouse were less than that of their background strain, while there was no significant difference between the dorsal skins of the two strains. This mutant phenotype was inherited as single-gene dominant inheritance, confirmed by genetic experiment, and there was no significant difference between VY(Slac) and B(6) for other biological parameters such as weight, anatomic and histological structures of major organs and blood physiology. When the linkage relationship between the genomic DNA samples of F(2) 48 mice (VY(Slac)D(2)F(1)×D(2)) and mutant phenotype were evaluated, the mutant gene was confirmed on chromosome 2 near D2Mit229. New microsatellite and SNP markers were selected to amplify genomic DNA samples of 196 F(2) mice and the mutant gene was narrowed down to 5.3 Mb region between rs13476833 and rs27310903 on chromosome 2. The preliminary results of our phenotype analysis and gene location provides a solid basis for further identification of this mutant gene.

  7. Comparison of the behavior of epiphytic fitness mutants of pseudomonas syringae under controlled and field conditions

    Energy Technology Data Exchange (ETDEWEB)

    Beattie, G.A.; Lindow, S.E. (Univ. of California, Berkeley, CA (United States))

    1994-10-01

    The epiphytic fitness of four Tn5 of Pseudomonas syringae that exhibited reduced epiphytic fitness in the laboratory was evaluated under field conditions. The mutants differed more from the parental strain under field conditions than under laboratory conditions, in their survival immediately following inoculation onto bean leaves and in the size of the epiphytic populations that they established at near-wild type rates, while the others exhibited reduced survival only in the warmest, driest conditions tested and grew epiphytically at reduced rates or, in the case of one mutant, not at all. The presence of the parental strain, B728a, did not influence the survival or growth of three of the mutants under field conditions; however, one mutant, an auxotroph, established larger populations in the presence of B728a than in its absence, possibly because of cross-feeding by By28a in planta. Experiments with B728a demonstrated that established epiphytic populations survived exposure of leaves to dry conditions better than newly inoculated cells did and that epiphytic survival was not dependent on the cell density in the inoculum. Three of the mutants behaved similarly to two nonpathogenic strains of P. syringae, suggesting that the mutants may be altered in traits that are missing or poorly expressed in naturally occurring nonpathogenic epiphytes. 58 refs., 5 figs., 3 tabs.

  8. Similarity in properties and mapping of three rec mutants of Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Kooistra, J.; Setlow, J.K.

    1976-07-01

    Three Rec/sup -/ mutants of Haemophilus influenzae have been studied with respect to their transformability, ultraviolet and mitomycin C sensitivities, spontaneous and ultraviolet-induced deoxyribonucleic acid breakdown, inducibility of lysogens, and the linkage of the three mutations to a streptomycin resistance marker. The data indicate that the three mutations cause the same phenotypic changes, and that they are all on the same gene. Transformability of the mutants is different when two different media are used for competence development, although transformability with the two competence methods is not different in a Rec/sup -/ strain that is mutant at another gene.

  9. Glycoprotein processing in mutants of HSV-1 that induce cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.; Kousoulas, K.G.; Knowles, R.W.; Read, G.S.; Holland, T.C.; Keller, P.M.; Warner, S.C.

    1982-01-01

    The synthesis of viral-specified glycoproteins, and their appearance on cell surfaces, were compared for cells infected either with syncytial mutants of HSV-1 or with the parental strains from which the mutants were derived. The mutants MP and tsB5, representatives of two different viral genes that affect fusion, were employed in these studies. Cells infected with either mutant gave rise to reduce surface labeling of major viral-specified glycoproteins throughout infection relative to the parental strains. Putative glycoprotein precursors were synthesized in similar amounts throughout infection in mutant and wild-type infected cells. However, during a chase period after a pulse of labeling, fully glycosylated species of glycoproteins accumulated more slowly in mutant than in parent infected cells. The effect was clearly visible early in MP-infected cells and by 9 hr after infection for tsB5-infected cells, and increased thereafter. Apparently the decrease in appearance of glycoproteins on the cell surface of mutant-infected cells is due to a decrease in their rate of processing. 26 references, 7 figures.

  10. Properties of mutants of haemophilus influenzae deficient in ATP-dependent deoxyribonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.

    1976-01-01

    Eight isogenic Haemophilus influenzae strains whose extracts lack ATP-dependent deoxyribonuclease activity (Add/sup -/ mutants) form three complementation and genetic linkage groups. Since there are known to be three subunits of the enzyme, these data suggest that each of the three genes specifies a different subunit. Gel electrophoresis of partially purified mutant extracts indicates that the smallest subunit is missing in one of the groups but is present in all the other mutants. The mutants are more sensitive to a variety of chemical agents than the wild type. The most sensitive mutants lack the ATPase activity associated with the enzyme. These strains exhibit aberrant incorporation of tritiated thymidine, which starts up more rapidly and shuts off sooner than in the wild type. An extracellular compound is responsible for most of this effect, in that wild type cells put into medium in which Add/sup -/ cells have been growing show a similar aberrant incorporation. The effect of these media can be mimicked by cyclic AMP and cyclic GMP, although millimolar concentrations are required. It is postulated that the Add/sup -/ mutants are more permeable to many substances than the wild type, partly because of the extracellular compound usually surrounding them, and the increased permeability might be responsible for the mutants' nonviability.

  11. Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Strazielle, C; Lefevre, A; Jacquelin, C; Lalonde, R

    2012-07-15

    Dab1(scm) mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1(scm) mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1(scm) mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar cortex affects grooming activity and is consequently involved in executing various components, but not in its sequential organization, which requires other brain regions such as cerebral cortices or basal ganglia.

  12. atp mutants of Escherichia coli fail to grow on succinate due to a transport deficiency

    DEFF Research Database (Denmark)

    Boogerd, Fred; Boe, Lars; Michelsen, Ole

    1998-01-01

    Escherichia coli atp mutants, which lack a functional Hf-ATPase complex, are capable of growth on glucose but not on succinate or other C-4-dicarboxylates (Suc(-) phenotype). Suc(+) revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack......) was expressed in trans, the Suc(-) phenotype of the atp deletion strain reverted to Suc(+), which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C-4-dicarboxylates is insufficient transport capacity for these substrates....

  13. atp mutants of Escherichia coli fail to grow on succinate due to a transport deficiency

    DEFF Research Database (Denmark)

    Boogerd, Fred; Boe, Lars; Michelsen, Ole

    1998-01-01

    Escherichia coli atp mutants, which lack a functional Hf-ATPase complex, are capable of growth on glucose but not on succinate or other C-4-dicarboxylates (Suc(-) phenotype). Suc(+) revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack......) was expressed in trans, the Suc(-) phenotype of the atp deletion strain reverted to Suc(+), which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C-4-dicarboxylates is insufficient transport capacity for these substrates....

  14. Bacterial mutants for enhanced succinate production

    NARCIS (Netherlands)

    Baart, G.J.E.; Beauprez, J.J.R.; Foulquie, M.M.R.; Heijnen, J.J.; Maertens, J.

    2010-01-01

    The present invention relates to a method for obtaining enhanced metabolite production in micro-organisms, and to mutants and/or transformants obtained with said method. More particularly, it relates to bacterial mutants and/or transformants for enhanced succinate production, especially mutants and/

  15. Problem-Solving Test: Tryptophan Operon Mutants

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  16. Ethanol production using nuclear petite yeast mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hutter, A.; Oliver, S.G. [Department of Biomolecular Sciences, UMIST, Manchester (United Kingdom)

    1998-12-31

    Two respiratory-deficient nuclear petites, FY23{Delta}pet191 and FY23{Delta}cox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23{rho}{sup 0}. Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant. K{sub i}, of 2.3% (w/v) and a specific rate of ethanol production, q{sub p}, of 0.90 g ethanol g dry cells{sup -1} h{sup -1}. FY23{rho}{sup 0} was the most sensitive to ethanol, exhibiting a K{sub i} of 1.71% (w/v) and q{sub p} of 0.87 g ethanol g dry cells{sup -1} h{sup -1}. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23{Delta}pet191, having a K{sub i} of 2.14% (w/v) and the 85% respiratory-deficient FY23{Delta}cox5a, having a K{sub i} of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23{rho}{sup 0} is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject of the Pasteur effect and so exhibit higher rates of fermentation. (orig.)

  17. [Breeding of Actinobacillus succiniogenes mutants with improved succinate production based on metabolic flux analysis].

    Science.gov (United States)

    Pan, Lijun; Li, Xingjiang; Jiang, Shaotong; Wei, Zhaojun; Chen, Xiaohui; Cai, Licheng; Wang, Hefeng; Jiang, Jijun

    2008-09-01

    It is very important to obtain high yield mutant strains on the base of metabolic flux analysis of Actinobacillus succinogenes S.JST for the industrial bioconversion of succinic acid. The metabolic pathway was analized at first and the flux of the metabolic networks was calculated by matrix. In order to decrease acetic acid flux, the strains mutated by soft X-ray of synchronous radiation were screened on the plates with high concentration of fluoroacetic acid. For decreasing the metabolic flux of ethanol the site-directed mutagenesis was carried out for the reduction of alcohol dehydrogenase(Adh) specific activity. Then the enzyme activity determination and the gene sequence analysis of the mutant strain was compared with those of the parent strain. Metabolic flux analysis of the parent strain indicated that the flux of succinic acid was 1.78(mmol/g/h) and that the flux of acetic acid and ethanol were 0.60 (mmol/g/h) and 1.04( mmol/g/h), respectively. Meanwhile the metabolic pathway analysis showed that the ethanol metabolism enhanced the lacking of H electron donor during the synthesis of succinic acid and that the succinic acid flux was weakened by the metabolism of byproducts ethanol and acetic acid. Compared with the parent strain, the acetic acid flux of anti-fluoroacetic mutant strain S.JST1 was 0.024 (mmol/g/h), decreasing by 96%. Then the enzyme determination showed that the specific activity unit of phosphotransacetylase(Pta) decreased from 602 to 74 and a mutated site was founded in the pta gene of the mutant strain S.JST1. Compared with that of the parent strain S.JST1 the ethanol flux of adh-site-directed mutant strain S.JST2 was 0.020 (mmol/g/h), decreasing by 98%. Then the enzyme determination showed that the specific activity unit of Adh decreased from 585 to 62 and the yield of end product succinic acid was 65.7 (g/L). The interdiction of Adh and Pta decreased the metabolism of byproducts and the H electron donor was well balanced, thus the succinic

  18. Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene.

    Science.gov (United States)

    Mérida, A; Flores, E; Florencio, F J

    1992-01-01

    The glnA gene from Synechocystis sp. strain PCC 6803 was cloned by hybridization with the glnA gene from Anabaena sp. strain PCC 7120, and a deletion-insertion mutation of the Synechocystis gene was generated in vitro. A strain derived from Synechocystis sp. strain PCC 6803 which contained integrated into the chromosome, in addition to its own glnA gene, the Anabaena glnA gene was constructed. From that strain, a Synechocystis sp. glnA mutant could be obtained by transformation with the inactivated Synechocystis glnA gene; this mutant grew by using Anabaena glutamine synthetase and was not a glutamine auxotroph. A Synechocystis sp. glnA mutant could not be obtained, however, from the wild-type Synechocystis sp. The Anabaena glutamine synthetase enzyme was subject to ammonium-promoted inactivation when expressed in the Synechocystis strain but not in the Anabaena strain itself.

  19. Gluconic acid production by Aspergillus niger mutant ORS-4.410 in submerged and solid state surface fermentation.

    Science.gov (United States)

    Singh, O V; Sharma, A; Singh, R P

    2001-07-01

    Aspergillus niger ORS-4.410, a mutant of Aspergillus niger ORS-4 was produced by repeated irradiation with UV rays. Treatments with chemical mutagnes also resulted into mutant strains. The mutants differed from the parent strain morphologically and in gluconic acid production. The relationship between UV treatment dosage, conidial survival and frequency of mutation showed the maximum frequency of positive mutants (25%) was obtained along with a conidial survival of 59% after second stage of UV irradiation. Comparison of gluconic acid production of the parent and mutant ORS-4.410 strain showed a significant increase in gluconic acid production that was 87% higher than the wild type strain. ORS-4.410 strain when transferred every 15 days and monitored for gluconic acid levels for a total period of ten months appeared stable. Mutant ORS-4.410 at 12% substrate concentration resulted into significantly higher i.e. 85-87 and 94-97% yields of gluconic acid under submerged and solid state surface conditions respectively. Further increase in substrate concentration appeared inhibitory. Maximum yield of gluconic acid was obtained after 6 days under submerged condition and decreased on further cultivation. Solid state surface culture condition on the other hand resulted into higher yield after 12 days of cultivation and similar levels of yields continued thereafter.

  20. JC polyomavirus mutants escape antibody-mediated neutralization.

    Science.gov (United States)

    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  1. Improved production of spiramycin by mutant Streptomyces ambofaciens

    Institute of Scientific and Technical Information of China (English)

    金志华; 岑沛霖

    2004-01-01

    Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S. ambofaciens XC 2-37 were studied. The potency of S. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation. The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.

  2. Improved production of spiramycin by mutant Streptomyces ambofaciens

    Institute of Scientific and Technical Information of China (English)

    金志华; 岑沛霖

    2004-01-01

    Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S.ambofaciens XC 2-37 were studied. The potency orS. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.

  3. Chemotyping of yeast mutants using robotics.

    Science.gov (United States)

    Rieger, K J; El-Alama, M; Stein, G; Bradshaw, C; Slonimski, P P; Maundrell, K

    1999-07-01

    By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed. Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories. Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547-1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics. The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors. Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae. In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype. The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper-pleiotropic phenotype with sensitivities to a dozen or more compounds. Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in-depth functional analysis and a more precise definition of molecular targets.

  4. GREEN FLUORESCENT PIGMENT ACCUMULATED BY A MUTANT OF CELLVIBRIO GILVUS.

    Science.gov (United States)

    LOVE, S H; HULCHER, F H

    1964-01-01

    Love, Samuel H. (Bowman Gray School of Medicine, Winston-Salem, N.C.), and Frank H. Hulcher. Green fluorescent pigment accumulated by a mutant of Cellvibrio gilvus. J. Bacteriol. 87:39-45. 1964.-A mutant of Cellvibrio gilvus, designated strain 139A, liberated a green, fluorescent pigment into the surrounding culture medium. A study of the factors which affected the accumulation of this pigment led to the development of a chemically defined medium which supported maximal pigment accumulation in aerated, liquid cultures. d-Glucose, glycine or l-serine, l-phenylalanine, l-proline, and l-lysine comprised the organic components of this medium. The visible absorption spectrum of the pigment showed a maximal band at 400 mmu (pH 7.0). A difference spectrum between reduced and oxidized pigment showed loss of the band at 400 mmu upon oxidation. However, a methanol-extractable, flavinelike compound occurred in the wild strain but not in the mutant. Ferric ions added to the defined medium stimulated growth, with a concomitant reduction of pigment accumulation. Pigment was formed at a maximal rate during the stationary growth phase, and the highest yield was obtained by 18 hr. Organic solvents did not extract the pigment from water solutions. One and sometimes two, compounds absorbing at 400 mmu could be eluted by ion-exchange chromatography on Cellex-P (H(+)), which was used to separate the pigment from other components in the culture supernatants so that the radioactivity of the pigment could be measured. The mutant synthesized C(14)-labeled pigment from d-glucose-U-C(14) and from each of four amino acids (glycine-1-C(14), l-phenylalanine-U-C(14), l-proline-U-C(14), and l-lysine-U-C(14). Delta-Amino-levulenic acid-4-C(14) did not contribute C(14) to the pigment.

  5. Mutants with Enhanced Nitrogenase Activity in Hydroponic Azospirillum brasilense-Wheat Associations

    Science.gov (United States)

    Pereg Gerk, Lily; Gilchrist, Kate; Kennedy, Ivan R.

    2000-01-01

    The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism. PMID:10788397

  6. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Hathroubi, S; Hancock, M A; Bossé, J T; Langford, P R; Tremblay, Y D N; Labrie, J; Jacques, M

    2015-10-19

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. The phenotype of a phospholipase C (plc-1) mutant in a filamentous fungus, Neurospora crassa.

    Science.gov (United States)

    Lew, Roger R; Giblon, Rachel E; Lorenti, Miranda S H

    2015-09-01

    In the filamentous fungus Neurospora crassa, phospholipase C may play a role in hyphal extension at the growing tips as part of a growth-sensing mechanism that activates calcium release from internal stores to mediate continued expansion of the hyphal tip. One candidate for a tip-localized phospholipase C is PLC-1. We characterized morphology and growth characteristics of a knockout mutant (KO plc-1) and a RIP mutated strain (RIP plc-1) (missense mutations and a nonsense mutation render the gene product non-functional). Growth and hyphal cytology of wildtype and KO plc-1 were similar, but the RIP plc-1 mutant grew slower and exhibited abnormal membrane structures at the hyphal tip, imaged using the fluorescence dye FM4-64. To test for causes of the slower growth of the RIP plc-1 mutant, we examined its physiological poise compared to wildtype and the KO plc-1 mutant. The electrical properties of all three strains and the electrogenic contribution of the plasma membrane H(+)-ATPase (identified by cyanide inhibition) were the same. Responses to high osmolarity were also similar. However, the RIP plc-1 mutant had a significantly lower turgor, a possible cause of its slower growth. While growth of all three strains was inhibited by the phospholipase C inhibitor 3-nitrocoumarin, the RIP plc-1 mutant did not exhibit hyphal bursting after addition of the inhibitor, observed in both wildtype and the KO plc-1 mutant. Although the plc-1 gene is not obligatory for tip growth, the phenotype of the RIP plc-1 mutant - abnormal tip cytology, lower turgor and resistance to inhibitor-induced hyphal bursting - suggest it does play a role in tip growth. The expression of a dysfunctional plc-1 gene may cause a shift to alternative mechanism(s) of growth sensing in hyphal extension. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis.

    Science.gov (United States)

    Valdés-Santiago, Laura; Cervantes-Chávez, José Antonio; Winkler, Robert; León-Ramírez, Claudia G; Ruiz-Herrera, José

    2012-03-01

    Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell.

  9. Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

    Science.gov (United States)

    Barbachano-Torres, Alejandra; Castelblanco-Matiz, Lina M; Ramos-Valdivia, Ana C; Cerda-García-Rojas, Carlos M; Salgado, Luis M; Flores-Ortiz, César M; Ponce-Noyola, Teresa

    2014-06-01

    The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297-417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.

  10. Identification of a Long Rice Spikelet Mutant

    Institute of Scientific and Technical Information of China (English)

    WU Xian-jun; WANG Bin; HAN Zan-ping; XIE Zhao-hui; MOU Chun-hong; WANG Xu-dong

    2004-01-01

    A spontaneously occurring rice (Oryza sativa L. ) mutant, characterized by homeotic conversion in glumes and stamens, was found in the progeny of a cross. The mutant showed long glumes and glumaceous lodicules and morphological transformation of stamens into pistils. Mutant florets consisted of 1 to 3 completely developed pistils, some pistilloid stamens with filaments, but tipped by bulged tissue and 0 to 3 stigmas. It seens that the mutant phenotype of the homeotic conversions in glumes and stamens is similar to that of the B loss-of-function mutants in Arabidopsis and Antirrhinum. The mutant is controlled by a single recessive gene as a segregation ratio of 3:1 (wild type to mutant plants) was observed in the F2 generation.

  11. A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

    Science.gov (United States)

    Bozue, Joel; Cote, Christopher K; Chance, Taylor; Kugelman, Jeffrey; Kern, Steven J; Kijek, Todd K; Jenkins, Amy; Mou, Sherry; Moody, Krishna; Fritz, David; Robinson, Camenzind G; Bell, Todd; Worsham, Patricia

    2014-01-01

    Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

  12. A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

    Directory of Open Access Journals (Sweden)

    Joel Bozue

    Full Text Available Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

  13. [Nature of membrane ATPase inactivation in an Escherichia coli mutant with genetically impaired ATPase].

    Science.gov (United States)

    Chetkauskaĭte, A V; Planutis, D L; Zimkus, A Z; Akimenko, V K; Grinius, L L

    1980-07-01

    Homogeneous preparations of F1 possessing identical subunit composition have been isolated from the mutant strain of E. coli AN 120 with genetically impaired membrane ATPase and from the wild strain of AN 180. Using ion-exchange chromatography, the subunits alpha and beta of F1 were isolated. It was shown that the alpha- and beta-subunits of both active and genetically impaired F1 have similar molecular weights and total electrical charges.

  14. Astaxanthin Synthesis by Yeast Xanthophyllomyces dendrorhous and its Mutants on Media Based on Plant Extracts.

    Science.gov (United States)

    Stachowiak, Barbara

    2012-12-01

    The study evaluated the effect of media based on plant extracts: potato, carrot and barley malt broth, on growth and astaxanthin synthesis by yeast Xanthophyllomyces dendrorhous DSM 5626 and its mutants. The carrot medium promoted carotenogenesis most effectively. In cultures on this medium the highest volumetric and cellular concentrations of astaxanthin were recorded for four out of five tested strains. Also the share of astaxanthin in the total carotenoids produced by the tested strains was the highest.

  15. Astaxanthin Synthesis by Yeast Xanthophyllomyces dendrorhous and its Mutants on Media Based on Plant Extracts

    OpenAIRE

    Stachowiak, Barbara

    2012-01-01

    The study evaluated the effect of media based on plant extracts: potato, carrot and barley malt broth, on growth and astaxanthin synthesis by yeast Xanthophyllomyces dendrorhous DSM 5626 and its mutants. The carrot medium promoted carotenogenesis most effectively. In cultures on this medium the highest volumetric and cellular concentrations of astaxanthin were recorded for four out of five tested strains. Also the share of astaxanthin in the total carotenoids produced by the tested strains wa...

  16. Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

    Directory of Open Access Journals (Sweden)

    de Montaigu Amaury

    2011-07-01

    Full Text Available Abstract A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker and in the strategies used to maintain and store transformants.

  17. Construction and Virulence of Filamentous Hemagglutinin Protein B1 Mutant of Pasteurella multocida in Chickens

    Institute of Scientific and Technical Information of China (English)

    GUO Dong-chun; QU Lian-dong; SUN Yan; ZHANG Ai-qin; LIU Jia-sen; LU Yan; LIU Pei-xin; YUAN Dong-wei; JIANG Qian; SI Chang-de

    2014-01-01

    Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowl cholera, bovine hemorrhagic septicemia, enzoonotic pneumonia and swine atropic rhinitis. Two iflamentous hemagglutinin genes, fhaB1 and fhaB2, are the potential virulence factors. In this study, an inactivation fhaB1 mutant of P. multocida in avian strain C48-102 was constructed by a kanamycin-resistance cassette. The virulence of the fhaB1 mutant and the wild type strain was assessed in chickens by intranasal and intramuscular challenge. The inactivation of fhaB1 resulted in a high degree of attenuation when the chickens were challenged intranasally and a lesser degree when challenged intramuscularly. The fhaB1 mutant and the wild type strain were investigated their sensitivity to the antibody-dependent classical complement-mediated killing pathway in 90%convalescent chicken serum. The fhaB1 mutant was serum sensitive as the viability has reduced between untreated serum and heat inactivated chicken serum (P<0.007). These results conifrmed that FhaB1 played the critical roles in the bacterial pathogenesis and further studies were needed to investigate the mechanism which caused reduced virulence of the fhaB1 mutant.

  18. pnp4a Is the Causal Gene of the Medaka Iridophore Mutant guanineless

    Directory of Open Access Journals (Sweden)

    Tetsuaki Kimura

    2017-04-01

    Full Text Available See-through medaka lines are suitable for observing internal organs throughout life. They were bred by crossing multiple color mutants. However, some of the causal genes for these mutants have not been identified. The medaka has four pigment cell types: black melanophores, yellow xanthophores, white leucophores, and silvery iridophores. The causal genes of melanophore, xanthophore, and leucophore mutants have been elucidated, but the causal gene for the iridophore mutant remains unknown. Here, we describe the iridophore mutant, guanineless (gu, which exhibits a strong reduction in visible iridophores throughout its larval to adult stages. The gu locus was previously mapped to chromosome 5, but was located near the telomeric region, making it difficult to integrate into the chromosome. We sought the causal gene of gu using synteny analysis with the zebrafish genome and found a strong candidate, purine nucleoside phosphorylase 4a (pnp4a. Gene targeting and complementation testing showed that pnp4a is the causal gene of gu. This result will allow the establishment of inbred medaka strains or other useful strains with see-through phenotypes without major disruption in the genetic background of each strain.

  19. Enhanced production of alkaline protease by a mutant of Bacillus licheniformis N-2 for dehairing

    Directory of Open Access Journals (Sweden)

    Muhammad Nadeem

    2010-10-01

    Full Text Available The purpose of the present investigations was to improve the yield of alkaline protease for leather dehairing by subjecting the indigenous proteolytic strain Bacillus licheniformis N-2 to various mutagenic treatments viz. UV irradiations, NTG (N-methyl-N-nitro-N-nitrosoguinidine and MMS (methyl methane sulfonate. After screening on skim milk agar plates, a total of nine positive mutants were selected for shake flask experiments. Among these, the best proteolytic mutant designated as UV-9 showed 1.4 fold higher alkaline protease activity in preoptimized growth medium than the parent strain. The fermentation profile and kinetic parameters such u(h-1, Yp/s, Yp/x, Yx/s, q s, Qs, q p and Qp also indicated the superiority of the selected mutant UV-9 for alkaline protease production over the parent strain and rest of the mutants. The dehairing capability of mutant UV-9 alkaline protease was analyzed by soaking goat skin pieces for different time intervals (3-15 h at 40 º C. A complete dehairing without degradation of collagen was achieved after 12 h, indicating its commercial exploitation in leather industry.

  20. Isolation and characterization of an Ashbya gossypii mutant for improved riboflavin production

    Directory of Open Access Journals (Sweden)

    Shiping Wei

    2012-06-01

    Full Text Available The use of the filamentous fungus, Ashbya gossypii, to improve riboflavin production at an industrial scale is described in this paper. A riboflavin overproducing strain was isolated by ultraviolet irradiation. Ten minutes after spore suspensions of A. gossypii were irradiated by ultraviolet light, a survival rate of 5.5% spores was observed, with 10% of the surviving spores giving rise to riboflavin-overproducing mutants. At this time point, a stable mutant of the wild strain was isolated. Riboflavin production of the mutant was two fold higher than that of the wild strain in flask culture. When the mutant was growing on the optimized medium, maximum riboflavin production could reach 6.38 g/l. It has even greater promise to increase its riboflavin production through dynamic analysis of its growth phase parameters, and riboflavin production could reach 8.12 g/l with pH was adjusted to the range of 6.0-7.0 using KH2PO4 in the later growth phase. This mutant has the potential to be used for industrial scale riboflavin production.

  1. Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants.

    Science.gov (United States)

    Quivey, R G; Grayhack, E J; Faustoferri, R C; Hubbard, C J; Baldeck, J D; Wolf, A S; MacGilvray, M E; Rosalen, P L; Scott-Anne, K; Santiago, B; Gopal, S; Payne, J; Marquis, R E

    2015-12-01

    A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.

  2. The mitochondrial genome of the fission yeast Schizosaccharomyces pombe : 5. Characterization of mitochondrial deletion mutants.

    Science.gov (United States)

    Ahne, F; Merlos-Lange, A M; Lang, B F; Wolf, K

    1984-09-01

    The three mutator strains ana (r)-8, ana (r)-14, and diu (r)-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe.

  3. Photooxidative stress stimulates illegitimate recombination and mutability in carotenoid-less mutants of Rubrivivax gelatinosus.

    Science.gov (United States)

    Ouchane, S; Picaud, M; Vernotte, C; Astier, C

    1997-08-01

    Carotenoids are essential to protection against photooxidative damage in photosynthetic and non-photosynthetic organisms. In a previous study, we reported the disruption of crtD and crtC carotenoid genes in the purple bacterium Rubrivivax gelatinosus, resulting in mutants that synthesized carotenoid intermediates. Here, carotenoid-less mutants have been constructed by disruption of the crtB gene. To study the biological role of carotenoids in photoprotection, the wild-type and the three carotenoid mutants were grown under different conditions. When exposed to photooxidative stress, only the carotenoid-less strains (crtB-) gave rise with a high frequency to four classes of mutants. In the first class, carotenoid biosynthesis was partially restored. The second class corresponded to photosynthetic-deficient mutants. The third class corresponded to mutants in which the LHI antenna level was decreased. In the fourth class, synthesis of the photosynthetic apparatus was inhibited only in aerobiosis. Molecular analyses indicated that the oxidative stress induced mutations and illegitimate recombination. Illegitimate recombination events produced either functional or non-functional chimeric genes. The R. gelatinosus crtB- strain could be very useful for studies of the SOS response and of illegitimate recombination induced by oxidants in bacteria.

  4. Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants of Ustilago maydis

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.

    1977-01-01

    The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungus Ustilago maydis after incubation at the restrictive temperature (32/sup 0/C) for eight hours. Mutants ts-220, ts-207, ts-432 and ts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutants ts-20, tsd 1-1, ts-84 and pol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutant pol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutant ts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32/sup 0/C. tsd 1-1 and ts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis which correlates to increasing UV sensitivity of these strains on incubation at 32/sup 0/C. A pol 1-1 ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.

  5. Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon.

    Science.gov (United States)

    Yano, Koichi; Masuda, Kenta; Akanuma, Genki; Wada, Tetsuya; Matsumoto, Takashi; Shiwa, Yuh; Ishige, Taichiro; Yoshikawa, Hirofumi; Niki, Hironori; Inaoka, Takashi; Kawamura, Fujio

    2016-01-01

    The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

  6. TagSmart: analysis and visualization for yeast mutant fitness data measured by tag microarrays

    Directory of Open Access Journals (Sweden)

    Xie Dan

    2007-04-01

    Full Text Available Abstract Background A nearly complete collection of gene-deletion mutants (96% of annotated open reading frames of the yeast Saccharomyces cerevisiae has been systematically constructed. Tag microarrays are widely used to measure the fitness of each mutant in a mutant mixture. The tag array experiments can have a complex experimental design, such as time course measurements and drug treatment with multiple dosages. Results TagSmart is a web application for analysis and visualization of Saccharomyces cerevisiae mutant fitness data measured by tag microarrays. It implements a robust statistical approach to assess the concentration differences among S. cerevisiae mutant strains. It also provides an interactive environment for data analysis and visualization. TagSmart has the following advantages over previously described analysis procedures: 1 it is user-friendly software rather than merely a description of analytical procedure; 2 It can handle complicated experimental designs, such as multiple time points and treatment with multiple dosages; 3 it has higher sensitivity and specificity; 4 It allows users to mask out "bad" tags in the analysis. Two biological tests were performed to illustrate the performance of TagSmart. First, we generated titration mixtures of mutant strains, in which the relative concentration of each strain was controlled. We used tag microarrays to measure the numbers of tag copies in each titration mixture. The data was analyzed with TagSmart and the result showed high precision and recall. Second, TagSmart was applied to a dataset in which heterozygous deletion strain mixture pools were treated with a new drug, Cincreasin. TagSmart identified 53 mutant strains as sensitive to Cincreasin treatment. We individually tested each identified mutant, and found 52 out of the 53 predicted mutants were indeed sensitive to Cincreasin. Conclusion TagSmart is provided "as is" to analyze tag array data produced by Affymetrix and Agilent

  7. Specific mistranslation in hisT mutants of Escherichia coli.

    Science.gov (United States)

    Parker, J

    1982-01-01

    Certain strains of Escherichia coli mistranslate at very high frequencies when starved for asparagine or histidine. This mistranslation is the result of misreading events on the ribosome. The introduction of a hisT mutation into such a strain decreases the frequency of mistranslation during histidine starvation but not during asparagine starvation. The most likely explanation is that the replacement of the pseudouridine residue in the anticodon loop of glutamine specific transfer ribonucleic acid by uridine in hisT mutants leads to an increase in fidelity of transfer ribonucleic acid function. The hisT gene in Escherichia coli has also been more accurately mapped, giving the gene order purF-hisT-aroC-fadL-dsdA.

  8. Growth and immunity conferred by a Plasmodium falciparum temperature sensitive mutant in Panamanian owl monkeys.

    Science.gov (United States)

    Inselburg, J; Rossan, R N; Escajadillo, A

    1989-05-01

    We have compared the growth of the wild type Plasmodium falciparum strain Honduras 1 and a previously isolated temperature sensitive mutant of it, AP1-16, in Panamanian owl monkeys. We examined serially infected splenectomized and normal animals that were initially infected with cultured parasites that had been grown in a mixture of owl monkey and human erythrocytes. Initial infections in splenectomized monkeys were marked by multiple recrudescences. The mutant grew less well than the wild type in the splenectomized monkeys, as determined by lower peak and total parasitemias. In the splenectomized monkeys tested by rechallenge with the wild type parasite, the mutant stimulated a comparable degree of protection. That protection was manifested in 2 ways. There was a marked reduction in the level of the primary parasitemia in the rechallenged monkeys and an absence of recrudescent parasitemias after the primary parasitemia. The potential value of generating and studying temperature sensitive P. falciparum strains that show attenuated growth is considered.

  9. Physiological and biochemical characteristics of laboratory induced mutants of Botrytis cinerea with resistance to fluazinam.

    Science.gov (United States)

    Shao, Wenyong; Zhang, Yu; Ren, Weichao; Chen, Changjun

    2015-01-01

    Botrytis cinerea is a necrotrophic and filamentous fungus with a high risk of developing resistance to fungicides. The pyridinamine fungicide fluazinam has been reported to have excellent activity against B. cinerea and better effect on controlling gray mold. In this study, the physiological and biochemical characteristics of laboratory-induced mutants of B. cinerea with resistance to fluazinam has been investigated. Compared to the wild-type strains, the fluazinam-resistant mutants had a significant decrease in respiratory rate, glycerol, oxalate, and ATP contents, and an increase in ATPase activity and sensitivity to osmotic pressure, but did not differ in cell membrane permeability. Sequencing indicated that two parental strains and four resistant mutants were identical in the nucleotide sequence of F-ATPase gene. These results will enrich our understanding of the resistance mechanism of B. cinerea to fluazinam.

  10. luxS Mutant Regulation: Quorum Sensing Impairment or Methylation Disorder?

    Directory of Open Access Journals (Sweden)

    Zhengwei Huang

    2012-05-01

    Full Text Available AI-2–mediated quorum sensing has been identified in various bacteria, including both Gram-negative and Gram-positive species, and numerous phenotypes have been reported to be regulated by this mechanism, using the luxS-mutant strain. But the AI-2 production process confused this regulatory function; some considered this regulation as the result of a metabolic change, which refers to an important metabolic cycle named activated methyl cycle (AMC, caused by luxS-mutant simultaneously with the defect of AI-2. Herein we hypothesized that the quorum sensing system—not the metabolic aspect—is responsible for such a regulatory function. In this study, we constructed plasmids infused with sahH and induced protein expression in the luxS-mutant strain to make the quorum-sensing system and metabolic system independent. The biofilm-related genes were investigated by real-time polymerase chain reaction (PCR, and the results demonstrated that the quorum-sensing completed strain restored the gene expression of the defective strain, but the metabolically completed one did not. This evidence supported our hypothesis that the autoinducer-2-mediated, quorum-sensing system, not the AMC, was responsible for luxS mutant regulation.

  11. In vitro capability of faropenem to select for resistant mutants of Streptococcus pneumoniae and Haemophilus influenzae.

    Science.gov (United States)

    Kosowska-Shick, Klaudia; Clark, Catherine; Credito, Kim; Dewasse, Bonifacio; Beachel, Linda; Ednie, Lois; Appelbaum, Peter C

    2008-02-01

    When tested against nine strains of pneumococci and six of Haemophilus influenzae of various resistotypes, faropenem failed to select for resistant mutants after 50 days of consecutive subculture in subinhibitory concentrations. Faropenem also yielded low rates of spontaneous mutations against all organisms of both species. By comparison, resistant clones were obtained with macrolides, ketolides, and quinolones.

  12. Combinatorial synthetic peptide vaccine strategy protects against hypervirulent CovR/S mutant streptococci

    DEFF Research Database (Denmark)

    Pandey, Manisha; Mortensen, Rasmus; Calcutt, Ainslie;

    2016-01-01

    Cluster of virulence responder/sensor (CovR/S) mutant group A streptococci (GAS) are serious human pathogens of multiple M protein strains that upregulate expression of virulence factors, including the IL-8 protease Streptococcus pyogenes cell envelope proteinase (SpyCEP), thus blunting neutrophi...

  13. Immuogenicity and safety of a natural rough mutant of Brucella suis as a vaccine for swine

    Science.gov (United States)

    The objective of the current study was to evaluate the safety, immunogenicity and clearance of the natural rough mutant of Brucella suis strain 353-1 (353-1) as a vaccine in domestic swine. In three studies encompassing 155 animals, pigs were inoculated with 353-1 by conjunctival (5 x 10**7 CFU), p...

  14. Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Deltaomp25 deletion mutant.

    Science.gov (United States)

    Edmonds, M D; Cloeckaert, A; Hagius, S D; Samartino, L E; Fulton, W T; Walker, J V; Enright, F M; Booth, N J; Elzer, P H

    2002-06-01

    The Brucella melitensis mutant BM 25, which lacks the major 25 kDa outer membrane protein Omp25, has previously been found to be attenuated in the murine brucellosis model. In the present study, the capacity of the Deltaomp25 mutant to colonise and cause abortions in the caprine host was evaluated. The vaccine potential of BM 25 was also investigated in goats. Inoculation of nine pregnant goats in late gestation with the B. melitensis mutant resulted in 0/9 abortions, while the virulent parental strain, B. melitensis 16M, induced 6/6 dams to abort (Pgoats for two weeks post-infection. Owing to the ability of BM 25 to colonise both non-pregnant and pregnant adults without inducing abortions, a vaccine efficacy study was performed. Vaccination of goats prior to breeding with either BM 25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in 100 per cent protection against abortion following challenge in late gestation with virulent strain 16M (Pmelitensis Deltaomp25 mutant, BM 25, may be a safe and efficacious alternative to strain Rev. 1 when dealing with goat herds of mixed age and pregnancy status.

  15. Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp.

    Science.gov (United States)

    Miao, Xiaoling; Wu, Qingyu; Wu, Guifang; Zhao, Nanming

    2003-03-01

    The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp-) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp- cells. The agp- mutant had 38% less photosynthetic capacity when grown in light over 600 micromol m(-2) s(-1). Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria.

  16. Ultrastructural Characterization of Infection and Colonization of Maize Leaves by Colletotrichum graminicola, and by a C. graminicola Pathogenicity Mutant.

    Science.gov (United States)

    Mims, C W; Vaillancourt, L J

    2002-07-01

    ABSTRACT Observations were made of the ultrastructure of infection and colonization of leaves of a susceptible maize inbred by Colletotrichum graminicola and by a C. graminicola pathogenicity mutant. The mutant causes no symptoms on either maize leaves or stalks. Prior evidence suggested that it is deficient in production of signal peptidase, responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum. There was no significant difference in the process of infection or colonization by the mutant and wild-type strains up to 48 h after inoculation. Both the mutant and the wild type produced globose, melanized appressoria within 24 h after inoculation on the host surface. By 36 h, both strains had penetrated the host epidermal cells directly. The host cells frequently formed papillae in response to appressoria, but these were not usually successful in preventing fungal ingress in either case. Penetration was followed by formation of irregularly shaped, swollen infection hyphae. Infection hyphae of both strains grew biotrophically for a relatively short time (less than 12 h). One or more hyphal branches was produced from each infection hypha, and these invaded adjacent mesophyll cells. Both strains of the fungus grew cell-to-cell, setting up new biotrophic interactions in each cell, between 36 and 48 h after inoculation. Papillae were frequently formed by the mesophyll cells, but these were not successful in preventing fungal ingress. The first noticeable difference between the mutant and the wild type was related to their interaction with mesophyll cells. Cells invaded by the wild type died relatively quickly, whereas those infected by the mutant appeared to survive longer. The most dramatic difference between the mutant and wild type occurred when the mutant completely failed to make a transition to necrotrophic growth, while the wild type made that switch at 48 to 72 h after inoculation. The mutant may be unable

  17. Early development of podocyte injury independently of hyperglycemia and elevations in arterial pressure in nondiabetic obese Dahl SS leptin receptor mutant rats.

    Science.gov (United States)

    McPherson, Kasi C; Taylor, Lateia; Johnson, Ashley C; Didion, Sean P; Geurts, Aron M; Garrett, Michael R; Williams, Jan M

    2016-10-01

    The current study examined the effect of obesity on the development of renal injury within the genetic background of the Dahl salt-sensitive rat with a dysfunctional leptin receptor derived from zinc-finger nucleases (SS(LepR)mutant strain). At 6 wk of age, body weight was 35% higher in the SS(LepR)mutant strain compared with SSWT rats and remained elevated throughout the entire study. The SS(LepR)mutant strain exhibited impaired glucose tolerance and increased plasma insulin levels at 6 wk of age, suggesting insulin resistance while SSWT rats did not. However, blood glucose levels were normal throughout the course of the study. Systolic arterial pressure (SAP) was similar between the two strains from 6 to 10 wk of age. However, by 18 wk of age, the development of hypertension was more severe in the SS(LepR)mutant strain compared with SSWT rats (201 ± 10 vs. 155 ± 3 mmHg, respectively). Interestingly, proteinuria was substantially higher at 6 wk of age in the SS(LepR)mutant strain vs. SSWT rats (241 ± 27 vs. 24 ± 2 mg/day, respectively) and remained elevated until the end of the study. The kidneys from the SS(LepR)mutant strain displayed significant glomerular injury, including podocyte foot process effacement and lipid droplets compared with SSWT rats as early as 6 wk of age. By 18 wk of age, plasma creatinine levels were twofold higher in the SS(LepR)mutant strain vs. SSWT rats, suggesting the presence of chronic kidney disease (CKD). Overall, these results indicate that the SS(LepR)mutant strain develops podocyte injury and proteinuria independently of hyperglycemia and elevated arterial pressure that later progresses to CKD. Copyright © 2016 the American Physiological Society.

  18. Genetics and complementation of Haemophilus influenzae mutants deficient in adenosine 5'-triphosphate-dependent nuclease

    Energy Technology Data Exchange (ETDEWEB)

    Kooistra, J.; Small, G.D.; Setlow, J.K.; Shapanka, R.

    1976-04-01

    Eight different mutations in Haemophilus influenzae leading to deficiency in adenosine 5'-triphosphate (ATP)-dependent nuclease have been investigated in strains in which the mutations of the originally mutagenized strains have been transferred into the wild type. Sensitivity to mitomycin C and deoxycholate and complementation between extracts and deoxyribonucleic acid (DNA)-dependent ATPase activity have been measured. Genetic crosses have provided information on the relative position of the mutations on the genome. There are three complementation groups, corresponding to three genetic groups. The strains most sensitive to mitomycin and deoxycholate, derived from mutants originally selected on the basis of sensitivity to mitomycin C or methyl methanesulfonate, are in one group. Apparently all these sensitive strains lack DNA-dependent ATPase activity, as does a strain intermediate in sensitivity to deoxycholate, which is the sole representative of another group. There are four strains that are relatively resistant to deoxycholate and mitomycin C, and all of these contain the ATPase activity.

  19. Construction, detection and microarray analysis on Shigella dysenteriae A1 IroN, ShuA single, double mutants

    Institute of Scientific and Technical Information of China (English)

    BIN; Wen; LIU; Moqing; PENG; Junping; SUN; Lilian; XU; Xingye; ZHANG; Jinghai; JIN; Qi

    2006-01-01

    In this study, we constructed single mutants MTS-1, MTS-2 of IroN and ShuA gene and double mutant MTS of them in Shigella dysenteriae A1 strain 51197 by insert and absence. The functional detection of every mutant was performed at the level of culture medium and cell experiment. The gene expression profiles of the mutants and the wild-type strains under iron- enriched and iron-limited conditions were analyzed by the SD51197 whole genomic microarray. The results showed that all the mutants grew obviously less well than the wild-type strains in L broth appending iron chelator DIP. The addition of iron to the cultures can stimulate the growth of mutants back to wild-type levels. In either the experiments on the ability of intracellular multiplication or the cell-to-cell spread in HeLa and U937 cell lines, mutants showed no obvious change in virulence compared with the parental strain SD51197. However when DIP was added to the cultured HeLa cells, the ability of intracellular multiplication of MTS-1, MTS-2, MTS has reduced about 23.4%, 25.2%, 43.6% respectively. The analysis of expression profiles under the iron-limited condition showed that the mutants were more sensitive for the changes of iron deficiency than the wild-type strains, many genes have been altered. Up-regulated genes mainly involved genes of transcription, coenzyme metabolism, amino acid transport and metabolism, and unknown functional genes, while down-regulated genes mainly involved genes of energy and carbohydrate metabolism and unknown function genes; the expression levels of known iron-transport associated genes generally showed up-regulated. The results demonstrated that iron-transport associated genes IroN, ShuA were likely to have some effects on the virulence and growth of S. dysenteriae.

  20. Loss of genetic accuracy in mutants of the thermoacidophile Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Greg D. Bell

    2002-01-01

    Full Text Available To investigate how hyperthermophilic archaea can propagate their genomes accurately, we isolated Sulfolobus acidocaldarius mutants exhibiting abnormally high rates of spontaneous mutation. Our isolation strategy involved enrichment for mutator lineages via alternating selections, followed by screening for the production of spontaneous, 5-fluoro-orotate-resistant mutants in micro-colonies. Several candidates were evaluated and found to have high frequencies of pyrE and pyrF mutation and reversion. Neither an increased efficiency of plating of mutants on selective medium, nor the creation of a genetically unstable pyrE allele, could be implicated as the cause of these high frequencies. The strains had elevated frequencies of other mutations, and exhibited certain phenotypic differences among themselves. A large increase in sensitivity to DNA-damaging agents was not observed, however. These properties generally resemble those of bacterial mutator mutants and suggest loss of functions specific to genetic accuracy.

  1. Protective properties of rifampin-resistant rough mutants of Brucella melitensis.

    Science.gov (United States)

    Adone, R; Ciuchini, F; Marianelli, C; Tarantino, M; Pistoia, C; Marcon, G; Petrucci, P; Francia, M; Riccardi, G; Pasquali, P

    2005-07-01

    Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.

  2. Pattern formation mechanisms in motility mutants of Myxococcus xanthus

    CERN Document Server

    Starruss, Joern; Jakovljevic, Vladimir; Sogaard-Andersen, Lotte; Deutsch, Andreas; Baer, Markus

    2016-01-01

    Formation of spatial patterns of cells is a recurring theme in biology and often depends on regulated cell motility. Motility of M. xanthus depends on two motility machineries: the S-engine and A-engine. Moving M. xanthus cells can organize into spreading colonies or spore-filled fruiting bodies depending on their nutritional status. To understand these two pattern formation processes and the contributions by the two motility machineries, as well as cell reversal, we analyze spatial self-organization in 3 strains: i) a mutant that moves unidirectionally without reversing by the A-motility system only, ii) a unidirectional mutant that is also equipped with the S-motility system, and iii) the wild-type that, in addition to the two motility systems, reverses its direction of movement. The mutant moving by the A-engine illustrates that collective motion in the form of large moving clusters can arise in gliding bacteria due to steric interactions of the rod-shaped cells, without the need of invoking any biochemica...

  3. Biochemical and histological characterization of tomato mutants

    Directory of Open Access Journals (Sweden)

    Carolina C. Monteiro

    2012-06-01

    Full Text Available Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT, we observed that the malondialdehyde (MDA content was enhanced in the diageotropica (dgt and lutescent (l mutants, whilst the highest levels of hydrogen peroxide (H2O2 were observed in high pigment 1 (hp1 and aurea (au mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT activity when compared to MT. Guaiacol peroxidase (GPOX was enhanced in both sitiens (sit and notabilis (not mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX. Based on PAGE analysis, the activities of glutathione reductase (GR isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD isoform III was reduced in leaves of sit, epi, Never ripe (Nr and green flesh (gf mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.Neste trabalho, analisamos as respostas bioquímicas inerentes ao sistema antioxidante, assim como propriedades morfológicas e anatômicas de mutantes fotomorfogenéticos e hormonais de tomateiro. Comparados ao não mutante Micro-Tom (MT, observamos que o conteúdo de malondialdeído (MDA aumentou nos mutantes diageotropica (dgt e lutescent (l, enquanto os maiores níveis de H2O2 foram encontrados nos mutantes high pigment 1 (hp1 e aurea (au. Análises de enzimas antioxidantes mostraram que todos os mutantes reduziram a atividade de catalase (CAT quando comparado a MT. A

  4. An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Li, Xiaobo; Zhang, Ru; Patena, Weronika; Gang, Spencer S; Blum, Sean R; Ivanova, Nina; Yue, Rebecca; Robertson, Jacob M; Lefebvre, Paul A; Fitz-Gibbon, Sorel T; Grossman, Arthur R; Jonikas, Martin C

    2016-02-01

    The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.

  5. Cariogenicity of a lactate dehydrogenase-deficient mutant of Streptococcus mutans serotype c in gnotobiotic rats.

    Science.gov (United States)

    Fitzgerald, R J; Adams, B O; Sandham, H J; Abhyankar, S

    1989-03-01

    A lactate dehydrogenase-deficient (Ldh-) mutant of a human isolate of Streptococcus mutans serotype c was tested in a gnotobiotic rat caries model. Compared with the wild-type Ldh-positive (Ldh+) strains, it was significantly (alpha less than or equal to 0.005) less cariogenic in experiments with two different sublines of Sprague-Dawley rats. The Ldh- mutant strain 044 colonized the oral cavity of the test animals to the same extent as its parent strain 041, although its initial implantation was slightly but not significantly (P greater than or equal to 0.2) less. Multiple oral or fecal samples plated on 2,3,5-triphenyltetrazolium indicator medium revealed no evidence of back mutation from Ldh- to Ldh+ in vivo. Both Ldh+ strain 041 and Ldh- strain 044 demonstrated bacteriocinlike activity in vitro against a number of human strains of mutans streptococci representing serotype a (S. cricetus) and serotypes c and e (S. mutans). Serotypes b (S. rattus) and f (S. mutans) and strains of S. mitior, S. sanguis, and S. salivarius were not inhibited. Thus, Ldh mutant strain 044 possesses a number of desirable traits that suggest it should be investigated further as a possible effector strain for replacement therapy of dental caries. These traits include its stability and low cariogenicity in the sensitive gnotobiotic rat caries model, its bacteriocinlike activity against certain other cariogenic S. mutans (but not against more inocuous indigenous oral streptococci), and the fact that it is a member of the most prevalent human serotype of cariogenic streptococci.

  6. Hepatitis B surface gene 145 mutant as a minor population in hepatitis B virus carriers

    Directory of Open Access Journals (Sweden)

    Komatsu Haruki

    2012-01-01

    Full Text Available Abstract Background Hepatitis B virus (HBV can have mutations that include the a determinant, which causes breakthrough infection. In particular, a single mutation at amino acid 145 of the surface protein (G145 is frequently reported in the failure of prophylactic treatment. The aim of this study was to evaluate the frequency of the a determinant mutants, especially the G145 variant, in Japan, where universal vaccination has not been adopted. Methods The present study was a retrospective study. The study cohorts were defined as follows: group 1, children with failure to prevent mother-to-child transmission despite immunoprophylaxis (n = 18, male/female = 8/10, age 1-14 years; median 6 years; group 2, HBV carriers who had not received vaccination or hepatitis B immunoglobulin (n = 107, male/female = 107, age 1-52 years; median 16 years. To detect the G145R and G145A mutants in patients, we designed 3 probes for real-time PCR. We also performed direct sequencing and cloning of PCR products. Results By mutant-specific real-time PCR, one subject (5.6% was positive for the G145R mutant in group 1, while the G145 mutant was undetectable in group 2. The a determinant mutants were detected in one (5.6% of the group 1 subjects and 10 (9.3% of the group 2 subjects using direct sequencing, but direct sequencing did not reveal the G145 mutant as a predominant strain in the two groups. However, the subject who was positive according to the mutant-specific real-time PCR in group 1 had overlapped peaks at nt 587 in the electropherogram. In group 2, 11 patients had overlapped peaks at nt 587 in the electropherogram. Cloning of PCR products allowed detection of the G145R mutant as a minor strain in 7 (group 1: 1 subject, group 2: 6 subjects of 12 subjects who had overlapped peaks at nt 587 in the electropherogram. Conclusions The frequency of the a determinant mutants was not high in Japan. However, the G145R mutant was often present as a minor population in

  7. The Effect of Vitamin E on the Survival Rate of unc-13 Caenorhabditis elegans mutants under Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Jessica Porcelan

    2012-01-01

    Full Text Available Caenorhabditis elegans unc-13 mutants express decreased neuronal activity and thus are a good model strain for examining defective nervous systems. These unc-13 mutants as well as wild type N2 strains, show rapid mortality when under oxidative stress. However, the antioxidant vitamin E may prolong survival in unc-13 mutant and N2 strains under oxidative stress. The addition of vitamin E to organisms under oxidative stress has a protective effect in both N2 and unc-13 C. elegans strains. Interestingly, vitamin E resulted in a greater increase in survival rate in N2 worms than with unc-13 mutant worms. While both strains displayed lower mortality rates with the addition of vitamin E, this finding suggests that vitamin E more efficiently increases survival rates of C. elegans with typical nervous system function. The efficacy of vitamin E implies that use of antioxidants may lessen the damage caused by oxidative stress in both N2 and mutant worms.

  8. Isolation and partial characterization of a mutant of Bacillus thuringiensis producing melanin Isolamento e caracterização parcial de um mutante de Bacillus thuringiensis produtor de melanina

    Directory of Open Access Journals (Sweden)

    Gislayne T. Vilas-Bôas

    2005-09-01

    Full Text Available A mutant (407-P of Bacillus thuringiensis subsp. thuringiensis strain 407 producing a melanin was obtained after treatment with the mutagenic agent ethyl-methane-sulfonate. Several microbiological and biochemical properties of the two strains were analyzed and the results were similar. The mutant 407-P was also incorporated into non-sterilized soil samples, recovered, easily identified, and quantified, what enables its use in ecology of B. thuringiensis.Um mutante (407-P da linhagem Bacillus thuringiensis subsp. thuringiensis 407 produtor de melanina foi obtido após tratamento com o agente mutagênico etil-metano-sulfonato. Diversas propriedades microbiológicas e bioquímicas das duas linhagens foram analisadas e os resultados foram similares. O mutante 407-P foi incorporado em amostras de solo não esterilizado, recuperado, facilmente identificado e quantificado, possibilitando seu uso em estudos de ecologia de B. thuringiensis.

  9. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA.

    OpenAIRE

    Ostroff, G. R.; Pène, J. J.

    1983-01-01

    Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.

  10. Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation

    DEFF Research Database (Denmark)

    Hammer, Karin; Jensen, Kaj Frank; Poulsen, Peter;

    1987-01-01

    Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage lambda cII protein were isolated. The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of c...

  11. Generation of astaxanthin mutants in Xanthophyllomyces dendrorhous using a double recombination method based on hygromycin resistance.

    Science.gov (United States)

    Niklitschek, Mauricio; Baeza, Marcelo; Fernández-Lobato, María; Cifuentes, Víctor

    2012-01-01

    Generally two selection markers are required to obtain homozygous mutations in a diploid background, one for each gene copy that is interrupted. In this chapter is described a method that allows the double gene deletions of the two copies of a gene from a diploid organism, a wild-type strain of the Xanthophyllomyces dendrorhous yeast, using hygromycin B resistance as the only selection marker. To accomplish this, in a first step, a heterozygous hygromycin B-resistant strain is obtained by a single process of transformation (carrying the inserted hph gene). Following, the heterozygous mutant is grown in media with increasing concentrations of the antibiotic. In this way, the strains that became homozygous (by mitotic recombination) for the antibiotic marker would able to growth at higher concentration of the antibiotic than the heterozygous. The method can be potentially applied for obtaining double mutants of other diploid organisms.

  12. CMPD: cancer mutant proteome database.

    Science.gov (United States)

    Huang, Po-Jung; Lee, Chi-Ching; Tan, Bertrand Chin-Ming; Yeh, Yuan-Ming; Julie Chu, Lichieh; Chen, Ting-Wen; Chang, Kai-Ping; Lee, Cheng-Yang; Gan, Ruei-Chi; Liu, Hsuan; Tang, Petrus

    2015-01-01

    Whole-exome sequencing, which centres on the protein coding regions of disease/cancer associated genes, represents the most cost-effective method to-date for deciphering the association between genetic alterations and diseases. Large-scale whole exome/genome sequencing projects have been launched by various institutions, such as NCI, Broad Institute and TCGA, to provide a comprehensive catalogue of coding variants in diverse tissue samples and cell lines. Further functional and clinical interrogation of these sequence variations must rely on extensive cross-platforms integration of sequencing information and a proteome database that explicitly and comprehensively archives the corresponding mutated peptide sequences. While such data resource is a critical for the mass spectrometry-based proteomic analysis of exomic variants, no database is currently available for the collection of mutant protein sequences that correspond to recent large-scale genomic data. To address this issue and serve as bridge to integrate genomic and proteomics datasets, CMPD (http://cgbc.cgu.edu.tw/cmpd) collected over 2 millions genetic alterations, which not only facilitates the confirmation and examination of potential cancer biomarkers but also provides an invaluable resource for translational medicine research and opportunities to identify mutated proteins encoded by mutated genes.

  13. Hip flexor strain - aftercare

    Science.gov (United States)

    ... flexor - aftercare; Hip flexor injury - aftercare; Hip flexor tear - aftercare; Iliopsoas strain - aftercare; Strained iliopsoas muscle - aftercare; Torn iliopsoas muscle - aftercare; Psoas strain - aftercare

  14. Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

    Science.gov (United States)

    Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

    2014-09-01

    Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

  15. Branched-chain fatty acid biosynthesis in a branched-chain amino acid aminotransferase mutant of Staphylococcus carnosus

    DEFF Research Database (Denmark)

    Beck, Hans Christian

    2005-01-01

    Fatty acid biosynthesis by a mutant strain of Staphylococcus carnosus deficient in branched-chain amino acid aminotransferase (IlvE) activity was analysed. This mutant was unable to produce the appropriate branched-chain alpha-ketoacid precursors for branched-chain fatty acid biosynthesis from...... for 2-methylpropanoic acid production, revealing that the IlvE protein plays an important, but not essential role in the biosynthesis of branched-chain fatty acids and secondary metabolites in S. carnosus....

  16. Factors contributing to the biofilm-deficient phenotype of Staphylococcus aureus sarA mutants.

    Directory of Open Access Journals (Sweden)

    Laura H Tsang

    Full Text Available Mutation of sarA in Staphylococcus aureus results in a reduced capacity to form a biofilm, but the mechanistic basis for this remains unknown. Previous transcriptional profiling experiments identified a number of genes that are differentially expressed both in a biofilm and in a sarA mutant. This included genes involved in acid tolerance and the production of nucleolytic and proteolytic exoenzymes. Based on this we generated mutations in alsSD, nuc and sspA in the S. aureus clinical isolate UAMS-1 and its isogenic sarA mutant and assessed the impact on biofilm formation. Because expression of alsSD was increased in a biofilm but decreased in a sarA mutant, we also generated a plasmid construct that allowed expression of alsSD in a sarA mutant. Mutation of alsSD limited biofilm formation, but not to the degree observed with the corresponding sarA mutant, and restoration of alsSD expression did not restore the ability to form a biofilm. In contrast, concomitant mutation of sarA and nuc significantly enhanced biofilm formation by comparison to the sarA mutant. Although mutation of sspA had no significant impact on the ability of a sarA mutant to form a biofilm, a combination of protease inhibitors (E-64, 1-10-phenanthroline, and dichloroisocoumarin that was shown to inhibit the production of multiple extracellular proteases without inhibiting growth was also shown to enhance the ability of a sarA mutant to form a biofilm. This effect was evident only when all three inhibitors were used concurrently. This suggests that the reduced capacity of a sarA mutant to form a biofilm involves extracellular proteases of all three classes (serine, cysteine and metalloproteases. Inclusion of protease inhibitors also enhanced biofilm formation in a sarA/nuc mutant, with the combined effect of mutating nuc and adding protease inhibitors resulting in a level of biofilm formation with the sarA mutant that approached that of the UAMS-1 parent strain. These results

  17. A galU mutant of francisella tularensis is attenuated for virulence in a murine pulmonary model of tularemia

    Directory of Open Access Journals (Sweden)

    Re Fabio

    2011-08-01

    Full Text Available Abstract Background A number of studies have revealed that Francisella tularensis (FT suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen. Results Here we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS. We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1β following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT. Conclusions Disruption of the galU gene of FTLVS has little (if any effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in

  18. Rat Strain Ontology: structured controlled vocabulary designed to facilitate access to strain data at RGD.

    Science.gov (United States)

    Nigam, Rajni; Munzenmaier, Diane H; Worthey, Elizabeth A; Dwinell, Melinda R; Shimoyama, Mary; Jacob, Howard J

    2013-11-22

    The Rat Genome Database (RGD) ( http://rgd.mcw.edu/) is the premier site for comprehensive data on the different strains of the laboratory rat (Rattus norvegicus). The strain data are collected from various publications, direct submissions from individual researchers, and rat providers worldwide. Rat strain, substrain designation and nomenclature follow the Guidelines for Nomenclature of Mouse and Rat Strains, instituted by the International Committee on Standardized Genetic Nomenclature for Mice. While symbols and names aid in identifying strains correctly, the flat nature of this information prohibits easy search and retrieval, as well as other data mining functions. In order to improve these functionalities, particularly in ontology-based tools, the Rat Strain Ontology (RS) was developed. The Rat Strain Ontology (RS) reflects the breeding history, parental background, and genetic manipulation of rat strains. This controlled vocabulary organizes strains by type: inbred, outbred, chromosome altered, congenic, mutant and so on. In addition, under the chromosome altered category, strains are organized by chromosome, and further by type of manipulations, such as mutant or congenic. This allows users to easily retrieve strains of interest with modifications in specific genomic regions. The ontology was developed using the Open Biological and Biomedical Ontology (OBO) file format, and is organized on the Directed Acyclic Graph (DAG) structure. Rat Strain Ontology IDs are included as part of the strain report (RS: ######). As rat researchers are often unaware of the number of substrains or altered strains within a breeding line, this vocabulary now provides an easy way to retrieve all substrains and accompanying information. Its usefulness is particularly evident in tools such as the PhenoMiner at RGD, where users can now easily retrieve phenotype measurement data for related strains, strains with similar backgrounds or those with similar introgressed regions. This

  19. Temperature-sensitive mutants of Pseudomonas aeruginosa: isolation and preliminary immunological evaluation.

    Science.gov (United States)

    Hooke, A M; Arroyo, P J; Oeschger, M P; Bellanti, J A

    1982-01-01

    The immunogenicity of two temperature-sensitive (ts) mutants of Pseudomonas aeruginosa immunotype 1, isolated and characterized for the development of a safe, live vaccine strain, was evaluated in a mouse protection model. One mutant, A/10/25, had a limited "coasting" property (i.e., continued replication for two divisions) at the nonpermissive temperature (36 degrees C), whereas the other mutant, E/9/9, continued replication for five generations after transfer to 36 degrees C. Groups of 3- to 5-week-old ICR mice were immunized intraperitoneally with various doses of the two ts mutants; at various times thereafter, the mice were challenged intraperitoneally with lethal doses of the parental wild type. The more extensive coaster, E/9/9, induced 100% protection at immunizing doses lower than those required for A/10/25 to induce the same protection (1 x 10(8) to 2 x 10(8) and 6 x 10(8) colony-forming units, respectively). Both ts strains induced significant protection for up to 5 weeks after immunization. The results of these studies suggest that the use of P. aeruginosa ts mutants might provide a novel approach to the prevention of P. aeruginosa colonization of patients with cystic fibrosis. PMID:6815088

  20. Selection of the Mutants with High Hydroquinone Degradation Ability of Serratia Marcesscen by Plasma Mutation

    Institute of Scientific and Technical Information of China (English)

    YAO Risheng; YOU Qidong; HE Weijing; ZHU Huixia

    2009-01-01

    In this study, an efficient way by plasma induced mutation was applied to improve the hydroquinone degradation capacity of Serratia marcescens AB 90027 (SM27). The results showed that combined with the selection of hydroquinone tolerance, the mutant with high hy-droquinone degradation ability induced by plasma could be achieved. The best dose for plasma mutation was 15 s, which showed a 47.0% higher positive mutation ratio. Besides, the aimed mutant was markedly different from the parent strain (SM27) in colonial traits while cultivated on Kings media. Finally, the hydroquinone degradation ratio reached 70.5% using the induced mutant strain with 1500 mg/L hydroquinone (HQ) after 15 days of cultivation as the selective conditions; however, it was only 46.7% for SM27. The improvement of the degradation capacity by the induced mutant with a high concentration of HQ selection was attributed to its faster growth and higher hydroquinone tolerance compared with that of the parent strain.

  1. λ N gene expression regulated by translation termination in ribosome L24 mutant

    Institute of Scientific and Technical Information of China (English)

    LI; Muyang; (李沐阳); HU; Qirui; (胡其锐); XUAN; Jinsong; (宣劲松); DENG; Daiyong; (邓代永); WENG; Manli; (翁曼丽)

    2003-01-01

    Besides transcription regulation, gene expression is also regulated at translation level. Although translation regulation is mainly mediated by translation initiation, an abundance of evidence shows that the termination phase of translation is also important for gene expression. The expression of λN gene is down regulated at translation level in L24 mutant, however the precise mechanism still remains unknown. We report here that in an L24 mutant strain, the expression of lac-λN and GST-λN is decreased to 25% and 50% of that in wild type T83 strain respectively. Strikingly, the yield of GST-λN fusion protein in L24 mutant can be restored to the level as in T83 wild type strain by changing the two codons upstream λN stop codon. These findings imply that the stop codon and its context are involved in the translation regulation. The possible reason is that the translation termination complex containing L24 mutant ribosome may not dissociate properly in stop code region. This failure of disengagement from mRNA will slow down the process of following ribosomes, and consequently decrease the efficiency of λN gene expression.

  2. High-throughput transformation method for Yarrowia lipolytica mutant library screening.

    Science.gov (United States)

    Leplat, Christophe; Nicaud, Jean-Marc; Rossignol, Tristan

    2015-09-01

    As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway® vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided.

  3. Exoprotein production correlates with morphotype changes of nonmotile Shewanella oneidensis mutants.

    Science.gov (United States)

    Shi, Miaomiao; Wu, Lin; Xia, Yu; Chen, Haijiang; Luo, Qixia; Sun, Linlin; Gao, Haichun

    2013-04-01

    We report a previously undescribed mechanism for the rugose morphotype in Shewanella oneidensis, a research model for investigating redox transformations of environmental contaminants. Bacteria may form smooth or rugose colonies on agar plates. In general, conversion from the smooth to rugose colony morphotype is attributed to increased production of exopolysaccharide (EPS). In this work, we discovered that aflagellate S. oneidensis mutants grew into rugose colonies, whereas those with nonfunctional flagellar filaments remained smooth. EPS production was not altered in either case, but mutants with the rugose morphotype showed significantly reduced exoprotein secretion. The idea that exoproteins at a reduced level correlate with rugosity gained support from smooth suppressor strains of an aflagellate rugose fliD (encoding the capping protein) mutant, which restored the exoprotein level to the levels of the wild-type and mutant strains with a smooth morphotype. Further analyses revealed that SO1072 (a putative GlcNAc-binding protein) was one of the highly upregulated exoproteins in these suppressor strains. Most intriguingly, this study identified a compensatory mechanism of SO1072 to flagellins possibly mediated by bis-(3'-5')-cyclic dimeric GMP.

  4. Mutation induction of Pleurotus ferulae by low-energy N+ ion implantation and characters of the selected mutant

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to obtain Pleurotus ferulae with high temperature tolerance,mycelium mono-cells of wild type strain ACK was treated by nitrogen ion(5~30 keV,1.5×1015~1.5×1016cm-12)implantation,and mutant CGMCC1762 was selected through auxotrophy screening method,which was Lys.VB6 auxotrophy stress with high temperature.We found that during riper period the surface layer mycelium of the mutant was not aging neither grew tegument even above 30℃.The mycelium endurable temperature of the mutant was increased 70℃ compared with that of the wild type strain.The fruiting bodies growth temperature of the mutant was 16-20℃ in daytime and was 6~12℃ at night.The highest growth temperature of fruiting bodies of the mutant Was increased by 5℃ than that of original strain.Through three generation investigation,we found that the mutant CGMCC1762 was stable with high temperature tolerance.

  5. Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent sulfite liquor and acetic acid.

    Science.gov (United States)

    Harner, Nicole K; Bajwa, Paramjit K; Habash, Marc B; Trevors, Jack T; Austin, Glen D; Lee, Hung

    2014-01-01

    A strain development program was initiated to improve the tolerance of the pentose-fermenting yeast Pachysolen tannophilus to inhibitors in lignocellulosic hydrolysates. Several rounds of UV mutagenesis followed by screening were used to select for mutants of P. tannophilus NRRL Y2460 with improved tolerance to hardwood spent sulfite liquor (HW SSL) and acetic acid in separate selection lines. The wild type (WT) strain grew in 50 % (v/v) HW SSL while third round HW SSL mutants (designated UHW301, UHW302 and UHW303) grew in 60 % (v/v) HW SSL, with two of these isolates (UHW302 and UHW303) being viable and growing, respectively, in 70 % (v/v) HW SSL. In defined liquid media containing acetic acid, the WT strain grew in 0.70 % (w/v) acetic acid, while third round acetic acid mutants (designated UAA301, UAA302 and UAA303) grew in 0.80 % (w/v) acetic acid, with one isolate (UAA302) growing in 0.90 % (w/v) acetic acid. Cross-tolerance of HW SSL-tolerant mutants to acetic acid and vice versa was observed with UHW303 able to grow in 0.90 % (w/v) acetic acid and UAA302 growing in 60 % (v/v) HW SSL. The UV-induced mutants retained the ability to ferment glucose and xylose to ethanol in defined media. These mutants of P. tannophilus are of considerable interest for bioconversion of the sugars in lignocellulosic hydrolysates to ethanol.

  6. Identification of a novel G2073A mutation in 23S rRNA in amphenicol-selected mutants of Campylobacter jejuni.

    Directory of Open Access Journals (Sweden)

    Licai Ma

    Full Text Available OBJECTIVES: This study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture. METHODS: Chloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations. RESULTS: C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides. CONCLUSIONS: This study identifies a novel point mutation (G2073A in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media.

  7. Lignocellulolytic mutants of Pleurotus ostreatus induced by gamma-ray radiation and their genetic similarities

    Science.gov (United States)

    Lee, Y.-K.; Chang, H.-H.; Kim, J.-S.; Kim, J. K.; Lee, K.-S.

    2000-02-01

    To induce the lignocellulolytic mutants of Pleurotus ostreatus, the mycelia were irradiated by gamma-ray radiation to doses of 1-2 kGy. Five strains were isolated by the criteria of clamp connection, fruiting body formation, growth rate and activities of extracellular enzymes. All isolated strains were able to form the fruiting bodies and grew similarly to the control. The extracellular enzymes activities in liquid media of isolated strains were up to 10 times higher than the control. Genetic similarities of the isolated strains ranged from 64.4% to 93.3% of the control. From these results, it seems that the genetic diversity of P. ostreatus could be changed and useful strains be induced by gamma-ray radiation to recycle or reuse biowastes.

  8. Lignocellulolytic mutants of Pleurotus ostreatus induced by gamma-ray radiation and their genetic similarities

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Y-K. E-mail: yklee@nanum.kaeri.re.kr; Chang, H-H.; Kim, J-S.; Kim, J.K.; Lee, K-S

    2000-02-01

    To induce the lignocellulolytic mutants of Pleurotus ostreatus, the mycelia were irradiated by gamma-ray radiation to doses of 1-2 kGy. Five strains were isolated by the criteria of clamp connection, fruiting body formation, growth rate and activities of extracellular enzymes. All isolated strains were able to form the fruiting bodies and grew similarly to the control. The extracellular enzymes activities in liquid media of isolated strains were up to 10 times higher than the control. Genetic similarities of the isolated strains ranged from 64.4% to 93.3% of the control. From these results, it seems that the genetic diversity of P. ostreatus could be changed and useful strains be induced by gamma-ray radiation to recycle or reuse biowastes. (author)

  9. A (p)ppGpp-null mutant of Haemophilus ducreyi is partially attenuated in humans due to multiple conflicting phenotypes.

    Science.gov (United States)

    Holley, Concerta; Gangaiah, Dharanesh; Li, Wei; Fortney, Kate R; Janowicz, Diane M; Ellinger, Sheila; Zwickl, Beth; Katz, Barry P; Spinola, Stanley M

    2014-08-01

    (p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species. Haemophilus ducreyi, the causative agent of chancroid, has homologs of both relA and spoT, which primarily synthesize and hydrolyze (p)ppGpp in Escherichia coli. We constructed relA and relA spoT deletion mutants to assess the contribution of (p)ppGpp to H. ducreyi pathogenesis. Both the relA single mutant and the relA spoT double mutant failed to synthesize (p)ppGpp, suggesting that relA is the primary synthetase of (p)ppGpp in H. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented in trans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription of lspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. In E. coli, overexpression of DksA compensates for the loss of (p)ppGpp; the H. ducreyi double mutant expressed higher transcript levels of dksA than the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.

  10. Degradation of Tectilon Yellow 2G by hybrid technique: combination of sonolysis and biodegradation using mutant Pseudomonas putida.

    Science.gov (United States)

    Srinivasan, Raman; Kathiravan, Mathur Nadarajan; Gopinath, Kannappan Panchamoorthy

    2011-02-01

    Degradation of Tectilon Yellow 2G (TY2G), an azo dye has been studied by hybrid technique involving pretreatment by sonochemical method and further biological treatment by Pseudomonas putida mutant. Pretreatment experiments were carried out by sonolysis of the dye solution at different concentrations (100-1000 mg/L). Wild type Gram-negative P. putida species isolated from the textile effluent contaminated soil, which was found to be effective towards dye degradation, has been acclimatized so as to consume TY2G as the sole source of nutrition. Mutant strain was obtained from the acclimatized species by random mutagenesis using the chemical mutagen ethidium bromide for various time intervals (6-30 min). The optimum mutagenesis exposure time for obtaining the most efficient species for dye degradation was found to be 18 min. An efficient mutant strain P. putida ACT 1 has been isolated and was used for growth experiments. The mutant strain showed a better growth compared to the wild strain. The substrate utilization kinetics has been modeled using Monod and Haldane model equations of which the Haldane model provided a better fit. The enzyme kinetics of the mutant and wild species was obtained using Michaelis-Menten equation. The mutated species showed better enzyme kinetics towards the degradation of TY2G.

  11. Conidiation color mutants of Aspergillus fumigatus are highly pathogenic to the heterologous insect host Galleria mellonella.

    Directory of Open Access Journals (Sweden)

    Jennifer C Jackson

    Full Text Available The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen

  12. Enhanced Carotenoid Production by a Mutant of the Marine Yeast Rhodotorula sp. hidai

    Institute of Scientific and Technical Information of China (English)

    CONG Li; CHI Zhenming; LI Jing; WANG Xianghong

    2007-01-01

    After a serial of UV, EMS and NTG mutagenesis, a mutant named MM of the red marine yeast strain Rhodotorula sp.hidai was obtained. The mutant MM could produce 603.93 μg g-1 of carotenoid within 5 days in the medium containing 4.0 g sucrose,1.5 g yeast extract, 0.1 g MgSO4, and 100 mL of sea water, with pH 6.0 and at 30 ℃, while only 213.18 μg g-1 of carotenoid was produced by the wild type under the same conditions.

  13. Reduced activity of glutamine synthetase in Rhodospirillum rubrum mutants lacking the adenylyltransferase GlnE.

    Science.gov (United States)

    Jonsson, Anders; Nordlund, Stefan; Teixeira, Pedro Filipe

    2009-10-01

    In the nitrogen-fixing bacterium Rhodospirillum rubrum, the GlnE adenylyltransferase (encoded by glnE) catalyzes reversible adenylylation of glutamine synthetase, thereby regulating nitrogen assimilation. We have generated glnE mutant strains that are unable to adenylylate glutamine synthetase (GS). Surprisingly, the activity of GS was lower in the mutants than in the wild type, even when grown in nitrogen-fixing conditions. Our results support the proposal that R. rubrum can only cope with the absence of an adenylylation system in the presence of lowered GS expression or activity. In general terms, this report also provides further support for the central role of GS in bacterial metabolism.

  14. Furfural and hydroxymethylfurfural tolerance in Escherichia coli ΔacrR regulatory mutants.

    Science.gov (United States)

    Luhe, Annette Lin; Lim, Chan Yuen; Gerken, Henri; Wu, Jinchuan; Zhao, Hua

    2015-01-01

    The presence of the highly toxic furfural and hydroxymethylfurfural (HMF) in the hydrolysate of lignocellulosic biomass prompted the investigation of the Escherichia coli ΔacrR regulatory mutant for higher tolerance to these compounds, to facilitate the production of biofuels and biochemicals, and further biocatalytic conversions. In comparison with the parental strain, the regulatory mutant with the upregulated efflux pump AcrAB-TolC produced moderately better growth and higher tolerance to concentrations of furfural and HMF between 1 and 2 g L(-1) .

  15. Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

    Science.gov (United States)

    Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

    2003-07-30

    Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation.

  16. Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

    Science.gov (United States)

    Ashour, Joseph; Hondalus, Mary K

    2003-04-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

  17. A suitable streptomycin-resistant mutant for constructing unmarked in-frame gene deletions using rpsL as a counter-selection marker.

    Directory of Open Access Journals (Sweden)

    Yu-Kuo Tsai

    Full Text Available The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD50 (the bacterial dose that caused 50% death assay, the K42N and K42T mutants were ∼ 1,000-fold less lethal (∼ 2 × 10(5 CFU and the K87R mutant was ∼ 50-fold less lethal (∼ 1 × 10(4 CFU than the parental strain (∼ 2 × 10(2 CFU. A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P < 0.01 in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker.

  18. Complete Genome Sequence of NC983, a Live Attenuated Strain of Salmonella enterica Serovar Typhimurium

    Science.gov (United States)

    Troxell, Bryan; Fink, Ryan C.; Dickey, Allison N.; Scholl, Elizabeth H.

    2016-01-01

    Foodborne infections caused by Salmonella enterica serovars are a significant problem worldwide. Presented here is the genome sequence of the nontyphoidal S. enterica serovar Typhimurium mutant strain NC983, a potential vaccine candidate. PMID:27738027

  19. Mutations in the lux operon of natural dark mutants in the genus Vibrio.

    Science.gov (United States)

    O'Grady, Elizabeth A; Wimpee, Charles F

    2008-01-01

    Bacterial bioluminescence can display a wide range of intensities among strains, from very bright to undetectable, and it has been shown previously that there are nonluminous vibrios that possess lux genes. In this paper, we report the isolation and characterization of completely dark natural mutants in the genus Vibrio. Screening of over 600 Vibrio isolates with a luxA gene probe revealed that approximately 5% carried the luxA gene. Bioluminescence assays of the luxA-positive isolates, followed by repetitive extragenic palindromic-PCR fingerprinting, showed three unique genotypes that are completely dark. The dark mutants show a variety of lesions, including an insertion sequence, point mutations, and deletions. Strain BCB451 has an IS10 insertion sequence in luxA, a mutated luxE stop codon, and a truncated luxH. Strain BCB494 has a 396-bp deletion in luxC, and strain BCB440 has a frameshift in luxC. This paper represents the first molecular characterization of natural dark mutants and the first demonstration of incomplete lux operons in natural isolates.

  20. Autoaggressive, multi-headed and other mutant phenotypes in Hydractinia echinata (Cnidaria: Hydrozoa).

    Science.gov (United States)

    Müller, Werner A

    2002-12-01

    In an inbreeding program conducted with the colonial hydroid Hydractinia echinata, each F1 mating produced up to 50% F2 offspring displaying an aberrant, clone-constant phenotype, hence referred to as mutant strain. In autoaggressive strains, in one or several areas of the colony autoreactive stolons direct their aggressive devices (stolon tips filled with cytotoxic stinging cells), normally used to kill allogeneic competitors for living space, towards neighboring stolons or polyps (hydranths) of their own colony. In these areas tumor-like masses of self-aggressive stolons were formed, in severe cases causing the death of the colony. Based on previous genetic studies, the interpretation proposed here attributes autoaggressive behavior to a mosaic-type alternative expression of arl (allorecognition) alleles in heterozygous individuals. Developmental mutant strains termed He-mh form supernumerary heads during regeneration and normal development as well. Common to all He-mh phenotypes isthe production of additional headsalong the bodycolumn of fully-grown polyps. The heads give rise to complete hydranths connected by a tube that derives from the gastric region of the original polyp and eventually transforms into a stolon. In bastol strains, polyps convert the basal region of their body column into a periderm-covered stolon from which the residual apical hydranth detaches. Colonies expressing both the He-mh and the bastol (bst) phenotype frequently lose detaching multi-headed hydranths and the colony disintegrates. The large number of mutant F2 offspring reveals high genetic variability in Hydractinia.

  1. Direct mating between diploid sake strains of Saccharomyces cerevisiae.

    Science.gov (United States)

    Hashimoto, Shinji; Aritomi, Kazuo; Minohara, Takafumi; Nishizawa, Yoshinori; Hoshida, Hisashi; Kashiwagi, Susumu; Akada, Rinji

    2006-02-01

    Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATalpha mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.

  2. Muscle development in mdx mutant mice.

    Science.gov (United States)

    Dangain, J; Vrbova, G

    1984-01-01

    Mechanical and contractile properties of tibialis anterior (TA) muscles from X-linked muscular dystrophic (mdx) mutant mice at different stages of development are compared to those of muscles from normal control animals. There is no difference between the tension output, speeds of contraction and relaxation, and weight of TA muscles from mutant adults and normal control animals. However, it is found that in 3-4-week-old mutant animals, tension output and muscle weight are very much reduced, and half relaxation time is prolonged. Thus, during this stage of development, muscles from mdx mice do not function properly. Histological examination of these muscles provides further evidence that, in these animals, rapid muscle destruction occurs at a particular time of development and that it is followed by complete recovery. This new mutant therefore presents an interesting case of muscle destruction and rapid regeneration. However, it is not an adequate model for Duchenne muscular dystrophy.

  3. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1-Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1-Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT...mutations found in breast cancer using both structural and cell based assays. We have now have evidence for the effects of the most recurrent

  4. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    Introduction Approximately 70% of ER+ breast cancers harbor expression of the estrogen receptor and are dependent upon its activity for various aspects of the...resistance to current FDA approved ER antagonists, but that more potent and selective estrogen receptor antagonists will be sufficiently active to...antagonists and their potency against ER mutants both in vitro and in vivo . Targeting ESR1-Mutant Breast Cancer W81XWH-14-1-0359 9 4. Impact A) Impact

  5. [Mutant alleles associated to chloroquine and sulfadoxine-pyrimethanime resistance in Plasmodium falciparum of the Ecuador-Peru and Ecuador-Colombia borders].

    Science.gov (United States)

    Arróspide, Nancy; Hijar-Guerra, Gisely; de Mora, Doménica; Diaz-Cortéz, César Eduardo; Veloz-Perez, Raúl; Gutierrez, Sonia; Cabezas-Sánchez, César

    2014-04-01

    The frequency of mutations in pfCRT and DHFR/DHPS genes of Plasmodium falciparum associated with resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated in 83 strains from the districts of Esmeralda and Machala, located on the borders of Ecuador-Peru and Ecuador-Colombia in 2002. Polymerase chain reaction (PCR), conventional and its variants, was used. Mutations in the pfCRT gene were found in more than 90% of the samples from Esmeralda and Machala. For the DHFR gene, 90% of the strains were mutant samples from Esmeralda, 3 were double mutations and 1 was a triple mutation. In Machala, 25% were simple mutant forms and 75% mixed mutant forms (wild forms/mutant). In conclusion, resistance to chloroquine has been fixed in strains carrying K76T pfCRT mutation, whereas genetic imprinting for resistance to pyrimethamine is evolving, particularly in the district of Esmeralda.

  6. Changes in patterns of ADP-ribosylated proteins during differentiation of Streptomyces coelicolor A3(2) and its development mutants.

    Science.gov (United States)

    Shima, J; Penyige, A; Ochi, K

    1996-07-01

    Mutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces coelicolor A3(2). One (strain 27) was analyzed in detail. Mutant 27 had a reduced ADP-ribosyl-transferase activity, exhibited substantial changes from the wild type in ADP-ribosylated protein profile during cell aging, and was defective in producing aerial mycelium and antibiotics. A 92-kDa ADP-ribosylated protein disappeared at the onset of differentiation in the parent strain but was present in mutant 27. Four ADP-ribosylated proteins (39, 41, 43, and 46 kDa) appeared at the onset of differentiation in the parent strain but were missing in mutant 27. Failure to ADP-ribosylate these four proteins was detected when the parent strain was grown in the presence of subinhibitory amounts of 3-aminobenzamide. Genetic analysis showed that the mutation, named brgA, conferring resistance to 3-aminobenzamide, cosegregated with the altered phenotypes (i.e., defects in ADP-ribosylation and aerial mycelium formation) and was mapped to a new locus near uraA. The brgA mutants were nonconditionally deficient in producing aerial mycelium and antibiotics, as determined by using various media, and had a morphological and physiological phenotype quite different from that of a bldG mutant carrying a mutation which was previously mapped near uraA. Among the known bld mutants, bldA, bldD, and bldG mutants exhibited a ADP-ribosylated protein profile similar to that of the wild type, while like mutant 27, bldB, bldC, and bldH mutants failed to ADP-ribosylate certain proteins.

  7. Helicobacter pylori arginase mutant colonizes arginase Ⅱ knockout mice

    Institute of Scientific and Technical Information of China (English)

    Songhee H Kim; Melanie L Langford; Jean-Luc Boucher; Traci L Testerman; David J McGee

    2011-01-01

    AIM: To investigate the role of host and bacterial argi-nases in the colonization of mice by Helicobacter pylori (H. Pylori).METHODS: H. Pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Urease is absolutely essential to H. Pylori pathogenesis; therefore, the urea substrate must be in ample supply for urease to work efficiently. The urea substrate is most likely provided by arginase activity, which hydrolyzes L-arginine to L-ornithine and urea. Previous work has demonstrated that H. Pylori arginase is surprisingly not required for colonization of wild-type mice. Hence, another in vivo source of the critical urea substrate must exist. We hypothesized that the urea source was provided by host arginase Ⅱ, since this enzyme is expressed in the stomach, and H. Pylori has previously been shown to induce the expres-sion of murine gastric arginase Ⅱ. To test this hypoth-esis, wild-type and arginase (rocF) mutant H. Pylori strain SS1 were inoculated into arginase Ⅱ knockout mice. RESULTS: Surprisingly, both the wild-type and rocF mutant bacteria still colonized arginase Ⅱ knock-out mice. Moreover, feeding arginase Ⅱ knockout mice the host arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC), while inhibiting > 50% of the host arginase Ⅰactivity in several tissues, did not block the ability of the rocF mutant H. Pylori to colonize. In con-trast, BEC poorly inhibited H. Pylori arginase activity. CONCLUSION: The in vivo source for the essential urea utilized by H. Pylori urease is neither bacterial arginase nor host arginase Ⅱ; instead, either residual host arginase Ⅰor agmatinase is probably responsible.

  8. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum.

    Science.gov (United States)

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V; Parks, Jerry M; Smolin, Nikolai; Yang, Shihui; Land, Miriam L; Klingeman, Dawn M; Bhandiwad, Ashwini; Rodriguez, Miguel; Raman, Babu; Shao, Xiongjun; Mielenz, Jonathan R; Smith, Jeremy C; Keller, Martin; Lynd, Lee R

    2011-08-16

    Clostridium thermocellum is a thermophilic, obligately anaerobic, gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  9. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Steven D [ORNL; Guss, Adam M [ORNL; Karpinets, Tatiana V [ORNL; Parks, Jerry M [ORNL; Smolin, Nikolai [ORNL; Yang, Shihui [ORNL; Land, Miriam L [ORNL; Klingeman, Dawn Marie [ORNL; Bhandiwad, Ashwini [Thayer School of Engineering at Dartmouth; Rodriguez, Jr., Miguel [ORNL; Raman, Babu [Dow Chemical Company, The; Shao, Xiongjun [Thayer School of Engineering at Dartmouth; Mielenz, Jonathan R [ORNL; Smith, Jeremy C [ORNL; Keller, Martin [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  10. Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes.

    Science.gov (United States)

    Hosted, T J; Baltz, R H

    1996-10-01

    Streptomyces roseosporus mutants that express enhanced recombination between partially homologous (homeologous) sequences were isolated by selection for recombination between the bacteriophage phi C31 derivative KC570 containing the Streptomyces coelicolor glucose kinase (glk) gene and the S. roseosporus chromosome. The frequencies of homeologous recombination in the ehr mutants were determined by measuring the chromosomal insertion frequencies of plasmids containing S. coelicolor glnA or whiG genes. S. roseosporus ehr mutants showed 10(2)- to 10(4)-fold increases in homeologous recombination relative to Ehr+ strains, but no increase in homologous recombination. Southern hybridization analysis revealed single unique sites for the insertion of each of the plasmids, and the crossovers occurred in frame and in proper translational register, yielding functional chimeric glnA and whiG genes.

  11. l-Serine Production by a Mutant of Sarcina albida Defective in l-Serine Degradation

    Science.gov (United States)

    Omori, Kenji; Kakimoto, Toshio; Chibata, Ichiro

    1983-01-01

    For improved l-serine production, an l-serine dehydratase-defective mutant of Sarcina albida IAM 1012 was obtained. In the mutant, the activities of the enzymes responsible for l-serine production were as high as those in the parent strain, and, at a low glycine concentration, the mutant accumulated l-serine more efficiently than the parent. Under optimum conditions, 21 mg of l-serine per ml accumulated from 100 mg of glycine per ml. l-Serine was isolated from a reaction mixture as l-serine m-xylene-4-sulfonate, and free amino acid was obtained in high yields by use of an ion-exchange resin. Residual glycine was recovered at a yield of 61%. PMID:16346305

  12. New and efficient method using Saccharomyces cerevisiae mutants for identification of siderophores produced by microorganisms.

    Science.gov (United States)

    Park, Yong-Sung; Kim, Ji-Hyun; Chang, Hyo-Ihl; Kim, Seung-Wook; Paik, Hyun-Dong; Kang, Chang-Won; Kim, Tae-Hyoung; Sung, Ha-Chin; Yun, Cheol-Won

    2007-09-01

    The separation and identification of siderophores produced by microorganisms is a time-consuming and an expensive procedure. We have developed a new and efficient method to identify siderophores using well-established Saccharomyces cerevisiae deletion mutants. The Deltafet3,arn strains fail to sustain growth, even when specific siderophores are supplied, and mutants are siderophore-specific: Deltafet3,arn2 for triacetylfusarinine C (TAFC), Deltafet3,arn1,sit1 for ferrichrome (FC), and Deltafet3,sit1 for ferrioxamine B (FOB). The culture broth of Fusarium graminearum was separated by HPLC, and each peak was subjected to a plate assay using S. cerevisiae mutants. We have found that each peak contained specific siderophores produced by F. graminearum, and these coincided with reference siderophores. This method is quite novel because nobody tried this method to identify the siderophores. Furthermore, this method will save time and cost in the identification of siderophores produced by microorganisms.

  13. Molecular and phenotypic traits of in-vitro-selected mutants of Bifidobacterium resistant to rifaximin.

    Science.gov (United States)

    Vitali, Beatrice; Turroni, Silvia; Serina, Stefania; Sosio, Margherita; Vannini, Lucia; Candela, Marco; Guerzoni, Maria Elisabetta; Brigidi, Patrizia

    2008-06-01

    Nucleotide mutations inside a core region of the rpoB gene, encoding the beta subunit of RNA polymerase, were found in rifaximin-resistant mutants of Bifidobacterium. Five different missense mutations of codons 513, 516, 522 and 529 were identified. Further aspects of rifaximin resistance were investigated, using Bifidobacterium infantis BI07 as a model strain. Partial resistance of RNA polymerase of a BI07 mutant at a rifaximin concentration >10 microg/mL was observed by cell-free transcription assay. Mass spectrometry detection of rifaximin in the cellular pellet of the BI07 resistant mutant, as well as changes in biosynthesis of saturated and cyclopropane fatty acids during growth, suggested a reduction in membrane permeability for the antibiotic moiety.

  14. Isolation of carotenoid hyperproducing mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by flow cytometry and cell sorting.

    Science.gov (United States)

    Brehm-Stecher, Byron F; Johnson, Eric A

    2012-01-01

    Approaches for improving astaxanthin yields in Xanthophyllomyces dendrorhous include optimization of fermentation conditions and generation of hyperproducing mutants through random mutagenesis using chemical or physical means. A key limitation of classical mutagenesis is the labor-intensive nature of the screening processes required to find relatively rare mutants having increased carotenoid content, as these are present against a high background of low-interest cells. Here, flow cytometry is described as a high-throughput, single-cell method for primary enrichment of mutagenized cells expressing high levels of astaxanthin. This approach improves the speed and productivity of classical strain selection, enhancing the chances for isolating the carotenoid hyperproducing mutants (CHMs) needed to enable high-titer, economical production of natural astaxanthin.

  15. Computational identification of adaptive mutants using the VERT system

    Directory of Open Access Journals (Sweden)

    Winkler James

    2012-04-01

    Full Text Available Background Evolutionary dynamics of microbial organisms can now be visualized using the Visualizing Evolution in Real Time (VERT system, in which several isogenic strains expressing different fluorescent proteins compete during adaptive evolution and are tracked using fluorescent cell sorting to construct a population history over time. Mutations conferring enhanced growth rates can be detected by observing changes in the fluorescent population proportions. Results Using data obtained from several VERT experiments, we construct a hidden Markov-derived model to detect these adaptive events in VERT experiments without external intervention beyond initial training. Analysis of annotated data revealed that the model achieves consensus with human annotation for 85-93% of the data points when detecting adaptive events. A method to determine the optimal time point to isolate adaptive mutants is also introduced. Conclusions The developed model offers a new way to monitor adaptive evolution experiments without the need for external intervention, thereby simplifying adaptive evolution efforts relying on population tracking. Future efforts to construct a fully automated system to isolate adaptive mutants may find the algorithm a useful tool.

  16. Simultaneous analysis of multiple Mycobacterium tuberculosis knockdown mutants in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Antje Blumenthal

    Full Text Available Mycobacterium tuberculosis (Mtb represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL, the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen

  17. Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains.

    Science.gov (United States)

    Wang, Junfeng; Liu, Changting; Liu, Jinyi; Fang, Xiangqun; Xu, Chen; Guo, Yinghua; Chang, De; Su, Longxiang

    2014-03-01

    The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis.

  18. Geobacteraceae strains and methods

    Science.gov (United States)

    Lovley, Derek R.; Nevin, Kelly P.; Yi, Hana

    2015-07-07

    Embodiments of the present invention provide a method of producing genetically modified strains of electricigenic microbes that are specifically adapted for the production of electrical current in microbial fuel cells, as well as strains produced by such methods and fuel cells using such strains. In preferred embodiments, the present invention provides genetically modified strains of Geobacter sulfurreducens and methods of using such strains.

  19. Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Dubini, A.; Mus, F.; Seibert, M.; Grossman, A. R.; Posewitz, M. C.

    2009-03-13

    The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.

  20. Mutagenesis and selection of high efficiency hydrogen producing mutants by ultraviolet radiation

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Hydrogen is an ideal, clean and sustainable energy source for the future because of its high conversion and nonpolluting nature. Biohydrogen production by dark-fermentation appears to have a great potential to be developed for practical application. However, one limiting factor affecting the development of hydrogen-production industrialization is that the hydrogen-producing capacity of bacteria is lower, so how to increase bacteria's hydrogen-producing ability will be an urgent issue. In this experiment, 2 mutants, namely UV3 and UV7,were obtained by ultra-violet radiation. They grew and produced hydrogen efficiently on iron-containing medium. The hydrogen evolution of UV3 and UV7 were 2 356. 68 ml/L and 2 219. 62 ml/L at a glucose concentration of 10 g/L, respectively. With wild parent strain Ethanoligenens sp. ZGX4, the hydrogen evohution was 1 806. 02 ml/L under the same conditions. Mutants' hydrogen-producing capacities were about 29. 71% and 22.22% higher than that of wild parent strain ZGX4. The maximum H2 production rate by mutants UV3 and UV7 were estimated to be 32. 57 mmol H2/g cell h and 31.19 mmol H2/g cell h, respectively, which were 38. 18% and 34. 78% higher than the control (23.57 mmol H2/g cell h). The abundant products of UV3 and UV7 were ethanol and acetic, which accounted for 95% -98% of total soluble microbial products. In each case, mutant strains UV3 and UV7 evolved hydrogen at a higher rate than the wild type, showing a possible potential for commercial hydrogen production. Another mutant named UV20' was also gained whose main end metabolites were butyric acid and acetic acid. This would provide researched material for a discussion of metabolic pathways of hydrogen-producing bacteria.

  1. Studies on the Mutant Systems of the Bombyx mori Gene Bank

    Institute of Scientific and Technical Information of China (English)

    LU Cheng; DAI Fang-yin; XIANG Zhong-huai

    2002-01-01

    Through over ten years of study, more than 1 000 genetic materials including mutant genes,chromosomal variation strains and special genetic materials of Bombyx mori, Linnaeus, collected, introduced or created since 1940s especially late 1980s, have been sorted out and put in order. After identifications and genetic analyses of their morphological, physiological and biochemical characters, the silkworm gene bank was constructed and the preservation system was perfected, and more than 600 silkworm strains were kept in this gene bank. The preserved silkworm mutant genes have covered more than 90% of existent ones across the world, in which, more than 100 are rare and precious mutant genes, and over 60 mutant genes were found and studied for the first time. Through hybrid analyses, linkage tests and three-point gene location tests, a perfect linkage retrieval labeling system of silkworm was established, which included 230 marker genes covering all the 28 linkage groups of Bombyx mori. The gene location system (composite system of recessive genes) of different linkage groups was set up. The intergenic complementation of mutant egg color and third type of maternal heredity egg color have been found, and indicated that the epistatic effect of mutant gene of white egg is universal. Twenty eight independent near isogenic lines murked with morphological mutation gene have been created and a series of novel breeding materials possessing great potential for application such as high feeding efficiency, special sex markers, natural colored silk, resistance to disease, wider feeding range and adjustable parthenogenesis, etc., have been developed. The sustainable maintenance and management technique system of silkworm gene resources were well established.

  2. Mutations in rpoBC suppress the defects of a Sinorhizobium meliloti relA mutant.

    Science.gov (United States)

    Wells, Derek H; Long, Sharon R

    2003-09-01

    The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa requires complex physiological adaptation by both partners. One method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the ability of RNA polymerase (RNAP) to initiate and elongate transcription and affect the affinity of the core enzyme for various sigma factors. An S. meliloti relA mutant which cannot produce ppGpp was previously shown to be defective in the ability to form nodules. This mutant also overproduces a symbiotically necessary exopolysaccharide called succinoglycan. The work presented here encompasses the analysis of suppressor mutants, isolated from host plants, that suppress the symbiotic defects of the relA mutant. All suppressor mutations are extragenic and map to either rpoB or rpoC, which encode the beta and beta' subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal that suppressor mutants can be divided into two classes; one is specific in its effect on stringent response-regulated genes and shares striking similarity with suppressor mutants of Escherichia coli strains that lack ppGpp, and another reduces transcription of all genes tested in comparison to that in the relA parent strain. Our findings indicate that the ability to successfully establish symbiosis is tightly coupled with the bacteria's ability to undergo global physiological adjustment via the stringent response.

  3. The electrospray ionization - mass spectra of erythromycin a obtained from a marine Streptomyces sp. mutant

    Directory of Open Access Journals (Sweden)

    El-Bondkly A

    2008-01-01

    Full Text Available In our ongoing search for production improvements of bioactive secondary metabolites from marine Streptomyces through the induction of mutations using UV light, out of 145 isolates, mutant 10/14 was able to produce potent antibacterial metabolites other than the parent strain as established by chromatographic analysis. Up-scaling fermentation of mutant 10/14, followed by working up and isolation delivered five metabolites, phenazine, 1-acetyl-β -carboline, perlolyrin and erythromycin A, along with an oily substance. The latter two compounds were responsible for the antibacterial activity of the strain. In this article, we discuss with the mutation of the marine Streptomyces sp. AH2, bioactivity evaluation, fermentation and isolation of the microbial metabolites. Moreover, we study to first time in detail the 1D and 2D NMR and ESI MS data including ESI MS 2 and MS 3 patterns combined with HRESI MS of erythromycin A.

  4. Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

    Science.gov (United States)

    Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

    2016-01-01

    We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

  5. Durations and frequencies of free locomotion in wild type and GABAergic mutants of Caenorhabditis elegans.

    Science.gov (United States)

    Shingai, R

    2000-09-01

    We investigated how much time wild-type Caenorhabditis elegans (Bristol N2) nematodes and the GABA-deficient unc25 mutant and the vesicular GABA transporter-deficient unc47 mutant spent moving. The worms were allowed to move freely on the surface of agarose plates either with or without the food bacterium OP50. We identified forward movement, backward movement, resting and turns by watching images on video and computer displays. Forward movement lasted longer and rests were briefer without, than with, bacteria. Frequency distributions except for backward movement fitted a sum of two exponential functions. The duration of backward movement was not strongly influenced by exposure to bacteria, whereas the frequency of backward movements increased in their presence. The duration of forward movement of unc25 nematodes had no long component, thus differing from that of N2 and unc47 strain nematodes in treatments with and without bacteria. The durations of resting in these mutants were much longer than in the N2 strain, especially in the absence of bacteria. The turn frequency of unc47 nematodes had a higher short component than that of the wild type N2 and unc25 nematodes, in the absence of bacteria. A neural network model is discussed in conjunction with the features of mutants and current knowledge of GABAergic neural transmission.

  6. Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice.

    Directory of Open Access Journals (Sweden)

    Tohru Matsuki

    Full Text Available Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.

  7. Changes of motor abilities during ontogenetic development in Lurcher mutant mice.

    Science.gov (United States)

    Markvartová, V; Cendelín, J; Vozeh, F

    2010-07-14

    Lurcher mutant mice represent a natural model of olivocerebellar degeneration. This degeneration is caused by a mutation of the gene for the delta2 glutamate receptor. Lurcher mutants suffer from cerebellar ataxia and cognitive functions deficiency as a consequence of excitotoxic apoptosis of Purkinje cells in the cerebellar cortex and a secondary decrease of granule cells and inferior olive neurons. This process finishes by the 90th day of postnatal life, but already by 14 days, the Purkinje cells are damaged and the ataxia is fully developed. Purkinje cells die by apoptosis within the first 3 weeks of life. The aim of our work was to study the development of motor functions in the course of the ontogenetic development in Lurcher mutant mice of the B6CBA strain and to compare it with wild type mice of the same strain. Mice aged 2, 3, 6, 9, and 22 weeks were used in our experiment. Motor skills were examined using four standard tests: the horizontal wire, rotating cylinder, footbridge and slanting ladder. Our findings in Lurcher mutant mice show a significant increase of motor abilities up to the sixth postnatal week and selective decrease early after this period. This improvement of motor skills is caused by the physiological development of musculature and the nervous system, probably with some contribution of plasticity of the maturing brain. The cause of the decline of these abilities immediately after the completion of the development is unknown.

  8. Chemostat cultivation and transcriptional analyses of Clostridium acetobutylicum mutants with defects in the acid and acetone biosynthetic pathways.

    Science.gov (United States)

    Hönicke, Daniel; Lütke-Eversloh, Tina; Liu, Ziyong; Lehmann, Dörte; Liebl, Wolfgang; Ehrenreich, Armin

    2014-12-01

    Clostridium acetobutylicum is a model organism for the biotechnologically important acetone-butanol-ethanol (ABE) fermentation. With the objective to rationally develop strains with improved butanol production, detailed insights into the physiological and genetic mechanisms of solvent production are required. Therefore, pH-controlled phosphate-limited chemostat cultivation and DNA microarray technology were employed for an in-depth analysis of knockout mutants with defects in the central fermentative metabolism. The set of studied mutants included strains with inactivated phosphotransacetylase (pta), phosphotransbutyrylase (ptb), and acetoacetate decarboxylase (adc) encoding genes, as well as an adc/pta double knockout mutant. A comprehensive physiological characterization of the mutants was performed by continuous cultivation, allowing for a well-defined separation of acidogenic and solventogenic growth, combined with the advantage of the high reproducibility of steady-state conditions. The ptb-negative strain C. acetobutylicum ptb::int(87) exhibited the most striking metabolite profile: Sizable amounts of butanol (29 ± 1.3 mM) were already produced during acidogenic growth. The product patterns of the mutants as well as accompanying transcriptomic data are presented and discussed.

  9. Mutant-prevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin.

    Science.gov (United States)

    Gebru, Elias; Choi, Myung-Jin; Lee, Seung-Jin; Damte, Dereje; Park, Seung Chun

    2011-10-01

    The antibacterial activity and selection of resistant bacteria, along with mechanisms of fluoroquinolone resistance, were investigated by integrating the static [MIC or mutant-prevention concentration (MPC)] and in vitro dynamic model approaches using Escherichia coli isolates from diseased dogs. Using the dynamic models, selected E. coli strains and enrofloxacin and marbofloxacin at a range of simulated area under concentration-time curve over a 24 h interval (AUC(24 h))/MIC ratios were investigated. Our results indicated increasing losses in susceptibility of E. coli upon continuous exposure to enrofloxacin and marbofloxacin in vitro. This effect was transferable to other fluoroquinolones, as well as to structurally unrelated drugs. Our results also confirmed an AUC(24 h)/MIC (AUC(24 h)/MPC)-dependent antibacterial activity and selection of resistant E. coli mutants, in which maximum losses in fluoroquinolone susceptibility occurred at simulated AUC(24 h)/MIC ratios of 40-60. AUC(24 h)/MPC ratios of 39 (enrofloxacin) and 32 (marbofloxacin) were considered protective against the selection of resistant mutants of E. coli. Integrating our MIC and MPC data with published pharmacokinetic information in dogs revealed a better effect of the conventional dosing regimen of marbofloxacin than that of enrofloxacin in restricting the selection of resistant mutants of E. coli. Target mutations, especially at codon 83 (serine to leucine) of gyrA, and overexpression of efflux pumps contributed to resistance development in both clinically resistant and in vitro-selected mutants of E. coli. We also report here a previously undescribed mutation at codon 116 of parC in two laboratory-derived resistant mutants of E. coli. Additional studies would determine the exact role of this mutation in fluoroquinolone susceptibility, as well as establish the importance of our findings in the clinical setting.

  10. Mechanisms responsible for a PhiX174 mutant's ability to infect Escherichia coli by phosphorylation.

    Science.gov (United States)

    Cox, Jennifer; Putonti, Catherine

    2010-05-01

    The ability for a virus to expand its host range is dependent upon a successful mode of viral entry. As such, the host range of the well-studied PhiX174 bacteriophage is dictated by the presence of a particular lipopolysaccharide (LPS) on the bacterial surface. The mutant PhiX174 strain JACS-K, unlike its ancestor, is capable of infecting both its native host Escherichia coli C and E. coli K-12, which does not have the necessary LPS. The conversion of an alanine to a very reactive threonine on its virion surface was found to be responsible for the strain's expanded host range.

  11. Enhanced nodulation and nodule development by nolR mutants of Sinorhizobium medicae on specific Medicago host genotypes.

    Science.gov (United States)

    Sugawara, Masayuki; Sadowsky, Michael J

    2014-04-01

    The nolR gene encodes a negatively acting, transcriptional regulatory protein of core Nod-factor biosynthetic genes in the sinorhizobia. Although previous reports showed that nolR modulates Nod-factor production and enhances nodulation speed of Sinorhizobium meliloti on alfalfa, there have been no reports for the symbiotic function of this gene in the S. medicae-Medicago truncatula symbiosis. Here, we constructed an nolR mutant of S. medicae WSM419 and evaluated mutant and wild-type strains for their nodulation ability, competitiveness, host specificity, and density-dependent nodulation phenotypes. When the mutant was inoculated at low and medium population densities, it showed enhanced nodule formation during the initial stages of nodulation. Results of quantitative competitive nodulation assays indicated that an nolR mutant had 2.3-fold greater competitiveness for nodulation on M. truncatula 'A17' than did the wild-type strain. Moreover, the nodulation phenotype of the nolR mutant differed among Medicago genotypes and showed significantly enhanced nodule development on M. tricycla. Taken together, these results indicated that mutation of nolR in S. medicae positively influenced nodule initiation, competitive nodulation, and nodule development at later nodulation stages. This may allow nolR mutants of S. medicae to have a selective advantage under field conditions.

  12. Characterization of changes in the short unique segment of pseudorabies virus BUK-TK900 (Suivac A) vaccine strain

    NARCIS (Netherlands)

    Ficinska, J.; Bienkowska-Szewczyk, K.; Jacobs, L.; Plucienniczak, G.; Plucienniczak, A.; Szewczyk, B.

    2003-01-01

    Mutant strains of pseudorabies virus (PRV) of reduced virulence, such as Bartha or BUK-TK900, have been used for vaccination purposes for many years. In contrast to the Bartha strain, BUK-TK900 has not been well characterised at the molecular level. The detailed analysis of this vaccine strain was u

  13. Characterization of bacteriophages infecting Streptomyces erythreus and properties of phage-resistant mutants.

    Science.gov (United States)

    Donadio, S; Paladino, R; Costanzi, I; Sparapani, P; Schreil, W; Iaccarino, M

    1986-06-01

    Three bacteriophages infecting Streptomyces erythreus, called G3, G4 and G5, were isolated and characterized. They contain double-stranded linear DNA molecules with cohesive ends. The restriction map of G3 DNA (48 kilobases long) for four restriction endonucleases and that of G4 DNA (43 kilobases long) for seven restriction endonucleases are reported. Restriction analysis and hybridization experiments showed that G3 and G4 share little DNA homology, while G4 and G5 are apparently identical except for an additional EcoRI site present in G5. The region containing this EcoRI site has been mapped on G4 DNA. Microbiological and serological data showed that G5 is very similar to G4. G3- and G4-resistant mutants of S. erythreus PS1 were isolated. The screening of phage-resistant mutants showed a high frequency of strains with increased erythromycin production. The mechanism of phage resistance of strain PS3 (G3 resistant) and of strain PS16 (G4 resistant) was examined. The DNA of the resistant strains contains no phage DNA, ruling out lysogeny as a cause of phage resistance. Transfection of strains PS1, PS3, and PS16 with DNA of the three phages showed the same efficiency, indicating that resistance is at the level of the bacterial wall.

  14. Isolation of Streptomyces globisporus and Blakeslea trispora mutants with increased carotenoid content.

    Science.gov (United States)

    Matselyukh, B P; Matselyukh, D Ya; Golembiovska, S L; Polishchuk, L V; Lavrinchuk, V Ya

    2013-01-01

    Hyperpigmented mutants of Streptomyces globisporus 1912-Hp7 and Blakeslea trispora 18(+), 184(-) were isolated by action of hydrogen peroxide and nitrosoguanidine, correspondingly, from initial strains S. globisporus 1912-4Lcp and B. trispora 72(-), 198(+). The carotenoids of dry biomass of obtained strains, rubbed thoroughly with glass powder by a pestle in porcelain mortar were extracted by acetone and purified by TLC. Identification of the individual carotenoids was performed by means of HPLC and LC/MS spectrometry. It was shown that strain S. globisporus 1912-4Crt produced beta-carotene/lycopene (6.91/3.24 mg/L), mutants 1912-4Lcp and 1912-7Hp synthesized only lycopene (26.05 and 50.9 mg/L, respectively), and strains B. trispora 18(+) and 184(-)-beta-carotene (6.2% in dry biomass or more 2.5 g/L) without illumination in shake flasks. It is the first example of high constitutive production of the carotenoids by the representative of genus Streptomyces without photoinduction or increased synthesis of sigma factor The improved strains of B. trispora 18(+) and 184(-) can be used for biotechnological production of beta-carotene in industrial conditions.

  15. In Vitro selection of Neisseria gonorrhoeae mutants with elevated MIC values and increased resistance to cephalosporins.

    Science.gov (United States)

    Johnson, Steven R; Grad, Yonatan; Ganakammal, Satishkumar Ranganathan; Burroughs, Mark; Frace, Mike; Lipsitch, Marc; Weil, Ryan; Trees, David

    2014-11-01

    Strains of Neisseria gonorrhoeae with mosaic penA genes bearing novel point mutations in penA have been isolated from ceftriaxone treatment failures. Such isolates exhibit significantly higher MIC values to third-generation cephalosporins. Here we report the in vitro isolation of two mutants with elevated MICs to cephalosporins. The first possesses a point mutation in the transpeptidase region of the mosaic penA gene, and the second contains an insertion mutation in pilQ.

  16. Sensitivity of Escherichia coli acrA Mutants to Psoralen plus Near-Ultraviolet Radiation

    DEFF Research Database (Denmark)

    Hansen, M. Trier

    1982-01-01

    The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage λ. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free λ phage...... specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens....

  17. Sprains and Strains

    Science.gov (United States)

    ... wrestling put people at risk for strains. Gymnastics, tennis, rowing, golf, and other sports that require extensive gripping can increase the risk of hand and forearm strains. Elbow strains sometimes occur in people who participate in ...

  18. Muscle strain treatment

    Science.gov (United States)

    Treatment - muscle strain ... Question: How do you treat a muscle strain ? Answer: Rest the strained muscle and apply ice for the first few days after the injury. Anti-inflammatory medicines or acetaminophen ( ...

  19. Muscle strain (image)

    Science.gov (United States)

    A muscle strain is the stretching or tearing of muscle fibers. A muscle strain can be caused by sports, exercise, a ... something that is too heavy. Symptoms of a muscle strain include pain, tightness, swelling, tenderness, and the ...

  20. Within-host stochastic emergence dynamics of immune-escape mutants.

    Directory of Open Access Journals (Sweden)

    Matthew Hartfield

    2015-03-01

    Full Text Available Predicting the emergence of new pathogenic strains is a key goal of evolutionary epidemiology. However, the majority of existing studies have focussed on emergence at the population level, and not within a host. In particular, the coexistence of pre-existing and mutated strains triggers a heightened immune response due to the larger total pathogen population; this feedback can smother mutated strains before they reach an ample size and establish. Here, we extend previous work for measuring emergence probabilities in non-equilibrium populations, to within-host models of acute infections. We create a mathematical model to investigate the emergence probability of a fitter strain if it mutates from a self-limiting strain that is guaranteed to go extinct in the long-term. We show that ongoing immune cell proliferation during the initial stages of infection causes a drastic reduction in the probability of emergence of mutated strains; we further outline how this effect can be accurately measured. Further analysis of the model shows that, in the short-term, mutant strains that enlarge their replication rate due to evolving an increased growth rate are more favoured than strains that suffer a lower immune-mediated death rate ('immune tolerance', as the latter does not completely evade ongoing immune proliferation due to inter-parasitic competition. We end by discussing the model in relation to within-host evolution of human pathogens (including HIV, hepatitis C virus, and cancer, and how ongoing immune growth can affect their evolutionary dynamics.

  1. Mutant breeding of ornamental trees for creating variations with high value using Proton Beam

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, H. J.; Lim, J. H.; Woo, S. M.; Hwang, M. J.; Pyo, S. H.; Woo, J. S. [Phygen Co., Daejeon (Korea, Republic of)

    2009-04-15

    It is necessary to induce the improved strains of ornamental plants with more disease-resistant and useful for landscape or phytoremediation. Mutation breeding has played an important role in crop improvement, and more than 2,000 mutant cultivars have been released. For the induction of mutation, gamma rays and X-rays are widely used as a mutagen. Proton beam had higher energy than -ray and worked with localized strength, so that proton-beam radiation could be valuable tool to induce useful strains of ornamental plants. Proton ion beam irradiation was used to induce a useful mutant in rice, chrysanthemum, carnation, and so on in Japan. Also, proton ion beam was used to select a useful host strain, in polyhydroxybutyrate (PHB), a member of biodegradable plastic, could be overproduced in Korea. Therefore, we surmise that the effects of proton beam is different from those of gamma rays and X-rays, and we expect proton beam to be a new mutagen. This research was conducted to investigate the proton-beam radiation sensitivity and seed germination rate of the various ornamental plants like as Albizia julibrissin, Ficus religiosa, Rhus chinensis, Sorbaria sorbilfolia and Spiraea chinensis, to survey the quantitative characteristics of proton beam induced strains. To induce the variants of ornamental plants, seeds were irradiated at the dose of 0{approx}2kGy of proton beam at room temperature. Proton beam energy level was 45 MeV and was irradiated at dose of 0{approx}2kGy by MC-50 Cyclotron. After irradiation, to assess the effects of proton beam on radiation sensitivity and morphological changes of the plants and the seed germination rate were analysed. By the proton beam radiation, the germination rate decreased at the higher dose. The other hand, the germination rate of Rhus chinensis increased the dose higher, so that it need to investigate the germination rate over 2kGy radiation. The effects of mutation induction by proton beam irradiation on seeds in Lagerstroemia

  2. Comparisons of Caenorhabditis Fucosyltransferase Mutants Reveal a Multiplicity of Isomeric N-Glycan Structures.

    Science.gov (United States)

    Yan, Shi; Jin, Chunsheng; Wilson, Iain B H; Paschinger, Katharina

    2015-12-01

    Recent studies have shown a remarkable degree of plasticity in the N-glycome of the model nematode Caenorhabditis elegans; ablation of glycosylation-relevant genes can result in radically altered N-glycan profiles despite only minor biological phenotypic effects. Up to four fucose residues and five different linkages of fucose are known on the N-glycans of C. elegans. Due to the complexity in the wild type, we established three mutant strains defective in two core fucosyltransferases each (fut-1;fut-6, fut-1;fut-8, and fut-6;fut-8). Enzymatically released N-glycans were subject to HPLC and MALDI-TOF MS/MS, in combination with various treatments, to verify structural details. The N-glycome of the fut-1;fut-6 mutant was the most complex of the three double-mutant strains due to the extension of the core α1,6-fucose as well as the presence of fucose on the bisecting galactose. In contrast, maximally two fucoses were found on N-glycans of the fut-1;fut-8 and fut-6;fut-8 strains. The different locations and capping of fucose meant that up to 13 isomeric structures, many highly galactosylated, were determined for some single masses. These data not only show the high variability of the N-glycomic capacity of a "simple" nematode but also exemplify the need for multiple approaches to reveal individual glycan structures within complex invertebrate glycomes.

  3. Breeding of clavulanic acid mutant%克拉维酸高产菌的选育

    Institute of Scientific and Technical Information of China (English)

    张汝玲

    2011-01-01

    以带小棒链霉菌bs1574为出发菌株,经γ射线照射,并结合甘油耐受性菌株的理性筛选,选育得到较佳诱变菌株bs2325,效价达2 569μg/mL.采用摇瓶发酵初筛和复筛,选育出甘油耐受性正向突变菌株,是出发菌株(效价为968 μg/mL)的2.65倍,该突变菌株在琼脂斜面培养基上连续转接传代4代,克拉维酸的产量保持稳定.%The method of screening and selecting better mutants of bs2325 by γ-ray irradiation onto streptomyces bs1574 is put forward, and the titer is 2 569 μg/mL. By fermentation screening and secondary screening, strains of mutant glycerol positive tolerance is selected, whose titer is 2. 65 times that of the original strain(968 μg/mL). The mutant strain grows well during four generations on the agar slant medium, and the yield of clavulanic acid remains stable.

  4. Screening of flocculant-producing strains by NTG mutagenesis

    Institute of Scientific and Technical Information of China (English)

    HUANG Xiao-wu; CHENG Wen; HU Yong-you

    2005-01-01

    Screening of new microorganism being able to produce efficiently flocculants was carried out. A new model for screening efficient flocculant-producing strains was designed and tested. The results showed that this model for screening efficient flocculant-producing strains is very reliable and can greatly shorten the screening period. 13 flocculant-producing strains were isolated from activated sludge by conventional method. A strain, designated as HHE6, produced the bioflocculant with the turbidity removal 98% for kaolin suspension. Six of 13 strains selected as the original strains were treated with NTG as mutagen, and five mutant strains(HHE-P7, HHE-A8, HHE-P21,HHE-P24, HHE-A26) with high flocculation efficiency was obtained by selection, which exhibited the flocculation rate for kaolin suspension above 90%. Strains HHE6, HHE-P7, and HHE-P24 were classified as Penicillium purpurogenum, HHE-P21 as Penicillium cyclopium,HHE-A26 as Aspergillus versicolor and HHE-A8 as Aspergillus fumigatus, and it is hitherto unreported for biofloccutant-producing strains of Penicillium. The growth of the six strains(HHE6, HHE-P7, HHE-A8, HHE-P21, HHE-P24, HHE-A26) had similar curves, i.e. firstly increasing rapidly, keeping relatively constant then and finally decreasing gradually with cultivation time. The production of bioflocculants by strains showed the similar pattern to strain growth.

  5. Evidence that mutant PfCRT facilitates the transmission to mosquitoes of chloroquine-treated Plasmodium gametocytes.

    Science.gov (United States)

    Ecker, Andrea; Lakshmanan, Viswanathan; Sinnis, Photini; Coppens, Isabelle; Fidock, David A

    2011-01-15

    Resistance of the human malarial parasite Plasmodium falciparum to the antimalarial drug chloroquine has rapidly spread from several independent origins and is now widely prevalent throughout the majority of malaria-endemic areas. Field studies have suggested that chloroquine-resistant strains might be more infective to mosquito vectors. To test the hypothesis that the primary chloroquine resistance determinant, mutations in PfCRT, facilitates parasite transmission under drug pressure, we have introduced a mutant or wild-type pfcrt allele into the rodent model malarial parasite Plasmodium berghei. Our results show that mutant PfCRT from the chloroquine-resistant 7G8 strain has no effect on asexual blood stage chloroquine susceptibility in vivo or ex vivo but confers a significant selective advantage in competitive mosquito infections in the presence of this drug, by protecting immature gametocytes from its lethal action. Enhanced infectivity to mosquitoes may have been a key factor driving the worldwide spread of mutant pfcrt.

  6. Effect of temperature on the production of cellulases, xylanases and lytic enzymes by selected Trichoderma reesei mutants

    Directory of Open Access Journals (Sweden)

    Piotr Janas

    2014-08-01

    Full Text Available The effect of temperature in the rangę of 26-38°C on the production of cellulases, xylanases and lytic enzymes by four mutant strains of Trichoderma reesei was analysed. On the basis of these investigations three thermosensitive strains (M-7. RUT C 30 and VTT-D-78085 which showed reduced excretion of the above mentioned enzymes as well as protein and a thermoresistant mutant (VTT-D-79I24 which grew within a temperature range of 26-34°C were characterized. Higher temperature caused an increase in the level of xylanolytic enzymes produced by the four mutants. In addition. it effected the complex composition of cellulolytic enzymes secreted by VTT-D-79l 24 (i.c. increased and reduced excertion of (β-glucosidase and β-1,4-endoglucanase respectively.

  7. Reduced insulin/IGF-1 signaling restores germ cell immortality to Caenorhabditis elegans Piwi mutants.

    Science.gov (United States)

    Simon, Matt; Sarkies, Peter; Ikegami, Kohta; Doebley, Anna-Lisa; Goldstein, Leonard D; Mitchell, Jacinth; Sakaguchi, Aisa; Miska, Eric A; Ahmed, Shawn

    2014-05-08

    Defects in the Piwi/piRNA pathway lead to transposon desilencing and immediate sterility in many organisms. We found that the C. elegans Piwi mutant prg-1 became sterile after growth for many generations. This phenotype did not occur for RNAi mutants with strong transposon-silencing defects and was separable from the role of PRG-1 in transgene silencing. Brief periods of starvation extended the transgenerational lifespan of prg-1 mutants by stimulating the DAF-16/FOXO longevity transcription factor. Constitutive activation of DAF-16 via reduced daf-2 insulin/IGF-1 signaling immortalized prg-1 strains via RNAi proteins and histone H3 lysine 4 demethylases. In late-generation prg-1 mutants, desilencing of repetitive segments of the genome occurred, and silencing of repetitive loci was restored in prg-1; daf-2 mutants. This study reveals an unexpected interface between aging and transgenerational maintenance of germ cells, where somatic longevity is coupled to a genome-silencing pathway that promotes germ cell immortality in parallel to the Piwi/piRNA system. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  8. A conditionally lethal mutant of Salmonella Typhimurium induces a protective response in mice.

    Science.gov (United States)

    Hidalgo, Alejandro A; Villagra, Nicolás A; Jerez, Sebastián A; Fuentes, Juan A; Mora, Guido C

    2016-02-01

    Here we present the design of a conditionally lethal mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) which growth depends on tetracycline (Tet). Four mutants of S. Typhimurium, with Tet-conditional growth, were created by inserting the tetRA cassette. Three of the mutants presented a conditional-lethal phenotype in vitro. One mutant in the yabB gene remained conditional inside cells and did not persisted after 24 h in cell cultures. The capacity of S. Typhimurium yabB::tetRA to invade deep organs was investigated in intraperitoneally (IP) infected mice fed with or without chlortetracycline (CTet), a Tet analog with lower antibiotic activity. The yabB::tetRA mutant was undetectable in liver or spleen of animals under normal diet, while in mice under diet including CTet, yabB::tetRA invaded at a level comparable to the WT in mice under normal diet. Moreover, yabB::tetRA produced a strong humoral-immunoresponse after one IP immunization with 10(6) bacteria, measured as serum reactivity against S. Typhimurium whole cell extract. By contrast, oral immunization with 10(6) bacteria was weaker and variable on inducing antibodies. Consistently, IP infected mice were fully protected in a challenge with 10(4) oral S. Typhimurium, while protection was partial in orally immunized mice. Our data indicate that S. Typhimurium yabB::tetRA is a conditionally attenuated strain capable of inducing a protective response in mice in non-permissive conditions.

  9. Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

    Science.gov (United States)

    Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

    2017-12-01

    The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr (a) ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr (a) has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr (a) significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

  10. Elevated oxidative membrane damage associated with genetic modifiers of Lyst-mutant phenotypes.

    Directory of Open Access Journals (Sweden)

    Colleen M Trantow

    2010-07-01

    Full Text Available LYST is a large cytosolic protein that influences the biogenesis of lysosome-related organelles, and mutation of the encoding gene, LYST, can cause Chediak-Higashi syndrome. Recently, Lyst-mutant mice were recognized to also exhibit an iris disease resembling exfoliation syndrome, a common cause of glaucoma in humans. Here, Lyst-mutant iris phenotypes were used in a search for genes that influence Lyst pathways. In a candidate gene-driven approach, albino Lyst-mutant mice homozygous for a mutation in Tyr, whose product is key to melanin synthesis within melanosomes, exhibited complete rescue of Lyst-mutant iris phenotypes. In a genetic background-driven approach using a DBA/2J strain of congenic mice, an interval containing Tyrp1 enhanced Lyst-dependent iris phenotypes. Thus, both experimental approaches implicated the melanosome, an organelle that is a potential source of oxidative stress, as contributing to the disease phenotype. Confirming an association with oxidative damage, Lyst mutation resulted in genetic context-sensitive changes in iris lipid hydroperoxide levels, being lowest in albino and highest in DBA/2J mice. Surprisingly, the DBA/2J genetic background also exposed a late-onset neurodegenerative phenotype involving cerebellar Purkinje-cell degeneration. These results identify an association between oxidative damage to lipid membranes and the severity of Lyst-mutant phenotypes, revealing a new mechanism that contributes to pathophysiology involving LYST.

  11. Mutational biosynthesis of neomycin analogs by a mutant of neomycin-producing Streptomyces fradiae.

    Science.gov (United States)

    Shi, Guanying; Zhang, Xingang; Wu, Lang; Xie, Jin; Tao, Ke; Hou, Taiping

    2011-11-01

    Neomycin, produced by Streptomyces fradiae, has been widely used for the treatment of bacterial infections in clinical and agricultural applications. In this study, a neomycin nonproducing mutant of S. fradiae was obtained by gene disruption technique for mutational biosynthesis. A crucial gene neoC (neo7) which encodes 2-deoxystreptamine (2-DOS) synthases was disrupted. The mutant could resume producing neomycin in the presence of 2-DOS. Salen derivatives of 2-DOS were synthesized and individually added to cultures of the mutant. Antibacterial activity of the mutasynthesis products against Staphylococcus aureus and four plant pathogenic bacteria (Pseudomonas solanacarum, Erwinia carotovora, Xanthomonas oryzae, and Xanthomonas campestris) was detected quantitatively by Oxford cup method. It is suggested that all 2-DOS derivatives were incorporated by the mutant into new active neomycin analogs except for 2-DOS derivative 2d ((1R,2r,3S,4R,6S)-4,6-bis((E)-3,5-di-tert-butyl-2-hydroxybenzylideneamino)cyclohexane-1,2,3-triol). Neomycin analogs produced by feeding 2-DOS derivative 2a ((1R,2r,3S,4R,6S)-4,6-bis((E)-2 hydroxybenzylideneamino)cyclohexane-1,2,3-triol) to cultures of the mutant displayed a similar antibacterial activity with neomycin produced by wild strain.

  12. Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

    Directory of Open Access Journals (Sweden)

    Christopher M Brennan

    Full Text Available The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA, the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

  13. Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

    Science.gov (United States)

    Brennan, Christopher M; Mazzucca, Nicholas Q; Mezoian, Taylor; Hunt, Taylor M; Keane, Meaghan L; Leonard, Jessica N; Scola, Shelby E; Beer, Emma N; Perdue, Sarah; Pellock, Brett J

    2014-01-01

    The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

  14. A mutant beta-tubulin confers resistance to the action of benzimidazole-carbamate microtubule inhibitors both in vivo and in vitro.

    Science.gov (United States)

    Foster, K E; Burland, T G; Gull, K

    1987-03-16

    The mutant BEN210 of Physarum polycephalum is highly resistant to a number of benzimidazole carbamate agents, including methylbenzimidazole-2-yl-carbamate and parbendazole. The resistance is conferred by the benD210 mutation in a structural gene for beta-tubulin. This mutant allele encodes a beta-tubulin with novel electrophoretic mobility. We have used this strain to determine whether the mutant beta-tubulin is used in microtubules and whether this usage permits microtubule polymerisation in the presence of drugs both in vivo and in vitro. In vitro assembly studies of tubulin purified from the mutant strain have shown that microtubules are formed both in the absence of drugs and in all drug concentrations tested (up to 50 microM parbendazole). In contrast, the assembly of microtubules from wild-type tubulin in vitro is totally inhibited by 2-5 microM parbendazole. Thus the resistance of BEN210 to parbendazole observed in vivo has been reproduced in vitro using tubulin purified from the mutant strain. Electrophoretic analysis of the microtubules formed in vitro has shown that both the wild-type and the mutant beta-tubulin are incorporated into the microtubules and that the proportion of mutant to wild-type beta-tubulin appears to remain constant with increasing drug concentration. This is the first demonstration of a single mutation in a tubulin structural gene causing an altered function of the gene product in vitro.

  15. In Vitro Capability of Faropenem To Select for Resistant Mutants of Streptococcus pneumoniae and Haemophilus influenzae▿ †

    Science.gov (United States)

    Kosowska-Shick, Klaudia; Clark, Catherine; Credito, Kim; Dewasse, Bonifacio; Beachel, Linda; Ednie, Lois; Appelbaum, Peter C.

    2008-01-01

    When tested against nine strains of pneumococci and six of Haemophilus influenzae of various resistotypes, faropenem failed to select for resistant mutants after 50 days of consecutive subculture in subinhibitory concentrations. Faropenem also yielded low rates of spontaneous mutations against all organisms of both species. By comparison, resistant clones were obtained with macrolides, ketolides, and quinolones. PMID:18086853

  16. Enhanced cellulase production in mutants of Thermomonospora

    Energy Technology Data Exchange (ETDEWEB)

    Fennington, G.; Lupo, D.; Stutzenberger, F.

    1982-01-01

    Thermomonospora curvata, a thermophilic actinomycete, secretes multiple forms of endo-beta, 1-4-glucanase (EG) when grown on cellulose-mineral salts liquid medium. The EG activity (measured as carboxymethyl cellulose hydrolysis) was separated by ion exchange chromatography into three distinct components which differed in their kinetic properties. Exposure of T. curvata to ultraviolet light, N-nitrosoguanidine, or ethane methyl sulfonate produced mutants with enhanced EG production. Selection of colonies which cleared cellulose agar plants containing 2-deoxyglucose or glycerol yielded mutants having 1.5 to 2.6 times the extracellular EG and saccharifying activity (measured by filter-paper and cotton-fiber hydrolysis). The secretion of extracellular protein was increased proportionally in mutant cultures. (Refs. 40).

  17. High Persister Mutants in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Heather L Torrey

    Full Text Available Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection.

  18. Characterization of antimicrobial activity against Listeria and cytotoxicity of native melittin and its mutant variants.

    Science.gov (United States)

    Wu, Xi; Singh, Atul K; Wu, Xiaoyu; Lyu, Yuan; Bhunia, Arun K; Narsimhan, Ganesan

    2016-07-01

    Antimicrobial peptides (AMPs) are relatively short peptides that have the ability to penetrate the cell membrane, form pores leading to cell death. This study compares both antimicrobial activity and cytotoxicity of native melittin and its two mutants, namely, melittin I17K (GIGAVLKVLTTGLPALKSWIKRKRQQ) with a higher charge and lower hydrophobicity and mutant G1I (IIGAVLKVLTTGLPALISWIKRKRQQ) of higher hydrophobicity. The antimicrobial activity against different strains of Listeria was investigated by bioassay, viability studies, fluorescence and transmission electron microscopy. Cytotoxicity was examined by lactate dehydrogenase (LDH) assay on mammalian Caco-2 cells. The minimum inhibitory concentration of native, mutant I17K, mutant G1I against Listeria monocytogenes F4244 was 0.315±0.008, 0.814±0.006 and 0.494±0.037μg/ml respectively, whereas the minimum bactericidal concentration values were 3.263±0.0034, 7.412±0.017 and 5.366±0.019μg/ml respectively. Lag time for inactivation of L. monocytogenes F4244 was observed at concentrations below 0.20 and 0.78μg/ml for native and mutant melittin I17K respectively. The antimicrobial activity against L. monocytogenes F4244 was in the order native>G1I>I17K. Native melittin was cytotoxic to mammalian Caco-2 cells above concentration of 2μg/ml, whereas the two mutants exhibited negligible cytotoxicity up to a concentration of 8μg/ml. Pore formation in cell wall/membrane was observed by transmission electron microscopy. Molecular dynamics (MD) simulation of native and its mutants indicated that (i) surface native melittin and G1I exhibited higher tendency to penetrate a mimic of bacterial cell membrane and (ii) transmembrane native and I17K formed water channel in mimics of bacterial and mammalian cell membranes.

  19. Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

    OpenAIRE

    Lorowitz, W; Clark, D.

    1982-01-01

    Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

  20. Altered expression of several genes in IIIManL-defective mutants of Streptococcus salivarius demonstrated by two-dimensional gel electrophoresis of cytoplasmic proteins.

    Science.gov (United States)

    Lapointe, R; Frenette, M; Vadeboncoeur, C

    1993-05-01

    Mannose, glucose and fructose are transported in Streptococcus salivarius by a phosphoenolpyruvate:mannose phosphotransferase system (PTS) which consists of a membrane-bound Enzyme II (EII) and two forms of IIIMan having molecular weights of 38,900 (IIIManH) and 35,200 (IIIManL), respectively. We have previously reported the isolation of spontaneous mutants lacking IIIManL and showed that they exhibit higher beta-galactosidase activity than the parental strain after growth on glucose, and that some of them constitutively express a fructose PTS which is induced by fructose in the parental strain. In an attempt to determine whether the expression of other genes is affected by the mutation and what the physiological link is between them, we examined three S. salivarius IIIManL-defective mutants (strains A37, B31 and G29) and the parental strain using two-dimensional gel electrophoresis after growth of the cells on a variety of sugars. After growth on glucose, five new proteins were detected in the cytoplasm of the three mutants. Two of these proteins were induced in the parental strain by galactose or oligosaccharides containing galactose, and one was specifically induced by melibiose. The other two proteins were not detected in the parental strain under any of the growth conditions tested. Two other proteins were only detected in glucose-grown cells of mutant A37, and a protein associated with the metabolism of fructose was constitutively expressed in mutants B31 and G29. Moreover, we have found that under identical growth conditions the amounts of several other proteins which were detected in the parental strain were either increased or decreased in the mutants. Globally, our results have indicated that (1) the expression of several genes was affected in the spontaneous IIIManL-defective mutants; (2) some of the proteins abnormally produced in the mutants were specifically induced in the parental strain by sugars; (3) the phenotypic modifications observed in the

  1. Buffering Capacity of Pigmented and Nonpigmented Strains of Serratia marcescens

    Science.gov (United States)

    Rius, Núria; Solé, Montserrat; Francia, Alicia; Lorén, José G.

    1994-01-01

    The pigmented strain Serratia marcescens ATCC 274 had a higher buffering capacity and a higher membrane H+ conductance than S. marcescens GP, a spontaneous nonpigmented mutant of ATCC 274. The data suggest that mutations which apparently affect only the synthesis of a secondary metabolite can modify buffering capacity and passive H+ conductance. PMID:16349300

  2. Native Mutant Huntingtin in Human Brain

    Science.gov (United States)

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  3. Aging Kit mutant mice develop cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Lei Ye

    Full Text Available Both bone marrow (BM and myocardium contain progenitor cells expressing the c-Kit tyrosine kinase. The aims of this study were to determine the effects of c-Kit mutations on: i. myocardial c-Kit(+ cells counts and ii. the stability of left ventricular (LV contractile function and structure during aging. LV structure and contractile function were evaluated (echocardiography in two groups of Kit mutant (W/Wv and W41/W42 and in wild type (WT mice at 4 and 12 months of age and the effects of the mutations on LV mass, vascular density and the numbers of proliferating cells were also determined. In 4 month old Kit mutant and WT mice, LV ejection fractions (EF and LV fractional shortening rates (FS were comparable. At 12 months of age EF and FS were significantly decreased and LV mass was significantly increased only in W41/W42 mice. Myocardial vascular densities and c-Kit(+ cell numbers were significantly reduced in both mutant groups when compared to WT hearts. Replacement of mutant BM with WT BM at 4 months of age did not prevent these abnormalities in either mutant group although they were somewhat attenuated in the W/Wv group. Notably BM transplantation did not prevent the development of cardiomyopathy in 12 month W41/W42 mice. The data suggest that decreased numbers and functional capacities of c-Kit(+ cardiac resident progenitor cells may be the basis of the cardiomyopathy in W41/W42 mice and although defects in mutant BM progenitor cells may prove to be contributory, they are not causal.

  4. [Repression of the enzyme inducible syntheses in Escherichia coli K12 mutant with a deleted ptsH gene].

    Science.gov (United States)

    Gershanovich, V N; Il'ina, T S; Rusina, O Iu; Iurovitskaia, N V; Bol'shakova, T N

    1977-01-01

    The genome of lambda phage with thermosensitive repressor was integrated into the pts region of the E. coli chromosome. Such a lysogenic culture behaves as a pts mutant at 30 degrees. Heating of cells of this strain leads to the induction of lambda prophage and formation of deletions in the pts region. A mutant with a deletion covering ptsH gene was isolated after prophage induction. The deletion nature of pts mutation was confirmed in genetic and biochemical experiments. It was shown that the deletion is small and does not involve ptsI and lig genes. The isolated deltaptsH mutant possesses all characteristics of pts mutants: pleiotropic impairment of transport and utilization of a number of carbohydrates, repression of the enzyme inducible synthesis and resistance to catabolite repression with glucose. These data (together with earlier ones) allow us to conclude that the phosphorylated form of HPr is involved (in direct of indirect manner/ in activation of DNA transcription.

  5. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    cell line, biochemical and structural biology techniques to uncover the best candidate drugs for the clinical targeting of these mutants. Targeting...ESR1-­‐Mutant  Breast  Cancer   W81XWH-­‐14-­‐1-­‐0360   4   2. Keywords Estrogen Receptor Acquired Drug Resistance Metastatic Breast...preparations for publication: 1) “ESR1 Somatic Mutations Y537S and D538G Confer Breast Cancer Endocrine Resistance by Stabilizing the Active AF-2 conformation

  6. Mutagenizing brewing yeast strain for improving fermentation property of beer.

    Science.gov (United States)

    Liu, Zengran; Zhang, Guangyi; Sun, Yunping

    2008-07-01

    A brewing yeast mutant with perfect sugar fermentation capacity was isolated by mutagenizing the Saccharomyces pastorianus transformant, which carries an integrated glucoamylase gene and has one copy of non-functional alpha-acetolactate synthase gene. The mutant was able to utilize maltotriose efficiently, and the maltotriose fermentability in YNB-2% maltotriose medium increased from 32.4% to 72.0% after 5 d in shaking culture. The wort fermentation test confirmed that the sugar fermentation property of the mutant was greatly improved, while its brewing performances were analogous to that of the wild-type strain and the characteristic trait of shortened beer maturation period was retained. Therefore, we believe that the brewing yeast mutant would benefit the beer industry and would be useful for low caloric beer production.

  7. Survival characteristics of environmental and clinically derived strains of Cronobacter sakazakii in infant milk formula (IMF) and ingredients.

    Science.gov (United States)

    Walsh, D; Molloy, C; Iversen, C; Carroll, J; Cagney, C; Fanning, S; Duffy, G

    2011-03-01

    The study aimed to compare survival of Cronobacter sakazakii strains in plant-derived infant milk formula (IMF) ingredients and their thermotolerance in reconstituted IMF. Inulin and lecithin were inoculated with isolates of C. sakazakii including the typed clinical strains, NCTC 11467(T) and BAA 894; a mutant strain in which the wcaD gene had been disrupted; and two environmental strains isolated from IMF processing facilities. Samples were stored and examined for C. sakazakii. All strains were still detectable in both matrices after 338 days storage, except for the mutant strain that was no longer detectable at that time. Higher numbers of the environmental strains were recoverable after 338 days than the clinical strains. The thermotolerance of the five strains was investigated in reconstituted IMF at 55, 60 and 65°C. The clinically derived type strain, NCTC 11467(T), and the mutant strain were shown to be significantly more thermotolerant than other strains tested. Environmental strains were more persistent than the clinical strains in inulin and lecithin, indicating that patho-adaptation may have contributed to a reduction in the desiccation tolerance phenotype. However, the thermotolerance results could indicate that the ability to produce extracellular polysaccharide decreases thermotolerance. These results indicate that desiccation resistance may play a role in survival of C. sakazakii in dry IMF ingredients and processing plants; however, this trait may be of less importance in clinical environs. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  8. Characterization and responses to environmental cues of a photosynthetic antenna-deficient mutant of the filamentous cyanobacterium Anabaena sp. PCC 7120.

    Science.gov (United States)

    Leganés, Francisco; Martínez-Granero, Francisco; Muñoz-Martín, M Ángeles; Marco, Eduardo; Jorge, Alberto; Carvajal, Laura; Vida, Teresa; González-Pleiter, Miguel; Fernández-Piñas, Francisca

    2014-07-01

    The cyanobacterial phycobilisome (PBS) is a giant pigment-protein complex which harvests light energy for photosynthesis and comprises two structures: a core and peripheral rods. Most studies on PBS structure and function are based on mutants of unicellular strains. In this report, we describe the phenotypic and genetic characterization of a transposon mutant of the filamentous Anabaena sp. strain PCC 7120, denoted LC1, which cannot synthesize the phycobiliprotein phycocyanin (PC), the main component of the rods; in this mutant, the transposon had inserted into the cpcB gene (orf alr0528) which putatively encodes PC-β chain. Mutant LC1 was able to synthesize phycoerythrocyanin (PEC), a phycobiliprotein (PBP) located at the terminal region of the rods; but in the absence of PC, PEC did not attach to the PBSs that only retained the allophycocyanin (APC) core; ferredoxin: NADP+-oxidoreductase (FNR) that is associated with the PBS in the wild type, was not found in isolated PBSs from LC1. The performance of the mutant exposed to different environmental conditions was evaluated. The mutant phenotype was successfully complemented by cloning and transfer of the wild type complete cpc operon to mutant LC1. Interestingly, LC1 compensated its mutation by significantly increasing the number of its core-PBS and the effective quantum yield of photosystem II (PSII) photochemistry; this feature suggests a more efficient energy conversion in the mutant which may be useful for biotechnological applications.

  9. Phenotype Microarray Profiling of Staphylococcus aureus menD and hemB Mutants with the Small-Colony-Variant Phenotype†

    Science.gov (United States)

    von Eiff, Christof; McNamara, Peter; Becker, Karsten; Bates, Donna; Lei, Xiang-He; Ziman, Michael; Bochner, Barry R.; Peters, Georg; Proctor, Richard A.

    2006-01-01

    Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in menD or hemB that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the hemB mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than those seen with the hemB mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in S. aureus. PMID:16385058

  10. Complete Genome Sequence of Pigmentation Negative Yersinia Pestis strain Cadman Running head: Complete Genome Sequence of Y. pestis strain Cadman

    Science.gov (United States)

    2016-10-27

    coding regions, 19 ribosomal RNAs, 68 tRNAs, 12 ncRNAs, and 2 CRISPR arrays. 64 Gene content was compared to Y. pestis CO92 (NC_003131, NC_003132...264. 89 3. Burrows TW, Jackson S. 1956. The virulence- enhancing effect of iron on 90 nonpigmented mutants of virulent strains of Pasteurella pestis

  11. Preferential repair in Saccharomyces cerevisiae rad mutants after induction of interstrand cross-links by 8-methoxypsoralen plus UVA.

    Science.gov (United States)

    Meniel, V; Magaña-Schwencke, N; Averbeck, D

    1995-11-01

    The gene specific induction and the incision step of the removal of 8-methoxypsoralen (8-MOP) plus UVA-induced interstrand cross-links (ICL) was measured in repair mutants of Saccharomyces cerevisiae. Events were examined at the MAT alpha and HML alpha loci in mutants deficient in the repair of ICL, namely rad1, rad2 delta, rad52, pso2 and the rad16 mutant which is impaired in the removal of UV-induced pyrimidine dimers from the silent HML alpha locus. Previously, we observed in a wild-type strain (K107) preferential repair concerning the incision of 8-MOP photo-induced ICL. The present study indicates that the two mutants rad1 and rad2 delta show no repair in either locus, due presumably to their deficiency in the incision step of ICL repair. The rad52 mutant which is defective in recombination, is proficient in the preferential incision of ICL at the MAT alpha locus versus the HML alpha locus. The same is true for the pso2 mutant which also lacks the ability to perform complete repair of ICL. The rad16 mutant is unable to repair ICL in the silent locus HML alpha but is proficient in repair (i.e. the incision of ICL) in the transcriptionally active MAT alpha locus.

  12. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  13. [The gene wxcA of Xanthomonas campestris pv. campestris 8004 strain involved in EPS yield].

    Science.gov (United States)

    Lu, Guang-Tao; Tang, Ji-Liang; Wei, Guang-Ning; He, Yong-Qiang; Chen, Bao-Shan

    2004-07-01

    Xanthomonas campestris pv. campestris (Xcc), the pathogenic agent of black rot disease in cruciferous plants, produces large amount of extracellular polysaccharide (EPS), which has found wide applications in industry. For the great commercial value of the xanthan gum, many of the genes involved in EPS biosynthesis have been cloned and the mechanism of EPS biosynthesis also has been studied. In order to clone genes involved in EPS biosynthesis, Xcc wild-type strain 8004 was mutagenized with transposon Tn5 gusA5, and a number of EPS-defective mutants were isolated in our previous work. The Tn5 gusA5 inserted sites of these mutants were located by using thermal asymmetric interlaced PCR, and results showed that two EPS-defective mutants were insertion mutants of the gene wxcA which involved in lipopolysaccharide (LPS) biosynthesis. The gene wxcA involved in lipopolysaccharide biosynthesis but dose not extracellular polysaccharide in others' report. wxcA::Tn5 gusA5 mutant 021C12, the polar mutant, was complemented with recombinant plasmid pLATC8570 harboring an intact wxcA gene in this work, but the yield of EPS of the wxcA::Tn5 gusA5 mutant was not restored. In order to identify the function of wxcA gene of Xcc 8004 strain, the gene wxcA was deleted by gene replacement strategy, and the no-polar mutant of wxcA was obtained. DeltawxcA mutant strain, named Xcc 8570, was confirmed by using both PCR and southern analysis. Beside the LPS biosynthesis of deltawxcA mutant was affected, The EPS yield of deltawxcA mutant strain reduced by 50% as compared with the wild-type strain 8004. DeltawxcA mutant could be complemented in trans with the intact wxcA gene, and the EPS yield of the mutant was restored. The combined data showed that wxcA gene not only involved in LPS biosynthesis but also EPS yield in Xcc 8004 strain.

  14. Morphological characterization of recombinant strains of Aspergillus oryzae producing alpha-amylase during batch cultivations

    DEFF Research Database (Denmark)

    Spohr, Anders Bendsen; Carlsen, Morten; Nielsen, Jens Bredal;

    1997-01-01

    Three alpha-amylase producing strains of Aspergillus oryzae used for recombinant protein production have been studied with respect to growth and protein production. By comparing the three strains with respect to morphology and protein production it is shown that a morphological mutant with a more...... dense mycelium is more efficient in producing alpha-amylase....

  15. Rapid Antibiotic Resistance Evolution of GASP Mutants

    Science.gov (United States)

    Zhang, Qiucen; Kim, Hyunsung; Pourmand, Nader; Austin, Robert

    2012-02-01

    The GASP phenotype in bacteria is due to a mutation which enables the bacteria to grow under high stress conditions where other bacteria stop growing. We probe using our Death Galaxy microenvironment how rapidly the GASP mutant can evolve resistance to mutagenic antibiotics compared to wild-type bacteria, and explore the genomic landscape changes due to the evolution of resistance.

  16. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    acer

    deleterious effects on living organisms (Shigaeva et al.,. 1994); they are also ... It was cultured at 25±0.5°C with a fluorescent light intensity of approximately 6 ... mutants) resulted in three new colonies characterized by different green colors ...

  17. A dominant semi dwarf mutant in rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ In the winter of 1997, a semi dwarf mutant was found in the F6 population of M9056/ R8018 xuan in Hainan Province. In the spring of 1998, the seeds were sown in Hefei, Anhui Province and the plant height of the population was measured at maturity.

  18. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    Science.gov (United States)

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  19. Mutant PTEN in Cancer : Worse Than Nothing

    NARCIS (Netherlands)

    Leslie, Nick R; den Hertog, Jeroen

    2014-01-01

    Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant

  20. Colored HOMFLY polynomials can distinguish mutant knots

    CERN Document Server

    Nawata, Satoshi; Singh, Vivek Kumar

    2015-01-01

    We illustrate from the viewpoint of braiding operations on WZNW conformal blocks how colored HOMFLY polynomials with multiplicity structure can detect mutations. As an example, we explicitly evaluate the (2,1)-colored HOMFLY polynomials that distinguish a famous mutant pair, Kinoshita-Terasaka and Conway knot.

  1. Characteristics of dibenzothiophene desulfurization by Rhodococcus erythropolis R1 and its Dsz-negative mutant

    Directory of Open Access Journals (Sweden)

    Zahra Etemadifar

    2014-01-01

    Full Text Available Introduction: Biodesulfurization is used as a selective method for lowering the sulfur content of petroleum products. Materials and methods: A sulfur-oxidation bacterial strain named Rhodococcus erythropolis R1 (NCBI GenBank Accession No. GU570564 was used in this study for desulfurization of dibenzothiophene (DBT. Results: The induced culture of strain R1 was able to produce 2-hydroxybiphenyl (2- HBP from DBT followed 4S pathway without further degrading carbon backbone. This process confirmed by gas chromatography (GC analysis. The specific activity of DBT desulfurization by R1 was 45 µM (g dry wt-1 h-1. The addition of Tween 80 as surfactant and glycerol as carbon source determines a 100% rate of DBT-desulfurization during 3 days. The heavy plasmid detected in R1 strain carries dsz genes responsible for biodesulfurization of DBT that was shown by PCR reaction. The mutant strains which had lost this plasmid also had lost desulfurization phenotype. Both mutant and wild strain were sensitive to high concentration of 2-HBP and some antibiotics. Discussion and conclusion: Strain R1 desulfurize DBT through the sulfur-specific degradation pathway or 4S pathway with the selective cleavage of carbon-sulfur (C-S bonds without reducing the energy content. Addition of surfactant enhanced the desulfurization of DBT by increasing its bioavailability and also could improve the growth and desulfurization rate. The location of desulfurization genes was on a heavy plasmid in strain R1. Based on the results of this study, R. erythropolis R1 could serve as a model system for efficient biodesulfurization of petroleum oil without reducing the energy value.

  2. Isolation and characterization of a newly isolated Pseudomonas mutant for protease production

    Directory of Open Access Journals (Sweden)

    Jayati Ray Dutta

    2006-01-01

    Full Text Available A potent bacterium for extracellular protease production was isolated from local soil and identified as Pseudomonas sp. RAJR 044. A mutant of this strain JNGR 242 with protease productivity 2.5 fold higher was obtained by ultraviolet irradiation under experimentally optimized conditions of pH 7.0, temperature of 34ºC, inoculum volume of 1.0 mL and incubation time of 24 hours. Comparative analysis of the chemical characteristics i.e. assimilation of carbon and nitrogen sources were also carried out. Maximum growth of the mutant strain in 2% gelatin agar plate was obtained in presence of dextrose (2%, maltose (2%, ammonium sulfate (2% and potassium nitrate (2% whereas, that of the parent strain was found in sucrose (2% and ammonium nitrate (2%. The purified proteases from both the strains (parent and mutant appeared as single homogeneous bands corresponding to 14.4 kDa molecular weight on SDS-PAGE. On studying the kinetic properties of both strains it was observed that the rate of casein hydrolysis was maximum at pH 8.0 and 7.0 and temperatures 45º C and 60º C for the parent and mutant strains respectively. It was also observed that both the extracellular proteases were inhibited by a serine protease inhibitor i.e. PMSF at 2mM concentration.Uma bactéria isolada do solo e identificada como Pseudomonas sp. RAJR 044 demonstrou ser uma potencial produtora de protéase extracelular. Um mutante JNGR 242 dessa espécie foi obtido mediante radiação ultravioleta sob condições experimentalmente otimizadas de pH 7,0, temperatura de 34ºC, volume de inóculo de 1,0 mL e tempo de incubação de 24 hora produziu 2,5 mais protease. Também foram realizadas análises comparativas das características química quanto assimilação de diferentes fontes de carbono e de nitrogênio. O crescimento máximo desse mutante na placa de ágar gelatina (2% foi obtido na presença de sacarose (2%, maltose (2%, sulfato de amônia (2% e nitrato de potássio (2%, ao

  3. Polydiacetylene-Based High-Throughput Screen for Surfactin Producing Strains of Bacillus subtilis

    Science.gov (United States)

    Zhu, Lingyan; Xu, Qing; Jiang, Ling; Huang, He; Li, Shuang

    2014-01-01

    Although traditional mutation is still an attractive approach for strain improvement, it is tedious, time-consuming, and inefficient to screen for surfactin producing strains. To overcome this, we developed a high-throughput screening method for surfactin producing mutants by applying polydiacetylene (PDA) vesicles as sensors with visible chromatic change from blue to red, detected as colorimetric response (CR%) signal, which can even semi-quantify the yields of surfactin. Bacillus subtilis 723 was used as parent strain and multiply mutated with atmospheric and room temperature plasma (ARTP). Mutants were cultured in MicroFlask by Duetz (24 square deepwell plates, Applikon Biotechnology) and surfactin titers were tested in 96-well plates with PDA vesicles. Mutants with surfactin titers above150 mg/L (CR% value above 26%) were selected as high-yield strains and further quantified by HPLC. By integrating MicroFlask cultivation and the PDA vesicles detection, we screened 27,000 mutants and found 37 high-yield strains. From these, one mutant produced 473.6 mg/L surfactin (including 353.1 mg/L C15 surfactin), which was 5.4-fold than that of the parent strain. This method is efficient, cost-effective and provides wider application in screening for various surfactants. PMID:24498439

  4. Flocculation of Saccharomyces cerevisiae tup1 mutants.

    OpenAIRE

    1984-01-01

    Strains of Saccharomyces cerevisiae carrying a mutation in the TUP1 locus exhibited calcium-dependent flocculation. The flocculation had none of the characteristics of sexual agglutination. The flocculation differed from that exhibited by a FLO1 strain in the effect of pH on cation dependence and sensitivity to chemical inactivation.

  5. GAMPMS: Genetic algorithm managed peptide mutant screening.

    Science.gov (United States)

    Long, Thomas; McDougal, Owen M; Andersen, Tim

    2015-06-30

    The prominence of endogenous peptide ligands targeted to receptors makes peptides with the desired binding activity good molecular scaffolds for drug development. Minor modifications to a peptide's primary sequence can significantly alter its binding properties with a receptor, and screening collections of peptide mutants is a useful technique for probing the receptor-ligand binding domain. Unfortunately, the combinatorial growth of such collections can limit the number of mutations which can be explored using structure-based molecular docking techniques. Genetic algorithm managed peptide mutant screening (GAMPMS) uses a genetic algorithm to conduct a heuristic search of the peptide's mutation space for peptides with optimal binding activity, significantly reducing the computational requirements of the virtual screening. The GAMPMS procedure was implemented and used to explore the binding domain of the nicotinic acetylcholine receptor (nAChR) α3β2-isoform with a library of 64,000 α-conotoxin (α-CTx) MII peptide mutants. To assess GAMPMS's performance, it was compared with a virtual screening procedure that used AutoDock to predict the binding affinity of each of the α-CTx MII peptide mutants with the α3β2-nAChR. The GAMPMS implementation performed AutoDock simulations for as few as 1140 of the 64,000 α-CTx MII peptide mutants and could consistently identify a set of 10 peptides with an aggregated binding energy that was at least 98% of the aggregated binding energy of the 10 top peptides from the exhaustive AutoDock screening.

  6. Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

    Science.gov (United States)

    Cusick, John K; Hager, Elizabeth; Gill, Ronald E

    2015-01-01

    The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program.

  7. A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs.

    Science.gov (United States)

    Feodorova, V A; Pan'kina, L N; Savostina, E P; Sayapina, L V; Motin, V L; Dentovskaya, S V; Shaikhutdinova, R Z; Ivanov, S A; Lindner, B; Kondakova, A N; Bystrova, O V; Kocharova, N A; Senchenkova, S N; Holst, O; Pier, G B; Knirel, Y A; Anisimov, A P

    2007-11-01

    The lpxM mutant of the live vaccine Yersinia pestis EV NIIEG strain synthesising a less toxic penta-acylated lipopolysaccharide was found to be avirulent in mice and guinea pigs, notably showing no measurable virulence in Balb/c mice which do retain some susceptibility to the parental strain itself. Twenty-one days after a single injection of the lpxM-mutant, 85-100% protection was achieved in outbred mice and guinea pigs, whereas a 43% protection rate was achieved in Balb/c mice given single low doses (10(3) to 2.5 x 10(4) CFU) of this vaccine. A subcutaneous challenge with 2000 median lethal doses (equal to 20,000 CFU) of fully virulent Y. pestis 231 strain, is a 6-10-fold higher dose than that which the EV NIIEG itself can protect against.

  8. Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants.

    Science.gov (United States)

    Tedde, Vittorio; Rosini, Roberto; Galeotti, Cesira L

    2016-01-01

    An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized null deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn2+ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and null mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the null adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

  9. Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant.

    Directory of Open Access Journals (Sweden)

    Anja Wartenberg

    2014-12-01

    Full Text Available Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

  10. Association of methionine requirement with methyl mercury resistant mutants of yeast

    Energy Technology Data Exchange (ETDEWEB)

    Singh, A.; Sherman, F.

    1974-01-25

    It has been known for several years that strains resistant to mercury can be obtained in several bacterial species. Soon after the correlation between resistance to antibiotics and to mercury was recognized, it was established that genetic elements conferring resistance to antibiotics, mercury and other heavy metals in Escherichia coli and Samonella typhimurium and Staphylococcus aureus reside on extrachromosomal resistance transfer factors or plasmids. Among fungi, mercury resistant strains of Botrytis cinerea, Penicillium notatum, Sclerotinia fructicola, Stemphylium sarcinaeforme, and Saccharomyces cerevisiae have been reported. In most cases, this was accomplished by training the normal strains for growth on media supplemented with successively increasing concentrations of mercury compounds, and in some cases the resistance was lost when subcultured on mercury-free media. It is noteworthy that in none of the mercury-adapted strains of fungi has the genetic basis of resistance been determined. In this report we describe a method of isolation and characterization of methyl mercury resistant mutants of S. cerevisiae. This study was undertaken with the view that the examination of physiological changes associated with genetically defined resistant mutants will be useful in studying the mechanisms of cellular detoxification of organic mercurials.

  11. Characterization of Emericella nidulans RodA and DewA hydrophobin mutants

    DEFF Research Database (Denmark)

    Jensen, Britt Guillaume; Nielsen, Jakob Blæsbjerg; Pedersen, Mona Højgaard

    Hydrophobins are small amphiphilic proteins containing an eight cysteine pattern only found in filamentous fungi. They are involved in the attachment of hyphae to hydrophobic structures and the formation of aerial structures. Five Emericella nidulans mutant strains were examined to study the two...... hydrophobins RodA and DewA. Individual knock-out mutants rodAΔ, dewAΔ and the double deletion strain rodAΔdewAΔ were constructed. Furthermore, two strains containing a point mutation in the first of the cysteines of RodA (rodA-C57G), where one was coupled to the dewA deletion, were included. The reference...... strain (NID1) and dewAΔ displayed green conidia. However, rodAΔ and rodAΔdewAΔ showed a dark green/brown conidial pigmentation, while rodA-C57G and rodAC57G dewAΔ displayed lighter brown conidia. rodAΔ and rodAΔdewAΔ displayed a higher degree of hülle cells compared to the moderate amount observed...

  12. Isolation and characterization of a Sinorhizobium fredii mutant that cannot utilize proline as the sole carbon and nitrogen source

    Institute of Scientific and Technical Information of China (English)

    HUANG Sheng; BAI Xueliang; MA Qingsheng; TANG Xianlai; WU Bo

    2004-01-01

    Sinorhizobium fredii strain HN01 can use proline as the sole carbon and nitrogen source. A mutant strain GXHN100 unable to catabolize proline was screened from 6000 Tn5gusA5 random insertional mutants of S.fredii strain HN01. Sequencing analysis showed that an open reading frame, named pmrA (proline metabolic relative), was inserted by the Tn5gusA5. A positive clone, named pGXHN100 which containing 3.3kb foreign DNA fragment of S.fredii strain HN01, was isolated from a partial gene library of S.fredii HN01 by colony in situ hybridization. Sequence analysis showed that pGXHN100 contained the entire pmrA gene. The 3.3kb DNA fragment of pGXHN100 was cloned into a broad-host-range cosmid vector pLAFR3 to form plasmid pGXHN200 which was subsequently introduced into GXHN100 to form a complemented strain GXHN200. Plant test showed that GXHN100 was effective and no obvious changes in nitrogenase activity comparing with parental strain. But GXHN100 nodulated 2 days later on soybean and its nodulation efficiency and competitiveness were decreased. The complemented strain GXHN200 restored the nodulation efficiency and competitiveness of GXHN100 to the wild type.

  13. [Mating success and courtship ritual in strains of Drosophila melanogaster carrying mutation flamenco].

    Science.gov (United States)

    Romanova, L G; Romanova, N I; Subocheva, E A; Kim, A I

    2000-04-01

    Mating success was examined in groups of Drosophila melanogaster carrying mutation flamMS (SS, MSn1-2, and MSn1-3) and in wild-type flies. The proportion of normally copulating males was significantly lower in the mutant strains. The reduction in mating efficiency was caused by changes in male behavior rather than in female attractiveness. Individual analyses showed that male mating behavior in strains carrying flamMS was qualitatively and quantitatively different from that in the wild-type strain Canton S. The proportion of males that performed consecutive courtship stages was significantly lower in the mutant strains. The sequence and duration of some courtship stages (in particular, orientation and wing vibration) in mutant flies was shown to be altered. The significance of the flamenco locus in regulation of processes occurring at the organismal level are discussed.

  14. Genetically Modified Vibrio harveyi Strains as Potential Bioindicators of Mutagenic Pollution of Marine Environments

    Science.gov (United States)

    Czyż, Agata; Jasiecki, Jacek; Bogdan, Adam; Szpilewska, Hanna; Węgrzyn, Grzegorz

    2000-01-01

    For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of V. harveyi mutants resistant to neomycin. We constructed genetically modified V. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, we consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments. PMID:10653723

  15. Genetically modified Vibrio harveyi strains as potential bioindicators of mutagenic pollution of marine environments

    Energy Technology Data Exchange (ETDEWEB)

    Czyz, A.; Jasiecki, J.; Bogdan, A.; Szpilewska, H.; Wegrzyn, G.

    2000-02-01

    For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. The authors found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, they developed a simple method for isolation of V. harveyi mutants resistant to neomycin. The authors constructed genetically modified V. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, the authors consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments.

  16. Altered cellular infiltration and cytokine levels during early Mycobacterium tuberculosis sigC mutant infection are associated with late-stage disease attenuation and milder immunopathology in mice

    Directory of Open Access Journals (Sweden)

    McMurray David N

    2008-09-01

    Full Text Available Abstract Background Mouse virulence assessments of certain Mycobacterium tuberculosis mutants have revealed an immunopathology defect in which high tissue CFU counts are observed but the tissue pathology and lethality are reduced. M. tuberculosis mutants which grow and persist in the mouse lungs, but have attenuated disease progression, have the immunopathology (imp phenotype. The antigenic properties of these strains may alter the progression of disease due to a reduction in host immune cell recruitment to the lungs resulting in disease attenuation and prolonged host survival. Results In this study we focused on the mouse immune response to one such mutant; the M. tuberculosis ΔsigC mutant. Aerosol infection of DBA/2 and SCID mice with the M. tuberculosis ΔsigC mutant, complemented mutant and wild type strain showed proliferation of mutant bacilli in mouse lungs, but with decreased inflammation and mortality in DBA/2 mice. SCID mice shared the same phenotype as the DBA/2 mice in response to the ΔsigC mutant, however, they succumbed to the infection faster. Bronchoalveolar lavage (BAL fluid analysis revealed elevated numbers of infiltrating neutrophils in the lungs of mice infected with wild type and complemented ΔsigC mutant strains but not in mice infected with the ΔsigC mutant. In addition, DBA/2 mice infected with the ΔsigC mutant had reduced levels of TNF-α, IL-1β, IL-6 and IFN-γ in the lungs. Similarly, there was a reduction in proinflammatory cytokines in the lungs of SCID mice. In contrast to the mouse model, the ΔsigC mutant had reduced initial growth in guinea pig lungs. A possible mechanism of attenuation in the ΔsigC mutant may be a reduction in neutrophilic-influx in the alveolar spaces of the lungs, and decreased proinflammatory cytokine secretion. In contrast to mouse data, the M. tuberculosis ΔsigC mutant proliferates slowly in guinea pig lungs, a setting characterized by caseating necrosis. Conclusion Our

  17. Improvement of bioinsecticides production by sporeless Bacillus thuringiensis strains in response to various stresses in low cost medium.

    Science.gov (United States)

    Ben Khedher, Saoussen; Jaoua, Samir; Zouari, Nabil

    2011-05-01

    The use of bioinsecticides, particularly those produced by sporeless Bacillus thuringiensis strains, has been shown to be a good alternative in pest management. Two types of sporeless mutants were distinguished. The asporogenic mutants which completely lack spores produce a regular bipyramidal crystal inclusion. The oligosporogenic mutants kept the ability to produce insecticidal crystal proteins. However, sporulation in such mutants was not totally blocked and very few of them could still produce spores. In order to improve bioinsecticides production, adaptation of sporeless strains to heat shock and osmotic stress was investigated. Delta-endotoxin production by 78% of sporeless mutants was significantly improved by osmotic stress with an overproduction of about 17%, compared to the wild strain BNS3. However, toxin production was improved by only 21% of mutants after heat shock, in low cost medium. The statistical analysis proved that delta-endotoxin production, cell growth, and spore formation of asporogenic and oligosporogenic mutants depended on the type of applied stress. Each strain has an important potential when applying the adequate stress. Moreover, adaptation of sporeless mutants to NaCl may allow the substitution of all minerals of the medium by diluted sea water which appeared to be a good alternative for the economic production of bioinsecticides at industrial scale which is of great importance from the practical point of view.

  18. Novel strategy to improve vanillin tolerance and ethanol fermentation performances of Saccharomycere cerevisiae strains.

    Science.gov (United States)

    Zheng, Dao-Qiong; Jin, Xin-Na; Zhang, Ke; Fang, Ya-Hong; Wu, Xue-Chang

    2017-05-01

    The aim of this work was to develop a novel strategy for improving the vanillin tolerance and ethanol fermentation performances of Saccharomyces cerevisiae strains. Isogeneic diploid, triploid, and tetraploid S. cerevisiae strains were generated by genome duplication of haploid strain CEN.PK2-1C. Ploidy increments improved vanillin tolerance and diminished proliferation capability. Antimitotic drug methyl benzimidazol-2-ylcarbamate (MBC) was used to introduce chromosomal aberrations into the tetraploid S. cerevisiae strain. Interestingly, aneuploid mutants with DNA contents between triploid and tetraploid were more resistant to vanillin and showed faster ethanol fermentation rates than all euploid strains. The physiological characteristics of these mutants suggest that higher bioconversion capacities of vanillin and ergosterol contents might contribute to improved vanillin tolerance. This study demonstrates that genome duplication and MBC treatment is a powerful strategy to improve the vanillin tolerance of yeast strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Characterization of P1-deficient isogenic mutant of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

    Science.gov (United States)

    Segada, L M; Carlone, G M; Gheesling, L L; Lesse, A J

    2000-03-01

    Haemophilus influenzae biogroup aegyptius (formerly H. aegyptius) is the etiologic agent of Brazilian purpuric fever (BPF). A surface-exposed epitope on the outer membrane protein P1 is present on most strains of H. influenzae biogroup aegyptius associated with BPF but is absent in almost all non-disease associated strains. The role of the outer membrane protein P1 in the pathogenesis of this disease was evaluated by utilizing an isogenic P1-deficient mutant. We compared the ability of the wild type and P1 isogenic mutant to grow under various conditions. The P1-deficient strain grew at a similar rate to the wild type in both complex and chemically defined medium. The P1-deficient mutant also had a similar growth rate to the wild type under anaerobic conditions. Anaerobic growth, however, resulted in up-regulation of the P1 protein in the wild type strain. Three assays were used to examine the pathophysiologic role of the P1 protein in BPF: 1) serum resistance; 2) sustained bacteremia in the infant rat model; and 3) the human microvascular endothelial cell (HMEC) cytotoxicity assay. Both the mutant and wild-type strains were resistant to killing in 95% normal human serum. The P1-deficient strain was also as virulent as the wild type in both the infant rat model of bacteremia and in the HMEC-1 tissue culture model. These results demonstrate that serum resistance, sustained bacteremia in the infant rat, and cytotoxicity of HMEC cells occur in the absence of P1. The P1 protein is not essential for the pathogenic potential identified by these assays. However, these results demonstrate that an anaerobic environment is a potent physiologic regulator of P1 protein expression. The impact of anaerobiosis on protein expression and pathogenesis will require further investigations.

  20. [Breeding and characterization of laccase-producing Phanerochaete chrysosporium mutant resistant to nutritional repression].

    Science.gov (United States)

    Qiu, Ailian; Li, Wenyan; Zheng, Yaotong; Fan, Xiaojing; Ye, Youxian; Meng, Yan

    2011-03-01

    To screen Phanerochaete chrysosporium mutants resisting nutritional repression and to characterize laccase produced by the mutants. We used repeated UV mutagenesis and screened the mutant strains by using the guaiacol nitrogen sufficient differential medium. We characterized enzymes production mechanism of the nutritional regulation through comparing the differences of cell growth and enzyme-production kinetics under different nutritional conditions; We validated production of laccase by Phanerochaete chrysosporium through measurements of the heat treatment, removal of manganese ion and addition of the catalase. Three different methods were validated that both strains of pcR5305 and pcR5324 can produce laccase under the nitrogen limitation (N-L) and nitrogen sufficient (N-S) conditions. Under the N-L conditions, pcR5305 can produce 203.5 U/L laccase and pcR5324 can produce 187.6 U/L laccase; Under the N-S conditions, pcR5305 can produce 220.6 U/L laccase and pcR5324 can produce 183.9 U/L laccase. The original strain pc530 only can produce very little laccase under either conditions. The laccase-production regulation mechanisms of the two strains are different: Production of laccase and the cell growth by pcR5305 are in synchronism. However production of the laccase by pcR5324 is repressed by nutrition. Both strains have the capacity of resisting nutritional repression and produce lignin peroxidase and manganese peroxidase with high yield. (LiP 1343.2, MnP 252.2 U/L and LiP 1169.5, MnP 172.4 U/L respectively). The mutants of Phanerochaete chrysosporium can produce laccase. At same time they showed the capacity of resisting nutritional repression and production of laccase, lignin peroxidase and manganese peroxidase. Our results possess high value for production, application and fundamental research. We provided new strains and established a very good foundation for the further research of metabolic regulation of ligninolytic enzymes production.

  1. Differentially expressed genes in the ovary of the sixth day of pupal "Ming" lethal egg mutant of silkworm, Bombyx mori.

    Science.gov (United States)

    Gao, Peng; Chen, An-Li; Zhao, Qiao-Ling; Shen, Xing-Jia; Qiu, Zhi-Yong; Xia, Ding-Guo; Tang, Shun-Ming; Zhang, Guo-Zheng

    2013-09-15

    The "Ming" lethal egg mutant (l-em) is a vitelline membrane mutant in silkworm, Bombyx mori. The eggs laid by the l-em mutant lose water, ultimately causing death within an hour. Previous studies have shown that the deletion of BmEP80 is responsible for the l-em mutation in silkworm, B. mori. In the current study, digital gene expression (DGE) was performed to investigate the difference of gene expression in ovaries between wild type and l-em mutant on the sixth day of the pupal stage to obtain a global view of gene expression profiles using the ovaries of three l-em mutants and three wild types. The results showed a total of 3,463,495 and 3,607,936 clean tags in the wild type and the l-em mutant libraries, respectively. Compared with those of wild type, 239 differentially expressed genes were detected in the l-em mutant, wherein 181 genes are up-regulated and 58 genes are down-regulated in the mutant strain. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that no pathway was significantly enriched and three pathways are tightly related to protein synthesis among the five leading pathways. Moreover, the expression profiles of eight important differentially expressed genes related to oogenesis changed. These results provide a comprehensive gene expression analysis of oogenesis and vitellogenesis in B. mori which facilitates understanding of both the specific molecular mechanism of the 1-em mutant and Lepidopteran oogenesis in general.

  2. Deficiency of antinociception and excessive grooming induced by acute immobilization stress in Per1 mutant mice.

    Science.gov (United States)

    Zhang, Jing; Wu, Zhouqiao; Zhou, Linglin; Li, Huili; Teng, Huajing; Dai, Wei; Wang, Yongqing; Sun, Zhong Sheng

    2011-01-14

    Acute stressors induce changes in numerous behavioral parameters through activation of the hypothalamic-pituitary-adrenal (HPA) axis. Several important hormones in paraventricular nucleus of the hypothalamus (PVN) play the roles in these stress-induced reactions. Corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP) and corticosterone are considered as molecular markers for stress-induced grooming behavior. Oxytocin in PVN is an essential modulator for stress-induced antinociception. The clock gene, Per1, has been identified as an effecter response to the acute stresses, but its function in neuroendocrine stress systems remains unclear. In the present study we observed the alterations in grooming and nociceptive behaviors induced by acute immobilization stress in Per1 mutant mice and other genotypes (wild types and Per2 mutant). The results displayed that stress elicited a more robust effect on grooming behavior in Per1 mutant mice than in other genotypes. Subsequently, the obvious stress-induced antinociception was observed in the wild-type and Per2 mutant mice, however, in Per1 mutant, this antinociceptive effects were partially-reversed (mechanical sensitivity), or over-reversed to hyperalgesia (thermal sensitivity). The real-time qPCR results showed that in PVN, there were stress-induced up-regulations of Crh, Avp and c-fos in all of genotypes; moreover, the expression change of Crh in Per1 mutant mice was much larger than in others. Another hormonal gene, Oxt, was up-regulated induced by stress in wild-type and Per2 mutant but not in Per1 mutant. In addition, the stress significantly elevated the serum corticosterone levels without genotype-dependent differences, and accordingly the glucocorticoid receptor gene, Nr3c1, expressed with a similar pattern in PVN of all strains. Taken together, the present study indicated that in acute stress treated Per1 mutant mice, there are abnormal hormonal responses in PVN, correlating with the aberrant

  3. Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17.

    Science.gov (United States)

    Kim, Dockyu; Kim, Young-Soo; Kim, Seong-Ki; Kim, Si Wouk; Zylstra, Gerben J; Kim, Young Min; Kim, Eungbin

    2002-07-01

    Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes