CRISPR-mediated control of the bacterial initiation of replication

NARCIS (Netherlands)

Wiktor, J.M.; Lesterlin, Christian; Sherratt, David J.; Dekker, C.

2016-01-01

Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is

Real-time observation of the initiation of RNA polymerase II transcription.

Science.gov (United States)

Fazal, Furqan M; Meng, Cong A; Murakami, Kenji; Kornberg, Roger D; Block, Steven M

2015-09-10

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

DEFF Research Database (Denmark)

Chen, Yun; Pai, Athma A; Herudek, Jan

2016-01-01

Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation s...... suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provide a framework with which evolution shapes transcriptomes.......Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation...

In vitro transcription in the presence of DNA oligonucleotides can generate strong anomalous initiation sites.

Science.gov (United States)

Chow, C W; Clark, M P; Rinaldo, J E; Chalkley, R

1996-03-01

In the present study, we have explored an unexpected observation in transcription initiation that is mediated by single-stranded oligonucleotides. Initially, our goal was to understand the function of different upstream regulatory elements/initiation sites in the rat xanthine dehydrogenase/oxidase (XDH/XO) promoter. We performed in vitro transcription with HeLa nuclear extracts in the presence of different double-stranded oligonucleotides against upstream elements as competitors. A new and unusual transcription initiation site was detected by primer extension. This new initiation site maps to the downstream region of the corresponding competitor. Subsequent analyses have indicated that the induction of a new transcription initiation site is anomalous which is due to the presence of a small amount of single-stranded oligonucleotide in the competitor. We found that this anomalous initiation site is insensitive to the orientation of the promoter and requires only a small amount of single-stranded oligonucleotide (< 2-fold molar excess relative to template). We surmise that a complementary interaction between the single-stranded oligonucleotide and transiently denatured promoter template may be responsible for this sequence-specific transcription initiation artifact. To study the regulation of transcription initiation by in vitro transcription approaches, we propose that one should probe the effect of removing transacting factors by adding an excess of a cognate oligonucleotide which does not bear exact sequence identity to the template.

D-amino acids inhibit initial bacterial adhesion: thermodynamic evidence.

Science.gov (United States)

Xing, Su-Fang; Sun, Xue-Fei; Taylor, Alicia A; Walker, Sharon L; Wang, Yi-Fu; Wang, Shu-Guang

2015-04-01

Bacterial biofilms are structured communities of cells enclosed in a self-produced hydrated polymeric matrix that can adhere to inert or living surfaces. D-Amino acids were previously identified as self-produced compounds that mediate biofilm disassembly by causing the release of the protein component of the polymeric matrix. However, whether exogenous D-amino acids could inhibit initial bacterial adhesion is still unknown. Here, the effect of the exogenous amino acid D-tyrosine on initial bacterial adhesion was determined by combined use of chemical analysis, force spectroscopic measurement, and theoretical predictions. The surface thermodynamic theory demonstrated that the total interaction energy increased with more D-tyrosine, and the contribution of Lewis acid-base interactions relative to the change in the total interaction energy was much greater than the overall nonspecific interactions. Finally, atomic force microscopy analysis implied that the hydrogen bond numbers and adhesion forces decreased with the increase in D-tyrosine concentrations. D-Tyrosine contributed to the repulsive nature of the cell and ultimately led to the inhibition of bacterial adhesion. This study provides a new way to regulate biofilm formation by manipulating the contents of D-amino acids in natural or engineered systems. © 2014 Wiley Periodicals, Inc.

Coordination of genomic structure and transcription by the main bacterial nucleoid-associated protein HU

Science.gov (United States)

Berger, Michael; Farcas, Anca; Geertz, Marcel; Zhelyazkova, Petya; Brix, Klaudia; Travers, Andrew; Muskhelishvili, Georgi

2010-01-01

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci—spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid. PMID:20010798

A transcript finishing initiative for closing gaps in the human transcriptome

DEFF Research Database (Denmark)

Sogayar, Mari Cleide; Camargo, Anamaria A; Bettoni, Fabiana

2004-01-01

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected...

CRISPR-mediated control of the bacterial initiation of replication.

Science.gov (United States)

Wiktor, Jakub; Lesterlin, Christian; Sherratt, David J; Dekker, Cees

2016-05-05

Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structures of transcription pre-initiation complex with TFIIH and Mediator.

Science.gov (United States)

Schilbach, S; Hantsche, M; Tegunov, D; Dienemann, C; Wigge, C; Urlaub, H; Cramer, P

2017-11-09

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

Science.gov (United States)

Fiume, Giuseppe; Rossi, Annalisa; de Laurentiis, Annamaria; Falcone, Cristina; Pisano, Antonio; Vecchio, Eleonora; Pontoriero, Marilena; Scala, Iris; Scialdone, Annarita; Masci, Francesca Fasanella; Mimmi, Selena; Palmieri, Camillo; Scala, Giuseppe; Quinto, Ileana

2013-01-01

Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control. PMID:23776612

Transcriptional start site turnover in the evolution of bacterial paralogous genes - the pelE-pelD virulence genes in Dickeya.

Science.gov (United States)

Duprey, Alexandre; Nasser, William; Léonard, Simon; Brochier-Armanet, Céline; Reverchon, Sylvie

2016-11-01

After a gene duplication event, the resulting paralogous genes frequently acquire distinct expression profiles, roles, and/or functions but the underlying mechanisms are poorly understood. While transcription start site (TSS) turnover, i.e., the repositioning of the TSS during evolution, is widespread in eukaryotes, it is less documented in bacteria. Using pelD and pelE, two closely related paralogous genes encoding key virulence factors in Dickeya, a gamma proteobacterial genus of phytopathogens, we show that pelE has been selected as an initiator of bacterial aggression, while pelD acts at a later stage, thanks to modifications in the transcriptional regulation of these two genes. This expression change is linked to a few mutations that caused a shift in the position of the pelETSS and the rapid divergence in the regulation of these genes after their duplication. Genomic surveys detected additional examples of putative turnovers in other bacteria. This first report of TSS shifting in bacteria suggests that this mechanism could play a major role in paralogous genes fixation in prokaryotes. © 2016 Federation of European Biochemical Societies.

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

Science.gov (United States)

Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

2018-03-29

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

An empirical strategy to detect bacterial transcript structure from directional RNA-seq transcriptome data.

Science.gov (United States)

Wang, Yejun; MacKenzie, Keith D; White, Aaron P

2015-05-07

As sequencing costs are being lowered continuously, RNA-seq has gradually been adopted as the first choice for comparative transcriptome studies with bacteria. Unlike microarrays, RNA-seq can directly detect cDNA derived from mRNA transcripts at a single nucleotide resolution. Not only does this allow researchers to determine the absolute expression level of genes, but it also conveys information about transcript structure. Few automatic software tools have yet been established to investigate large-scale RNA-seq data for bacterial transcript structure analysis. In this study, 54 directional RNA-seq libraries from Salmonella serovar Typhimurium (S. Typhimurium) 14028s were examined for potential relationships between read mapping patterns and transcript structure. We developed an empirical method, combined with statistical tests, to automatically detect key transcript features, including transcriptional start sites (TSSs), transcriptional termination sites (TTSs) and operon organization. Using our method, we obtained 2,764 TSSs and 1,467 TTSs for 1331 and 844 different genes, respectively. Identification of TSSs facilitated further discrimination of 215 putative sigma 38 regulons and 863 potential sigma 70 regulons. Combining the TSSs and TTSs with intergenic distance and co-expression information, we comprehensively annotated the operon organization in S. Typhimurium 14028s. Our results show that directional RNA-seq can be used to detect transcriptional borders at an acceptable resolution of ±10-20 nucleotides. Technical limitations of the RNA-seq procedure may prevent single nucleotide resolution. The automatic transcript border detection methods, statistical models and operon organization pipeline that we have described could be widely applied to RNA-seq studies in other bacteria. Furthermore, the TSSs, TTSs, operons, promoters and unstranslated regions that we have defined for S. Typhimurium 14028s may constitute valuable resources that can be used for

Frequent dual initiation of reverse transcription in murine leukemia virus-based vectors containing two primer-binding sites

International Nuclear Information System (INIS)

Voronin, Yegor A.; Pathak, Vinay K.

2003-01-01

Retroviruses package two copies of viral RNA into each virion. Although each RNA contains a primer-binding site for initiation of DNA synthesis, it is unknown whether reverse transcription is initiated on both RNAs. To determine whether a single virion is capable of initiating reverse transcription more than once, we constructed a murine leukemia virus-based vector containing a second primer-binding site (PBS) derived from spleen necrosis virus and inserted the green fluorescent protein gene (GFP) between the two PBSs. Initiation of reverse transcription at either PBS results in a provirus that expresses GFP. However, initiation at both PBSs can result in the deletion of GFP, which can be detected by flow cytometry and Southern blotting analysis. Approximately 22-29% of the proviruses formed deleted the GFP in a single replication cycle, indicating the minimum proportion of virions that initiated reverse transcription on both PBSs. These results show that a significant proportion of MLV-based vectors containing two PBSs have the capacity to initiate reverse transcription more than once

High-density transcriptional initiation signals underline genomic islands in bacteria.

Directory of Open Access Journals (Sweden)

Qianli Huang

Full Text Available Genomic islands (GIs, frequently associated with the pathogenicity of bacteria and having a substantial influence on bacterial evolution, are groups of "alien" elements which probably undergo special temporal-spatial regulation in the host genome. Are there particular hallmark transcriptional signals for these "exotic" regions? We here explore the potential transcriptional signals that underline the GIs beyond the conventional views on basic sequence composition, such as codon usage and GC property bias. It showed that there is a significant enrichment of the transcription start positions (TSPs in the GI regions compared to the whole genome of Salmonella enterica and Escherichia coli. There was up to a four-fold increase for the 70% GIs, implying high-density TSPs profile can potentially differentiate the GI regions. Based on this feature, we developed a new sliding window method GIST, Genomic-island Identification by Signals of Transcription, to identify these regions. Subsequently, we compared the known GI-associated features of the GIs detected by GIST and by the existing method Islandviewer to those of the whole genome. Our method demonstrates high sensitivity in detecting GIs harboring genes with biased GI-like function, preferred subcellular localization, skewed GC property, shorter gene length and biased "non-optimal" codon usage. The special transcriptional signals discovered here may contribute to the coordinate expression regulation of foreign genes. Finally, by using GIST, we detected many interesting GIs in the 2011 German E. coli O104:H4 outbreak strain TY-2482, including the microcin H47 system and gene cluster ycgXEFZ-ymgABC that activates the production of biofilm matrix. The aforesaid findings highlight the power of GIST to predict GIs with distinct intrinsic features to the genome. The heterogeneity of cumulative TSPs profiles may not only be a better identity for "alien" regions, but also provide hints to the special

Regulation, initiation, and termination of the cenA and cex transcripts of Cellulomonas fimi

International Nuclear Information System (INIS)

Greenberg, N.M.; Warren, R.A.J.; Kilburn, D.G.; Miller, R.C. Jr.

1987-01-01

The authors characterized the in vivo transcripts of two Cellulomonas fimi genes, which encodes an extracellular endo-β-1,4-glucanase. By Northern blot analysis, cenA mRNA was detected in C. fimi RNA preparations from glycerol- and carboxymethyl cellulose-grown cells but not from glucose-grown cells. In contrast, cex mRNA was detected only in the preparations from carboxymethyl cellulose-grown cells. Therefore, the transcription of these genes is subject to regulation by the carbon source provided to C. fimi. By nuclease SI protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, 5' termini were found 51 and 62 bases before the cenA translational initiation codon and 28 bases before the cex translational initiation codon. S1 mapping with unlabeled DNA probes and C. fimi RNA which had been isolated in vivo but which had been 5' labeled in vitro with guanylyltransferase and [α- 32 P]GTP confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. Comparative analysis of the DNA sequences immediately upstream of the initiation sites of the cenA and cex mRNAs revealed a 30-base-pair region where these two sequences display at least 66% homology. S1 mapping was also used to locate the 3' termini of the cenA and cex transcripts. Three 3' termini were found for cenA messages, whereas only one 3' terminus was identified for cex mRNA. The transcripts of both genes terminate in regions where their corresponding DNA sequences contain inverted repeats

A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

DEFF Research Database (Denmark)

Damgaard, Christian Kroun; Kahns, Søren; Lykke-Andersen, Søren

2008-01-01

RNAs, harboring wild-type or various 5′ splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5′ splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced...... a promoter-proximal 5′ splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing preinitiation complex assembly....

Genomic context drives transcription of insertion sequences in the bacterial endosymbiont Wolbachia wVulC.

Science.gov (United States)

Cerveau, Nicolas; Gilbert, Clément; Liu, Chao; Garrett, Roger A; Grève, Pierre; Bouchon, Didier; Cordaux, Richard

2015-06-10

Transposable elements (TEs) are DNA pieces that are present in almost all the living world at variable genomic density. Due to their mobility and density, TEs are involved in a large array of genomic modifications. In eukaryotes, TE expression has been studied in detail in several species. In prokaryotes, studies of IS expression are generally linked to particular copies that induce a modification of neighboring gene expression. Here we investigated global patterns of IS transcription in the Alphaproteobacterial endosymbiont Wolbachia wVulC, using both RT-PCR and bioinformatic analyses. We detected several transcriptional promoters in all IS groups. Nevertheless, only one of the potentially functional IS groups possesses a promoter located upstream of the transposase gene, that could lead up to the production of a functional protein. We found that the majority of IS groups are expressed whatever their functional status. RT-PCR analyses indicate that the transcription of two IS groups lacking internal promoters upstream of the transposase start codon may be driven by the genomic environment. We confirmed this observation with the transcription analysis of individual copies of one IS group. These results suggest that the genomic environment is important for IS expression and it could explain, at least partly, copy number variability of the various IS groups present in the wVulC genome and, more generally, in bacterial genomes. Copyright © 2015 Elsevier B.V. All rights reserved.

ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth

DEFF Research Database (Denmark)

Zhao, Jian; Yuan, Xuejun; Frödin, Morten

2003-01-01

-specific transcription initiation factor TIF-IA. Activation of TIF-IA and ribosomal gene transcription is sensitive to PD98059, indicating that TIF-IA is targeted by MAPK in vivo. Phosphopeptide mapping and mutational analysis reveals two serine residues (S633 and S649) that are phosphorylated by ERK and RSK kinases....... Replacement of S649 by alanine inactivates TIF-IA, inhibits pre-rRNA synthesis, and retards cell growth. The results provide a link between growth factor signaling, ribosome production, and cell growth, and may have a major impact on the mechanism of cell transformation....

The relationship between transcription initiation RNAs and CCCTC-binding factor (CTCF localization

Directory of Open Access Journals (Sweden)

Taft Ryan J

2011-08-01

Full Text Available Abstract Background Transcription initiation RNAs (tiRNAs are nuclear localized 18 nucleotide RNAs derived from sequences immediately downstream of RNA polymerase II (RNAPII transcription start sites. Previous reports have shown that tiRNAs are intimately correlated with gene expression, RNA polymerase II binding and behaviors, and epigenetic marks associated with transcription initiation, but not elongation. Results In the present work, we show that tiRNAs are commonly found at genomic CCCTC-binding factor (CTCF binding sites in human and mouse, and that CTCF sites that colocalize with RNAPII are highly enriched for tiRNAs. To directly investigate the relationship between tiRNAs and CTCF we examined tiRNAs originating near the intronic CTCF binding site in the human tumor suppressor gene, p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A. Inhibition of CTCF-proximal tiRNAs resulted in increased CTCF localization and increased p21 expression, while overexpression of CTCF-proximal tiRNA mimics decreased CTCF localization and p21 expression. We also found that tiRNA-regulated CTCF binding influences the levels of trimethylated H3K27 at the alternate upstream p21 promoter, and affects the levels of alternate p21 (p21alt transcripts. Extending these studies to another randomly selected locus with conserved CTCF binding we found that depletion of tiRNA alters nucleosome density proximal to sites of tiRNA biogenesis. Conclusions Taken together, these data suggest that tiRNAs modulate local epigenetic structure, which in turn regulates CTCF localization.

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

OpenAIRE

Brun, R P; Ryan, K; Sollner-Webb, B

1994-01-01

Factor C* is the component of the RNA polymerase I holoenzyme (factor C) that allows specific transcriptional initiation on a factor D (SL1)- and UBF-activated rRNA gene promoter. The in vitro transcriptional capacity of a preincubated rDNA promoter complex becomes exhausted very rapidly upon initiation of transcription. This is due to the rapid depletion of C* activity. In contrast, C* activity is not unstable in the absence of transcription, even in the presence of nucleoside triphosphates ...

Efficacy of a mouthrinse based on hydroxyapatite to reduce initial bacterial colonisation in situ.

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Kensche, A; Holder, C; Basche, S; Tahan, N; Hannig, C; Hannig, M

2017-08-01

The present in situ - investigation aimed to specify the impact of pure hydroxyapatite microclusters on initial bioadhesion and bacterial colonization at the tooth surface. Pellicle formation was carried out in situ on bovine enamel slabs (9 subjects). After 1min of pellicle formation rinses with 8ml of hydroxyapatite (HA) microclusters (5%) in bidestilled water or chlorhexidine 0.2% were performed. As negative control no rinse was adopted. In situ biofilm formation was promoted by the intraoral slab exposure for 8h overnight. Afterwards initial bacterial adhesion was quantified by DAPI staining and bacterial viability was determined in vivo/in vitro by live/dead-staining (BacLight). SEM analysis evaluated the efficacy of the mouthrinse to accumulate hydroxyapatite microclusters at the specimens' surface and spit-out samples of the testsolution were investigated by TEM. Compared to the control (2.36×10 6 ±2.01×10 6 bacteria/cm 2 ), significantly reduced amounts of adherent bacteria were detected on specimens rinsed with chlorhexidine 0.2% (8.73×10 4 ±1.37×10 5 bacteria/cm 2 ) and likewise after rinses with the hydroxyapatite testsolution (2.08×10 5 ±2.85×10 5 bacteria/cm 2 , phydroxyapatite microclusters at the tooth surface. Adhesive interactions of HA-particles with oral bacteria were shown by TEM. Hydroxyapatite microclusters reduced initial bacterial adhesion to enamel in situ considerably and could therefore sensibly supplement current approaches in dental prophylaxis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases.

Science.gov (United States)

Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, Ivan; Krásný, Libor; Hocek, Michal

2016-04-20

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Gene and transcript abundances of bacterial type III secretion systems from the rumen microbiome are correlated with methane yield in sheep.

Science.gov (United States)

Kamke, Janine; Soni, Priya; Li, Yang; Ganesh, Siva; Kelly, William J; Leahy, Sinead C; Shi, Weibing; Froula, Jeff; Rubin, Edward M; Attwood, Graeme T

2017-08-08

Ruminants are important contributors to global methane emissions via microbial fermentation in their reticulo-rumens. This study is part of a larger program, characterising the rumen microbiomes of sheep which vary naturally in methane yield (g CH 4 /kg DM/day) and aims to define differences in microbial communities, and in gene and transcript abundances that can explain the animal methane phenotype. Rumen microbiome metagenomic and metatranscriptomic data were analysed by Gene Set Enrichment, sparse partial least squares regression and the Wilcoxon Rank Sum test to estimate correlations between specific KEGG bacterial pathways/genes and high methane yield in sheep. KEGG genes enriched in high methane yield sheep were reassembled from raw reads and existing contigs and analysed by MEGAN to predict their phylogenetic origin. Protein coding sequences from Succinivibrio dextrinosolvens strains were analysed using Effective DB to predict bacterial type III secreted proteins. The effect of S. dextrinosolvens strain H5 growth on methane formation by rumen methanogens was explored using co-cultures. Detailed analysis of the rumen microbiomes of high methane yield sheep shows that gene and transcript abundances of bacterial type III secretion system genes are positively correlated with methane yield in sheep. Most of the bacterial type III secretion system genes could not be assigned to a particular bacterial group, but several genes were affiliated with the genus Succinivibrio, and searches of bacterial genome sequences found that strains of S. dextrinosolvens were part of a small group of rumen bacteria that encode this type of secretion system. In co-culture experiments, S. dextrinosolvens strain H5 showed a growth-enhancing effect on a methanogen belonging to the order Methanomassiliicoccales, and inhibition of a representative of the Methanobrevibacter gottschalkii clade. This is the first report of bacterial type III secretion system genes being associated with high

Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots.

Science.gov (United States)

Yamada, Shintaro; Okamura, Mika; Oda, Arisa; Murakami, Hiroshi; Ohta, Kunihiro; Yamada, Takatomi

2017-06-01

Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to the cyclic AMP response element and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049 , and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full-length messenger RNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously known recombination-activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcript production at ade6-3049 These results point to a possibility that the two events may be related to each other at ade6-3049 In fact, comparison of published maps of meiotic transcripts and hotspots suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions. Copyright © 2017 by the Genetics Society of America.

Symbiosis initiation in the bacterially luminous sea urchin cardinalfish Siphamia versicolor.

Science.gov (United States)

Dunlap, P V; Gould, A L; Wittenrich, M L; Nakamura, M

2012-09-01

To determine how each new generation of the sea urchin cardinalfish Siphamia versicolor acquires the symbiotic luminous bacterium Photobacterium mandapamensis, and when in its development the S. versicolor initiates the symbiosis, procedures were established for rearing S. versicolor larvae in an aposymbiotic state. Under the conditions provided, larvae survived and developed for 28 days after their release from the mouths of males. Notochord flexion began at 8 days post release (dpr). By 28 dpr, squamation was evident and the caudal complex was complete. The light organ remained free of bacteria but increased in size and complexity during development of the larvae. Thus, aposymbiotic larvae of the fish can survive and develop for extended periods, major components of the luminescence system develop in the absence of the bacteria and the bacteria are not acquired directly from a parent, via the egg or during mouth brooding. Presentation of the symbiotic bacteria to aposymbiotic larvae at 8-10 dpr, but not earlier, led to initiation of the symbiosis. Upon colonization of the light organ, the bacterial population increased rapidly and cells forming the light-organ chambers exhibited a differentiated appearance. Therefore, the light organ apparently first becomes receptive to colonization after 1 week post-release development, the symbiosis is initiated by bacteria acquired from the environment and bacterial colonization induces morphological changes in the nascent light organ. The abilities to culture larvae of S. versicolor for extended periods and to initiate the symbiosis in aposymbiotic larvae are key steps in establishing the experimental tractability of this highly specific vertebrate and microbe mutualism. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis

NARCIS (Netherlands)

C. Nepal (Chirag); Y. Hadzhiev (Yavor); C. Previti (Christopher); V. Haberle (Vanja); N. Li (Nan); H. Takahashi (Hiroyuki); A.M. Suzuki (Ana Maria); Y. Sheng (Ying); R.F. Abdelhamid (Rehab); S. Anand (Santosh); P.A. Gehrig (Paola A.); A. Akalin (Altuna); C. Kockx (Christel); A. Van Der Sloot (Antoine); W.F.J. van IJcken (Wilfred); O. Armant (Olivier); S. Rastegar (Sepand); C. Watson (Craig); U. Strähle (Uwe); E. Stupka (Elia); P. Carninci (Piero); B. Lenhard (Boris); F. Müller (Ferenc)

2013-01-01

textabstractSpatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we

Structural basis for the initiation of eukaryotic transcription-coupled DNA repair.

Science.gov (United States)

Xu, Jun; Lahiri, Indrajit; Wang, Wei; Wier, Adam; Cianfrocco, Michael A; Chong, Jenny; Hare, Alissa A; Dervan, Peter B; DiMaio, Frank; Leschziner, Andres E; Wang, Dong

2017-11-30

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

The role of bacterial antizyme: From an inhibitory protein to AtoC transcriptional regulator

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Kyriakidis Dimitrios A

2004-06-01

Full Text Available Abstract This review considers the role of bacterial antizyme in the regulation of polyamine biosynthesis and gives new perspectives on the involvement of antizyme in other significant cellular mechanisms. Antizyme is a protein molecule induced by the end product of the enzymic reaction that it inhibits, in a non-competitive manner. The bacterial ornithine decarboxylase is regulated by nucleotides, phosphorylation and antizyme. The inhibition of ornithine decarboxylase by antizyme can be relieved to different degrees by DNA or by a variety of synthetic nucleic acid polymers, attributed to a specific interaction between nucleic acid and antizyme. Recently, this interplay between bacterial antizyme and nucleic acid was determined by discerning an additional function to antizyme that proved to be the atoC gene product, encoding the response regulator of the bacterial two-component system AtoS-AtoC. The gene located just upstream of atoC encodes the sensor kinase, named AtoS, that modulates AtoC activity. AtoC regulates expression of atoDAEB operon which is involved in short-chain fatty acid metabolism. Antizyme is thus referred to as AtoC, functioning both as a post-translational and transcriptional regulator. Also, the AtoS-AtoC signal transduction system in E. coli has a positive regulatory role on poly-(R-3-hydroxybutyrate biosynthesis. The properties and gene structural similarities of antizymes from different organisms were compared. It was revealed that conserved domains are present mostly in the C-domain of all antizymes. BLAST analysis of the E. coli antizyme protein (AtoC showed similarities around 69–58% among proteobacteria, g-proteobacteria, enterobacteria and the thermophilic bacterium Thermus thermophilus. A working hypothesis is proposed for the metabolic role of antizyme (AtoC describing the significant biological implications of this protein molecule. Whether antizymes exist to other enzymes in different tissues, meeting the

In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

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Janet L Smith

2015-05-01

Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

Alternate capping mechanisms for transcription of spring viremia of carp virus: evidence for independent mRNA initiation.

Science.gov (United States)

Gupta, K C; Roy, P

1980-01-01

Two alternate mechanisms of mRNA capping for spring viremia of carp virus have been observed. Under normal reaction conditions, a ppG residue of the capping GTP is transferred to a pA moiety of the 5' termini of mRNA transcripts. However, in reaction conditions where GppNHp is used instead of GTP, an alternate capping mechanism occurs whereby a pG residue of the capping GTP is transferred to a ppA moiety of the transcripts. The first mechanism is identical to that described previously for vesicular stomatitis virus (G. Abraham, D. P. Rhodes, and A. K. Banerjee, Nature [London] 255:37-40, 1975; A. K. Banerjee, S. A. Moyer, and D. P. Rhodes, Virology 61:547-558, 1974), and thus appears to be a conserved function during the evolution of rhabdoviruses. The alternate mechanism of capping indicates not only that capping can take place by two procedures, but also that the substrate termini have di- or triphosphate 5' ends, indicating that they are probably independently initiated. An analog of ATP, AppNHp, has been found to completely inhibit the initiation of transcription by spring viremia of carp virus, suggesting that a cleavage between the beta and gamma phosphates of ATP is essential for the initiation of transcription. However, in the presence of GppNHp, uncapped (ppAp and pppAp), capped (GpppAp), and capped methylated (m7GpppAmpAp and GpppAmpAp) transcripts are detected. Size analyses of oligodeoxythymidylic acid-cellulose-bound transcripts resolved by formamide gel electrophoresis demonstrated that full-size mRNA transcripts are synthesized as well as larger RNA species. The presence of GppNHp and S-adenosylhomocysteine in reaction mixtures did not have any effect on the type of unmethylated transcription products. Our results favor a transcription model postulated previously (D. H. L. Bishop, in H. Fraenkel-Conrat and R. R. Wagner, ed., Comprehensive Virology, vol. 10, Plenum Press, New York, 1977; D. H. L. Bishop and A. Flamand, in D. C. Burke and W. C. Russell

Initial insights into bacterial succession during human decomposition.

Science.gov (United States)

Hyde, Embriette R; Haarmann, Daniel P; Petrosino, Joseph F; Lynne, Aaron M; Bucheli, Sibyl R

2015-05-01

Decomposition is a dynamic ecological process dependent upon many factors such as environment, climate, and bacterial, insect, and vertebrate activity in addition to intrinsic properties inherent to individual cadavers. Although largely attributed to microbial metabolism, very little is known about the bacterial basis of human decomposition. To assess the change in bacterial community structure through time, bacterial samples were collected from several sites across two cadavers placed outdoors to decompose and analyzed through 454 pyrosequencing and analysis of variable regions 3-5 of the bacterial 16S ribosomal RNA (16S rRNA) gene. Each cadaver was characterized by a change in bacterial community structure for all sites sampled as time, and decomposition, progressed. Bacteria community structure is variable at placement and before purge for all body sites. At bloat and purge and until tissues began to dehydrate or were removed, bacteria associated with flies, such as Ignatzschineria and Wohlfahrtimonas, were common. After dehydration and skeletonization, bacteria associated with soil, such as Acinetobacter, were common at most body sites sampled. However, more cadavers sampled through multiple seasons are necessary to assess major trends in bacterial succession.

Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

Science.gov (United States)

Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

2004-01-30

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.

X-ray Crystal Structures Elucidate the Nucleotidyl Transfer Reaction of Transcript Initiation Using Two Nucleotides

Energy Technology Data Exchange (ETDEWEB)

M Gleghorn; E Davydova; R Basu; L Rothman-Denes; K Murakami

2011-12-31

We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.

Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

DEFF Research Database (Denmark)

Lauritsen, Ida; Martinez, Virginia; Ronda, Carlotta

2018-01-01

In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest....... The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously...... been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool....

Transcriptional responses of resistant and susceptible fish clones to the bacterial pathogen Flavobacterium psychrophilum.

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Christelle Langevin

Full Text Available Flavobacterium psychrophilum is a bacterial species that represents one of the most important pathogens for aquaculture worldwide, especially for salmonids. To gain insights into the genetic basis of the natural resistance to F. psychrophilum, we selected homozygous clones of rainbow trout with contrasted susceptibility to the infection. We compared the transcriptional response to the bacteria in the pronephros of a susceptible and a resistant line by micro-array analysis five days after infection. While the basal transcriptome of healthy fish was significantly different in the resistant and susceptible lines, the transcriptome modifications induced by the bacteria involved essentially the same genes and pathways. The response to F. psychrophilum involved antimicrobial peptides, complement, and a number of enzymes and chemokines. The matrix metalloproteases mmp9 and mmp13 were among the most highly induced genes in both genetic backgrounds. Key genes of both pro- and anti-inflammatory response such as IL1 and IL10, were up-regulated with a greater magnitude in susceptible animals where the bacterial load was also much higher. While higher resistance to F. psychrophilum does not seem to be based on extensive differences in the orientation of the immune response, several genes including complement C3 showed stronger induction in the resistant fish. They may be important for the variation of susceptibility to the infection.

Riboregulation of the bacterial actin-homolog MreB by DsrA small noncoding RNA.

Science.gov (United States)

Cayrol, Bastien; Fortas, Emilie; Martret, Claire; Cech, Grzegorz; Kloska, Anna; Caulet, Stephane; Barbet, Marion; Trépout, Sylvain; Marco, Sergio; Taghbalout, Aziz; Busi, Florent; Wegrzyn, Grzegorz; Arluison, Véronique

2015-01-01

The bacterial actin-homolog MreB is a key player in bacterial cell-wall biosynthesis and is required for the maintenance of the rod-like morphology of Escherichia coli. However, how MreB cellular levels are adjusted to growth conditions is poorly understood. Here, we show that DsrA, an E. coli small noncoding RNA (sRNA), is involved in the post-transcriptional regulation of mreB. DsrA is required for the downregulation of MreB cellular concentration during environmentally induced slow growth-rates, mainly growth at low temperature and during the stationary phase. DsrA interacts in an Hfq-dependent manner with the 5' region of mreB mRNA, which contains signals for translation initiation and thereby affects mreB translation and stability. Moreover, as DsrA is also involved in the regulation of two transcriptional regulators, σ(S) and the nucleoid associated protein H-NS, which negatively regulate mreB transcription, it also indirectly contributes to mreB transcriptional down-regulation. By using quantitative analyses, our results evidence the complexity of this regulation and the tangled interplay between transcriptional and post-transcriptional control. As transcription factors and sRNA-mediated post-transcriptional regulators use different timescales, we propose that the sRNA pathway helps to adapt to changes in temperature, but also indirectly mediates long-term regulation of MreB concentration. The tight regulation and fine-tuning of mreB gene expression in response to cellular stresses is discussed in regard to the effect of the MreB protein on cell elongation.

R-loops in bacterial transcription: their causes and consequences.

Science.gov (United States)

Gowrishankar, J; Leela, J Krishna; Anupama, K

2013-01-01

Nascent untranslated transcripts in bacteria are prone to generating RNA-DNA hybrids (R-loops); Rho-dependent transcription termination acts to reduce their prevalence. Here we discuss the mechanisms of R-loop formation and growth inhibition in bacteria.

Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

DEFF Research Database (Denmark)

Jakobsen, Janus S; Bagger, Frederik O; Hasemann, Marie S

2015-01-01

BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low...... transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre...... cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from...

The used of formalin, borax and initial bacterial contamination on otak-otak

International Nuclear Information System (INIS)

Harsojo; Kadir I

2013-01-01

A research has been conducted to identify the used of formalin and borax content and also study the initial bacterial contamination on otak-otak. The wrapped and unwrapped samples were irradiated with a dose of 3 kGy Further, the samples were stored at room temperature (± 30°C) and low temperature (± 4°C) up to 4 weeks. The irradiation was done at a Multipurpose Panoramic Batch Irradiator (IRPASENA) with a dose rate of 1.149 kGy/h. Those samples were stored 4 weeks at 2 different temperatures and the total bacteria were observe every week. The measured parameter were formalin and borax content in otak-otak, the amount of total aerob bacteria, total coliforms, Escherichia coli, Staphylococcus spp., identification of Salmonella. The results showed all samples used formalin but borax was not detected. Initial contamination of total aerob bacteria for unwrapped and wrapped samples were 4.3 x 10"7 and 2.0 x 10"7 cfu/g, respectively. Irradiation dose up to 3 kGy showed no bacterial growth on unwrapped and wrapped samples. Combination treatment of irradiation and storage at low temperature could eliminate all aerobic bacteria at the first week. Initial contamination of coliform bacteria on unwrapped and wrapped samples were 1.9 x 10"5 and 5.7 x 10"5 cfu/g, respectively. Initial contamination of E. coli on unwrapped and wrapped samples were 1.2 x 10"5 cfu/g. The total amount of Staphylococcus spp. on unwrapped and wrapped samples were 3.3 x 10"5 and 4.8 x 10"6 cfu/g, respectively. Irradiation at a dose of 3 kGy could eliminate coliform bacteria, E. coli and Staphylococcus spp in all samples observed. No Salmonella was detected in all samples observed. (author)

Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

Science.gov (United States)

Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha; Jhingan, Gagan Deep

2016-03-01

Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

Quantification of 16S rRNAs in complex bacterial communities by multiple competitive reverse transcription-PCR in temperature gradient gel electrophoresis fingerprints.

Science.gov (United States)

Felske, A; Akkermans, A D; De Vos, W M

1998-11-01

A novel approach was developed to quantify rRNA sequences in complex bacterial communities. The main bacterial 16S rRNAs in Drentse A grassland soils (The Netherlands) were amplified by reverse transcription (RT)-PCR with bacterium-specific primers and were separated by temperature gradient gel electrophoresis (TGGE). The primer pair used (primers U968-GC and L1401) was found to amplify with the same efficiency 16S rRNAs from bacterial cultures containing different taxa and cloned 16S ribosomal DNA amplicons from uncultured soil bacteria. The sequence-specific efficiency of amplification was determined by monitoring the amplification kinetics by kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs resulted in identical amplicon yields. The sequence-specific detection system used for competitive amplifications was TGGE, which also has been found to be suitable for simultaneous quantification of more than one sequence. We demonstrate that this approach can be applied to TGGE fingerprints of soil bacteria to estimate the ratios of the bacterial 16S rRNAs.

A transcript cleavage factor of Mycobacterium tuberculosis important for its survival.

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Arnab China

Full Text Available After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP. Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

Core Mediator structure at 3.4 Å extends model of transcription initiation complex.

Science.gov (United States)

Nozawa, Kayo; Schneider, Thomas R; Cramer, Patrick

2017-05-11

Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos

DEFF Research Database (Denmark)

Jakobsen, Anne Sørig; Avery, Birthe; Dieleman, Steph J.

2006-01-01

Transcription from the embryos own ribosomal genes is initiated in most species at the same time as the maternal-embryonic transition. Recently data have indicated that a minor activation may take place during the third embryonic cell cycle in the bovine, one cell cycle before the major activation...

Initial Bacterial Adhesion on Different Yttria-Stabilized Tetragonal Zirconia Implant Surfaces in Vitro

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Lamprini Karygianni

2013-12-01

Full Text Available Bacterial adhesion to implant biomaterials constitutes a virulence factor leading to biofilm formation, infection and treatment failure. The aim of this study was to examine the initial bacterial adhesion on different implant materials in vitro. Four implant biomaterials were incubated with Enterococcus faecalis, Staphylococcus aureus and Candida albicans for 2 h: 3 mol % yttria-stabilized tetragonal zirconia polycrystal surface (B1a, B1a with zirconium oxide (ZrO2 coating (B2a, B1a with zirconia-based composite coating (B1b and B1a with zirconia-based composite and ZrO2 coatings (B2b. Bovine enamel slabs (BES served as control. The adherent microorganisms were quantified and visualized using scanning electron microscopy (SEM; DAPI and live/dead staining. The lowest bacterial count of E. faecalis was detected on BES and the highest on B1a. The fewest vital C. albicans strains (42.22% were detected on B2a surfaces, while most E. faecalis and S. aureus strains (approximately 80% were vital overall. Compared to BES; coated and uncoated zirconia substrata exhibited no anti-adhesive properties. Further improvement of the material surface characteristics is essential.

Study of the variation of the nuclear transcriptional map during de initial development of Drosophyla melanogaster embryos

International Nuclear Information System (INIS)

Alonso, C.E.V.

1987-01-01

The variation of nuclear transcriptional map during the initial development of Drosophyla melanogaster embryos were studied. Thermic treatment, chromatographic techniques and liquid scintilation in embryos inoculated with radioactive uridine were used. (L.J.C.)

Transcription initiation patterns indicate divergent strategies for gene regulation at the chromatin level.

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Elizabeth A Rach

2011-01-01

Full Text Available The application of deep sequencing to map 5' capped transcripts has confirmed the existence of at least two distinct promoter classes in metazoans: "focused" promoters with transcription start sites (TSSs that occur in a narrowly defined genomic span and "dispersed" promoters with TSSs that are spread over a larger window. Previous studies have explored the presence of genomic features, such as CpG islands and sequence motifs, in these promoter classes, but virtually no studies have directly investigated the relationship with chromatin features. Here, we show that promoter classes are significantly differentiated by nucleosome organization and chromatin structure. Dispersed promoters display higher associations with well-positioned nucleosomes downstream of the TSS and a more clearly defined nucleosome free region upstream, while focused promoters have a less organized nucleosome structure, yet higher presence of RNA polymerase II. These differences extend to histone variants (H2A.Z and marks (H3K4 methylation, as well as insulator binding (such as CTCF, independent of the expression levels of affected genes. Notably, differences are conserved across mammals and flies, and they provide for a clearer separation of promoter architectures than the presence and absence of CpG islands or the occurrence of stalled RNA polymerase. Computational models support the stronger contribution of chromatin features to the definition of dispersed promoters compared to focused start sites. Our results show that promoter classes defined from 5' capped transcripts not only reflect differences in the initiation process at the core promoter but also are indicative of divergent transcriptional programs established within gene-proximal nucleosome organization.

Bacterial meningitis

NARCIS (Netherlands)

Roos, Karen L.; van de Beek, Diederik

2010-01-01

Bacterial meningitis is a neurological emergency. Empiric antimicrobial and adjunctive therapy should be initiated as soon as a single set of blood cultures has been obtained. Clinical signs suggestive of bacterial meningitis include fever, headache, meningismus, vomiting, photophobia, and an

Transcription control engineering and applications in synthetic biology

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Michael D. Engstrom

2017-09-01

Full Text Available In synthetic biology, researchers assemble biological components in new ways to produce systems with practical applications. One of these practical applications is control of the flow of genetic information (from nucleic acid to protein, a.k.a. gene regulation. Regulation is critical for optimizing protein (and therefore activity levels and the subsequent levels of metabolites and other cellular properties. The central dogma of molecular biology posits that information flow commences with transcription, and accordingly, regulatory tools targeting transcription have received the most attention in synthetic biology. In this mini-review, we highlight many past successes and summarize the lessons learned in developing tools for controlling transcription. In particular, we focus on engineering studies where promoters and transcription terminators (cis-factors were directly engineered and/or isolated from DNA libraries. We also review several well-characterized transcription regulators (trans-factors, giving examples of how cis- and trans-acting factors have been combined to create digital and analogue switches for regulating transcription in response to various signals. Last, we provide examples of how engineered transcription control systems have been used in metabolic engineering and more complicated genetic circuits. While most of our mini-review focuses on the well-characterized bacterium Escherichia coli, we also provide several examples of the use of transcription control engineering in non-model organisms. Similar approaches have been applied outside the bacterial kingdom indicating that the lessons learned from bacterial studies may be generalized for other organisms.

Transcription control engineering and applications in synthetic biology.

Science.gov (United States)

Engstrom, Michael D; Pfleger, Brian F

2017-09-01

In synthetic biology, researchers assemble biological components in new ways to produce systems with practical applications. One of these practical applications is control of the flow of genetic information (from nucleic acid to protein), a.k.a. gene regulation. Regulation is critical for optimizing protein (and therefore activity) levels and the subsequent levels of metabolites and other cellular properties. The central dogma of molecular biology posits that information flow commences with transcription, and accordingly, regulatory tools targeting transcription have received the most attention in synthetic biology. In this mini-review, we highlight many past successes and summarize the lessons learned in developing tools for controlling transcription. In particular, we focus on engineering studies where promoters and transcription terminators ( cis -factors) were directly engineered and/or isolated from DNA libraries. We also review several well-characterized transcription regulators ( trans- factors), giving examples of how cis- and trans -acting factors have been combined to create digital and analogue switches for regulating transcription in response to various signals. Last, we provide examples of how engineered transcription control systems have been used in metabolic engineering and more complicated genetic circuits. While most of our mini-review focuses on the well-characterized bacterium Escherichia coli , we also provide several examples of the use of transcription control engineering in non-model organisms. Similar approaches have been applied outside the bacterial kingdom indicating that the lessons learned from bacterial studies may be generalized for other organisms.

Composing a Tumor Specific Bacterial Promoter.

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Igor V Deyneko

Full Text Available Systemically applied Salmonella enterica spp. have been shown to invade and colonize neoplastic tissues where it retards the growth of many tumors. This offers the possibility to use the bacteria as a vehicle for the tumor specific delivery of therapeutic molecules. Specificity of such delivery is solely depending on promoter sequences that control the production of a target molecule. We have established the functional structure of bacterial promoters that are transcriptionally active exclusively in tumor tissues after systemic application. We observed that the specific transcriptional activation is accomplished by a combination of a weak basal promoter and a strong FNR binding site. This represents a minimal set of control elements required for such activation. In natural promoters, additional DNA remodeling elements are found that alter the level of transcription quantitatively. Inefficiency of the basal promoter ensures the absence of transcription outside tumors. As a proof of concept, we compiled an artificial promoter sequence from individual motifs representing FNR and basal promoter and showed specific activation in a tumor microenvironment. Our results open possibilities for the generation of promoters with an adjusted level of expression of target proteins in particular for applications in bacterial tumor therapy.

A Transcriptional Regulatory Mechanism Finely Tunes the Firing of Type VI Secretion System in Response to Bacterial Enemies.

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Lazzaro, Martina; Feldman, Mario F; García Véscovi, Eleonora

2017-08-22

The ability to detect and measure danger from an environmental signal is paramount for bacteria to respond accordingly, deploying strategies that halt or counteract potential cellular injury and maximize survival chances. Type VI secretion systems (T6SSs) are complex bacterial contractile nanomachines able to target toxic effectors into neighboring bacteria competing for the same colonization niche. Previous studies support the concept that either T6SSs are constitutively active or they fire effectors in response to various stimuli, such as high bacterial density, cell-cell contact, nutrient depletion, or components from dead sibling cells. For Serratia marcescens , it has been proposed that its T6SS is stochastically expressed, with no distinction between harmless or aggressive competitors. In contrast, we demonstrate that the Rcs regulatory system is responsible for finely tuning Serratia T6SS expression levels, behaving as a transcriptional rheostat. When confronted with harmless bacteria, basal T6SS expression levels suffice for Serratia to eliminate the competitor. A moderate T6SS upregulation is triggered when, according to the aggressor-prey ratio, an unbalanced interplay between homologous and heterologous effectors and immunity proteins takes place. Higher T6SS expression levels are achieved when Serratia is challenged by a contender like Acinetobacter , which indiscriminately fires heterologous effectors able to exert lethal cellular harm, threatening the survival of the Serratia population. We also demonstrate that Serratia 's RcsB-dependent T6SS regulatory mechanism responds not to general stress signals but to the action of specific effectors from competitors, displaying an exquisite strategy to weigh risks and keep the balance between energy expenditure and fitness costs. IMPORTANCE Serratia marcescens is among the health-threatening pathogens categorized by the WHO as research priorities to develop alternative antimicrobial strategies, and it was

Transcriptional regulators of legume-rhizobia symbiosis: nuclear factors Ys and GRAS are two for tango.

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Rípodas, Carolina; Clúa, Joaquín; Battaglia, Marina; Baudin, Maël; Niebel, Andreas; Zanetti, María Eugenia; Blanco, Flavio

2014-01-01

Transcription factors are DNA binding proteins that regulate gene expression. The nitrogen fixing symbiosis established between legume plants and soil bacteria is a complex interaction, in which plants need to integrate signals derived from the symbiont and the surrounding environment to initiate the developmental program of nodule organogenesis and the infection process. Several transcription factors that play critical roles in these processes have been reported in the past decade, including proteins of the GRAS and NF-Y families. Recently, we reported the characterization of a new GRAS domain containing-protein that interacts with a member of the C subunit of the NF-Y family, which plays an important role in nodule development and the progression of bacterial infection during the symbiotic interaction. The connection between transcription factors of these families highlights the significance of multimeric complexes in the fabulous capacity of plants to integrate and respond to multiple environmental stimuli.

Differential Modulation of Transcription Factors and Cytoskeletal Proteins in Prostate Carcinoma Cells by a Bacterial Lactone

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Senthil R. Kumar

2018-01-01

Full Text Available The present study tested the effect of a bacterial lactone N-(3-oxododecanoyl-homoserine lactone (C12-HSL on the cytoskeletal and transcriptional genes and proteins in prostate adenocarcinoma (PA cells (DU145 and LNCaP and prostate small cell neuroendocrine carcinoma (SCNC PC3 cells including their cellular viability and apoptosis. Our data indicate that cell migration and colony formation were affected in the presence of C12-HSL. C12-HSL induced apoptosis and altered viability of both PA and SCNC cells in a concentration dependent manner as measured by fluorescence and chemiluminescence assays. Compared to PCa cells, noncancerous prostate epithelial cells (RWPE1 were resistant to modification by C12-HSL. Further, the viability of PC3 cells in 3D matrix was suppressed by C12-HSL treatment as detected using calcein AM fluorescence in situ. C12-HSL treatment induced cytoskeletal associated protein expression of vinculin and RhoC, which may have implications in cancer cell motility, adhesion, and metastasis. IQGAP protein expression was reduced in DU145 and RWPE1 cells in the presence of C12-HSL. C12-HSL decreased STAT3 phosphorylation in DU145 cells but increased STAT1 protein phosphorylation in PC3 and LNCaP cells. Overall, these studies indicate that C12-HSL can trigger changes in transcription factors and cytoskeletal proteins and thereby modulate growth and migration properties of PCa cells.

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

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Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism

DEFF Research Database (Denmark)

Koul, A.; Vranckx, L.; Dhar, N.

2014-01-01

Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using...... microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3-4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional...... and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non...

A cysteine protease (cathepsin Z) from disk abalone, Haliotis discus discus: Genomic characterization and transcriptional profiling during bacterial infections.

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Godahewa, G I; Perera, N C N; Lee, Sukkyoung; Kim, Myoung-Jin; Lee, Jehee

2017-09-05

Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone. Copyright © 2017. Published by Elsevier

RNA-binding proteins involved in post-transcriptional regulation in bacteria

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Elke eVan Assche

2015-03-01

Full Text Available Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RNA-binding proteins, which include (i adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii modulating the accessibility of the ribosome binding site of mRNAs, (iii recruiting and assisting in the interaction of mRNAs with other molecules and (iv regulating transcription terminator / antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future.

Pesticide side effects in an agricultural soil ecosystem as measured by amoA expression quantification and bacterial diversity changes

DEFF Research Database (Denmark)

Feld, Louise; Hjort Hjelmsø, Mathis; Schostag, Morten

2015-01-01

, but only transiently. The bacterial and archaeal amoA transcripts were both sensitive bioindicators of pesticide side effects. Additionally, the numbers of bacterial amoA transcripts correlated with nitrate production in N-amended microcosms. Dazomet reduced the total bacterial numbers by one log unit...

A structural model of the E. coli PhoB Dimer in the transcription initiation complex

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Tung Chang-Shung

2012-03-01

Full Text Available Abstract Background There exist > 78,000 proteins and/or nucleic acids structures that were determined experimentally. Only a small portion of these structures corresponds to those of protein complexes. While homology modeling is able to exploit knowledge-based potentials of side-chain rotomers and backbone motifs to infer structures for new proteins, no such general method exists to extend our understanding of protein interaction motifs to novel protein complexes. Results We use a Motif Binding Geometries (MBG approach, to infer the structure of a protein complex from the database of complexes of homologous proteins taken from other contexts (such as the helix-turn-helix motif binding double stranded DNA, and demonstrate its utility on one of the more important regulatory complexes in biology, that of the RNA polymerase initiating transcription under conditions of phosphate starvation. The modeled PhoB/RNAP/σ-factor/DNA complex is stereo-chemically reasonable, has sufficient interfacial Solvent Excluded Surface Areas (SESAs to provide adequate binding strength, is physically meaningful for transcription regulation, and is consistent with a variety of known experimental constraints. Conclusions Based on a straightforward and easy to comprehend concept, "proteins and protein domains that fold similarly could interact similarly", a structural model of the PhoB dimer in the transcription initiation complex has been developed. This approach could be extended to enable structural modeling and prediction of other bio-molecular complexes. Just as models of individual proteins provide insight into molecular recognition, catalytic mechanism, and substrate specificity, models of protein complexes will provide understanding into the combinatorial rules of cellular regulation and signaling.

Post-transcriptional regulation of gene expression in Yersinia species

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Chelsea A Schiano

2012-11-01

Full Text Available Proper regulation of gene expression is required by bacterial pathogens to respond to continually changing environmental conditions and the host response during the infectious process. While transcriptional regulation is perhaps the most well understood form of controlling gene expression, recent studies have demonstrated the importance of post-transcriptional mechanisms of gene regulation that allow for more refined management of the bacterial response to host conditions. Yersinia species of bacteria are known to use various forms of post-transcriptional regulation for control of many virulence-associated genes. These include regulation by cis- and trans-acting small non-coding RNAs, RNA-binding proteins, RNases, and thermoswitches. The effects of these and other regulatory mechanisms on Yersinia physiology can be profound and have been shown to influence type III secretion, motility, biofilm formation, host cell invasion, intracellular survival and replication, and more. In this review, we will discuss these and other post-transcriptional mechanisms and their influence on virulence gene regulation, with a particular emphasis on how these processes influence the virulence of Yersinia in the host.

Theory of site-specific interactions of the combinatorial transcription factors with DNA

International Nuclear Information System (INIS)

Murugan, R

2010-01-01

We derive a functional relationship between the mean first passage time associated with the concurrent binding of multiple transcription factors (TFs) at their respective combinatorial cis-regulatory module sites (CRMs) and the number n of TFs involved in the regulation of the initiation of transcription of a gene of interest. Our results suggest that the overall search time τ s that is required by the n TFs to locate their CRMs which are all located on the same DNA chain scales with n as τ s ∼n α where α ∼ (2/5). When the jump size k that is associated with the dynamics of all the n TFs along DNA is higher than that of the critical jump size k c that scales with the size of DNA N as k c ∼ N 2/3 , we observe a similar power law scaling relationship and also the exponent α. When k c , α shows a strong dependence on both n and k. Apparently there is a critical number of combinatorial TFs n c ∼ 20 that is required to efficiently regulate the initiation of transcription of a given gene below which (2/5) 1. These results seem to be independent of the initial distances between the TFs and their corresponding CRMs and also suggest that the maximum number of TFs involved in a given combinatorial regulation of the initiation of transcription of a gene of interest seems to be restricted by the degree of condensation of the genomic DNA. The optimum number m opt of roadblock protein molecules per genome at which the search time associated with these n TFs to locate their binding sites is a minimum seems to scale as m opt ∼Ln α/2 where L is the sliding length of TFs whose maximum value seems to be such that L ≤ 10 4 bps for the E. coli bacterial genome.

Bacterial meningitis

NARCIS (Netherlands)

Heckenberg, Sebastiaan G. B.; Brouwer, Matthijs C.; van de Beek, Diederik

2014-01-01

Bacterial meningitis is a neurologic emergency. Vaccination against common pathogens has decreased the burden of disease. Early diagnosis and rapid initiation of empiric antimicrobial and adjunctive therapy are vital. Therapy should be initiated as soon as blood cultures have been obtained,

Hippo, TGF-β, and Src-MAPK pathways regulate transcription of the upd3 cytokine in Drosophila enterocytes upon bacterial infection.

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Houtz, Philip; Bonfini, Alessandro; Liu, Xi; Revah, Jonathan; Guillou, Aurélien; Poidevin, Mickael; Hens, Korneel; Huang, Hsin-Yi; Deplancke, Bart; Tsai, Yu-Chen; Buchon, Nicolas

2017-11-01

Cytokine signaling is responsible for coordinating conserved epithelial regeneration and immune responses in the digestive tract. In the Drosophila midgut, Upd3 is a major cytokine, which is induced in enterocytes (EC) and enteroblasts (EB) upon oral infection, and initiates intestinal stem cell (ISC) dependent tissue repair. To date, the genetic network directing upd3 transcription remains largely uncharacterized. Here, we have identified the key infection-responsive enhancers of the upd3 gene and show that distinct enhancers respond to various stresses. Furthermore, through functional genetic screening, bioinformatic analyses and yeast one-hybrid screening, we determined that the transcription factors Scalloped (Sd), Mothers against dpp (Mad), and D-Fos are principal regulators of upd3 expression. Our study demonstrates that upd3 transcription in the gut is regulated by the activation of multiple pathways, including the Hippo, TGF-β/Dpp, and Src, as well as p38-dependent MAPK pathways. Thus, these essential pathways, which are known to control ISC proliferation cell-autonomously, are also activated in ECs to promote tissue turnover the regulation of upd3 transcription.

DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation

OpenAIRE

Alexandrov, Boian S.; Gelev, Vladimir; Yoo, Sang Wook; Alexandrov, Ludmil B.; Fukuyo, Yayoi; Bishop, Alan R.; Rasmussen, Kim ?.; Usheva, Anny

2009-01-01

We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding–DNA co...

Modeling the integration of bacterial rRNA fragments into the human cancer genome.

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Sieber, Karsten B; Gajer, Pawel; Dunning Hotopp, Julie C

2016-03-21

Cancer is a disease driven by the accumulation of genomic alterations, including the integration of exogenous DNA into the human somatic genome. We previously identified in silico evidence of DNA fragments from a Pseudomonas-like bacteria integrating into the 5'-UTR of four proto-oncogenes in stomach cancer sequencing data. The functional and biological consequences of these bacterial DNA integrations remain unknown. Modeling of these integrations suggests that the previously identified sequences cover most of the sequence flanking the junction between the bacterial and human DNA. Further examination of these reads reveals that these integrations are rich in guanine nucleotides and the integrated bacterial DNA may have complex transcript secondary structures. The models presented here lay the foundation for future experiments to test if bacterial DNA integrations alter the transcription of the human genes.

TIP48/Reptin and H2A.Z requirement for initiating chromatin remodeling in estrogen-activated transcription.

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Mathieu Dalvai

2013-04-01

Full Text Available Histone variants, including histone H2A.Z, are incorporated into specific genomic sites and participate in transcription regulation. The role of H2A.Z at these sites remains poorly characterized. Our study investigates changes in the chromatin environment at the Cyclin D1 gene (CCND1 during transcriptional initiation in response to estradiol in estrogen receptor positive mammary tumour cells. We show that H2A.Z is present at the transcription start-site and downstream enhancer sequences of CCND1 when the gene is poorly transcribed. Stimulation of CCND1 expression required release of H2A.Z concomitantly from both these DNA elements. The AAA+ family members TIP48/reptin and the histone variant H2A.Z are required to remodel the chromatin environment at CCND1 as a prerequisite for binding of the estrogen receptor (ERα in the presence of hormone. TIP48 promotes acetylation and exchange of H2A.Z, which triggers a dissociation of the CCND1 3' enhancer from the promoter, thereby releasing a repressive intragenic loop. This release then enables the estrogen receptor to bind to the CCND1 promoter. Our findings provide new insight into the priming of chromatin required for transcription factor access to their target sequence. Dynamic release of gene loops could be a rapid means to remodel chromatin and to stimulate transcription in response to hormones.

Preliminary structural studies of the transcriptional regulator CmeR from Campylobacter jejuni

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Su, Chih-Chia [Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 (United States); Shi, Feng [Department of Veterinary Microbiology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (United States); Gu, Ruoyu; Li, Ming [Department of Physics and Astronomy, Iowa State University, Ames, IA 50011 (United States); McDermott, Gerry [Department of Anatomy, School of Medicine, University of California, San Francisco, CA 94143 (United States); Yu, Edward W., E-mail: ewyu@iastate.edu [Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 (United States); Department of Physics and Astronomy, Iowa State University, Ames, IA 50011 (United States); Zhang, Qijing [Department of Veterinary Microbiology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 (United States); Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 (United States)

2007-01-01

The transcriptional regulator CmeR from C. jejuni has been purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.2 Å. In Campylobacter jejuni, a Gram-negative bacterial pathogen causing gastroenteritis in humans, the CmeR regulatory protein controls transcription of the multidrug transporter gene operon cmeABC. CmeR belongs to the TetR family of transcriptional regulators. The 210-residue CmeR consists of two functional motifs: an N-terminal DNA-binding domain and a C-terminal ligand-binding domain. It is predicted that the DNA-binding domain interacts directly with target promoters, while the C-terminal motif interacts with inducing ligands (such as bile salts). As an initial step towards confirming this structural model, recombinant CmeR protein containing a 6×His tag at the N-terminus was crystallized. Crystals of ligand-free CmeR belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 37.4, b = 57.6, c = 93.3 Å. Diffraction was observed to at least 2.2 Å at 100 K. Analysis of the detailed CmeR structure is currently in progress.

Fungal Innate Immunity Induced by Bacterial Microbe-Associated Molecular Patterns (MAMPs

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Simon Ipcho

2016-06-01

Full Text Available Plants and animals detect bacterial presence through Microbe-Associated Molecular Patterns (MAMPs which induce an innate immune response. The field of fungal–bacterial interaction at the molecular level is still in its infancy and little is known about MAMPs and their detection by fungi. Exposing Fusarium graminearum to bacterial MAMPs led to increased fungal membrane hyperpolarization, a putative defense response, and a range of transcriptional responses. The fungus reacted with a different transcript profile to each of the three tested MAMPs, although a core set of genes related to energy generation, transport, amino acid production, secondary metabolism, and especially iron uptake were detected for all three. Half of the genes related to iron uptake were predicted MirA type transporters that potentially take up bacterial siderophores. These quick responses can be viewed as a preparation for further interactions with beneficial or pathogenic bacteria, and constitute a fungal innate immune response with similarities to those of plants and animals.

ITPI: Initial Transcription Process-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula

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Baixia Zhang

2016-01-01

Full Text Available Identification of bioactive components is an important area of research in traditional Chinese medicine (TCM formula. The reported identification methods only consider the interaction between the components and the target proteins, which is not sufficient to explain the influence of TCM on the gene expression. Here, we propose the Initial Transcription Process-based Identification (ITPI method for the discovery of bioactive components that influence transcription factors (TFs. In this method, genome-wide chip detection technology was used to identify differentially expressed genes (DEGs. The TFs of DEGs were derived from GeneCards. The components influencing the TFs were derived from STITCH. The bioactive components in the formula were identified by evaluating the molecular similarity between the components in formula and the components that influence the TF of DEGs. Using the formula of Tian-Zhu-San (TZS as an example, the reliability and limitation of ITPI were examined and 16 bioactive components that influence TFs were identified.

Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

Energy Technology Data Exchange (ETDEWEB)

Hubin, Elizabeth A.; Lilic, Mirjana; Darst, Seth A.; Campbell, Elizabeth A.

2017-07-13

The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the αCTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σA, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.

An Ancient Transcription Factor Initiates the Burst of piRNA Production During Early Meiosis in Mouse Testes

Science.gov (United States)

Li, Xin Zhiguo; Roy, Christian K.; Dong, Xianjun; Bolcun-Filas, Ewelina; Wang, Jie; Han, Bo W.; Xu, Jia; Moore, Melissa J.; Schimenti, John C.; Weng, Zhiping; Zamore, Phillip D.

2013-01-01

SUMMARY Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. PMID:23523368

Effect of immediate initiation of antiretroviral therapy on risk of severe bacterial infections in HIV-positive people with CD4 cell counts of more than 500 cells per μL

DEFF Research Database (Denmark)

O'Connor, Jemma; Vjecha, Michael J; Phillips, Andrew N

2017-01-01

BACKGROUND: The effects of antiretroviral therapy on risk of severe bacterial infections in people with high CD4 cell counts have not been well described. In this study, we aimed to quantify the effects of immediate versus deferred ART on the risk of severe bacterial infection in people with high...... μL. We used Cox proportional hazards regression to model time to severe bacterial infection, which was defined as a composite endpoint of bacterial pneumonia (confirmed by the endpoint review committee), pulmonary or extrapulmonary tuberculosis, or any bacterial infectious disorder of grade 4...... had severe bacterial infections (immediate-initiation group n=34, deferred-initiation group n=86; median 2·8 years of follow-up). Immediate ART was associated with a reduced risk of severe bacterial infection compared with deferred ART (hazard ratio [HR] 0·39, 95% CI 0·26-0·57, p

RNA Transcriptional Biosignature Analysis for Identifying Febrile Infants With Serious Bacterial Infections in the Emergency Department

Science.gov (United States)

Mahajan, Prashant; Kuppermann, Nathan; Suarez, Nicolas; Mejias, Asuncion; Casper, Charlie; Dean, J. Michael; Ramilo, Octavio

2015-01-01

Objectives To develop the infrastructure and demonstrate the feasibility of conducting microarray-based RNA transcriptional profile analyses for the diagnosis of serious bacterial infections in febrile infants 60 days and younger in a multicenter pediatric emergency research network. Methods We designed a prospective multicenter cohort study with the aim of enrolling more than 4000 febrile infants 60 days and younger. To ensure success of conducting complex genomic studies in emergency department (ED) settings, we established an infrastructure within the Pediatric Emergency Care Applied Research Network, including 21 sites, to evaluate RNA transcriptional profiles in young febrile infants. We developed a comprehensive manual of operations and trained site investigators to obtain and process blood samples for RNA extraction and genomic analyses. We created standard operating procedures for blood sample collection, processing, storage, shipping, and analyses. We planned to prospectively identify, enroll, and collect 1 mL blood samples for genomic analyses from eligible patients to identify logistical issues with study procedures. Finally, we planned to batch blood samples and determined RNA quantity and quality at the central microarray laboratory and organized data analysis with the Pediatric Emergency Care Applied Research Network data coordinating center. Below we report on establishment of the infrastructure and the feasibility success in the first year based on the enrollment of a limited number of patients. Results We successfully established the infrastructure at 21 EDs. Over the first 5 months we enrolled 79% (74 of 94) of eligible febrile infants. We were able to obtain and ship 1 mL of blood from 74% (55 of 74) of enrolled participants, with at least 1 sample per participating ED. The 55 samples were shipped and evaluated at the microarray laboratory, and 95% (52 of 55) of blood samples were of adequate quality and contained sufficient RNA for expression

Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

DEFF Research Database (Denmark)

1998-01-01

RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex...... to the action of a DNA dependant RNA polymerase in the presence of nucleoside triphosphates. Equal length transcripts may be obtained by placing a block to transcription downstream from the initiation site or by cutting the template at such a selected location. The initiation site is formed by displacement...... of one strand of the DNA locally by the PNA hybridization....

HFR1 Sequesters PIF1 to Govern the Transcriptional Network Underlying Light-Initiated Seed Germination in Arabidopsis[C][W][OPEN

Science.gov (United States)

Shi, Hui; Zhong, Shangwei; Mo, Xiaorong; Liu, Na; Nezames, Cynthia D.; Deng, Xing Wang

2013-01-01

Seed germination is the first step for seed plants to initiate a new life cycle. Light plays a predominant role in promoting seed germination, where the initial phase is mediated by photoreceptor phytochrome B (phyB). Previous studies showed that PHYTOCHROME-INTERACTING FACTOR1 (PIF1) represses seed germination downstream of phyB. Here, we identify a positive regulator of phyB-dependent seed germination, LONG HYPOCOTYL IN FAR-RED1 (HFR1). HFR1 blocks PIF1 transcriptional activity by forming a heterodimer with PIF1 that prevents PIF1 from binding to DNA. Our whole-genomic analysis shows that HFR1 and PIF1 oppositely mediate the light-regulated transcriptome in imbibed seeds. Through the HFR1–PIF1 module, light regulates expression of numerous genes involved in cell wall loosening, cell division, and hormone pathways to initiate seed germination. The functionally antagonistic HFR1–PIF1 pair constructs a fail-safe mechanism for fine-tuning seed germination during low-level illumination, ensuring a rapid response to favorable environmental changes. This study identifies the HFR1–PIF1 pair as a central module directing the whole genomic transcriptional network to rapidly initiate light-induced seed germination. PMID:24179122

Cecocentral scotoma as the initial manifestation of subacute bacterial endocarditis

Directory of Open Access Journals (Sweden)

Danielle Savitsky Strauss

2011-03-01

Full Text Available Danielle Savitsky Strauss, Samuel Baharestani, Julia Nemiroff, Kiran Amesur, David HowardNew York University Langone Medical Center, New York, NY, USAIntroduction: We report a case of a 67-year-old male who presented with a cecocentral scotoma caused by a septic embolus from subacute bacterial endocarditis (SBE.Methods: A 67-year-old man presented with sudden, painless decreased vision in the left eye. A dilated fundoscopic exam, Humphrey visual field test, transthoracic echocardiogram, abdominal computed tomography (CT, and blood cultures were all performed.Results: A dilated fundoscopic exam revealed temporal segmental optic disc pallor on the left, and Humphrey visual field testing demonstrated a dense left cecocentral scotoma. When the patient developed fever (103.9°F and palpitations, transthoracic echocardiogram revealed valvular vegetations, and contrast CT of the abdomen revealed an abscess in the dome of the liver likely due to an infectious thrombus. Blood cultures grew viridians group streptococci in three separate peripheral collections.Conclusion: This case illustrates that a sudden cecocentral scotoma may be the initial manifestation of SBE. Keywords: endocarditis, scotoma, streptococcal infections, visual fields

Insulin increases transcription of rat gene 33 through cis-acting elements in 5[prime]-flanking DNA

Energy Technology Data Exchange (ETDEWEB)

Cadilla, C.; Isham, K.R.; Lee, K.L.; Ch' ang, L.Y.; Kenney, F.T. (Oak Ridge National Lab., TN (United States)); Johnson, A.C. (National Cancer Institute, Bethesda, MD (United States). Lab. of Molecular Biology)

1992-01-01

Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, the authors have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequence in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme. This expression was increased by insulin treatment in a fashion resembling the effect of this hormone on transcription of the native gene. In vitro transcription assays in nuclear extracts also revealed increased transcription of the chimeric plasmids when the extracts were prepared from insulin-treated rat hepatoma cells. The results demonstrate that induction by insulin is mediated by cis-acting nucleotide sequences located between bp [minus]480 to +27 relative to the tsp.

The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses

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Ozan eGundogdu

2015-07-01

Full Text Available The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2 stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA expression. However a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2 stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the Galleria mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2 stress responses, enhancing bacterial survival in vivo and in the environment.

RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

KAUST Repository

Piatek, Agnieszka Anna

2014-11-14

Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

KAUST Repository

Piatek, Agnieszka Anna; Ali, Zahir; Baazim, Hatoon; Li, Lixin; Abulfaraj, Aala A.; Alshareef, Sahar; Aouida, Mustapha; Mahfouz, Magdy M.

2014-01-01

Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering

DEFF Research Database (Denmark)

Dossani, Zain Y.; Apel, Amanda Reider; Szmidt-Middleton, Heather

2018-01-01

regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein....... Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes...... levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain....

Novel Functions for TAF7, a Regulator of TAF1-independent Transcription

OpenAIRE

Devaiah, Ballachanda N.; Lu, Hanxin; Gegonne, Anne; Sercan, Zeynep; Zhang, Hongen; Clifford, Robert J.; Lee, Maxwell P.; Singer, Dinah S.

2010-01-01

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TA...

In vitro transcription of a torsionally constrained template

DEFF Research Database (Denmark)

Bentin, Thomas; Nielsen, Peter E

2002-01-01

RNA polymerase (RNAP) and the DNA template must rotate relative to each other during transcription elongation. In the cell, however, the components of the transcription apparatus may be subject to rotary constraints. For instance, the DNA is divided into topological domains that are delineated...... of torsionally constrained DNA by free RNAP. We asked whether or not a newly synthesized RNA chain would limit transcription elongation. For this purpose we developed a method to immobilize covalently closed circular DNA to streptavidin-coated beads via a peptide nucleic acid (PNA)-biotin conjugate in principle...... constrained. We conclude that transcription of a natural bacterial gene may proceed with high efficiency despite the fact that newly synthesized RNA is entangled around the template in the narrow confines of torsionally constrained supercoiled DNA....

Ribonuclease E modulation of the bacterial SOS response.

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Robert Manasherob

Full Text Available Plants, animals, bacteria, and Archaea all have evolved mechanisms to cope with environmental or cellular stress. Bacterial cells respond to the stress of DNA damage by activation of the SOS response, the canonical RecA/LexA-dependent signal transduction pathway that transcriptionally derepresses a multiplicity of genes-leading to transient arrest of cell division and initiation of DNA repair. Here we report the previously unsuspected role of E. coli endoribonuclease RNase E in regulation of the SOS response. We show that RNase E deletion or inactivation of temperature-sensitive RNase E protein precludes normal initiation of SOS. The ability of RNase E to regulate SOS is dynamic, as down regulation of RNase E following DNA damage by mitomycin C resulted in SOS termination and restoration of RNase E function leads to resumption of a previously aborted response. Overexpression of the RraA protein, which binds to the C-terminal region of RNase E and modulates the actions of degradosomes, recapitulated the effects of RNase E deficiency. Possible mechanisms for RNase E effects on SOS are discussed.

Ribonuclease E modulation of the bacterial SOS response.

Science.gov (United States)

Manasherob, Robert; Miller, Christine; Kim, Kwang-sun; Cohen, Stanley N

2012-01-01

Plants, animals, bacteria, and Archaea all have evolved mechanisms to cope with environmental or cellular stress. Bacterial cells respond to the stress of DNA damage by activation of the SOS response, the canonical RecA/LexA-dependent signal transduction pathway that transcriptionally derepresses a multiplicity of genes-leading to transient arrest of cell division and initiation of DNA repair. Here we report the previously unsuspected role of E. coli endoribonuclease RNase E in regulation of the SOS response. We show that RNase E deletion or inactivation of temperature-sensitive RNase E protein precludes normal initiation of SOS. The ability of RNase E to regulate SOS is dynamic, as down regulation of RNase E following DNA damage by mitomycin C resulted in SOS termination and restoration of RNase E function leads to resumption of a previously aborted response. Overexpression of the RraA protein, which binds to the C-terminal region of RNase E and modulates the actions of degradosomes, recapitulated the effects of RNase E deficiency. Possible mechanisms for RNase E effects on SOS are discussed.

Land use type significantly affects microbial gene transcription in soil.

Science.gov (United States)

Nacke, Heiko; Fischer, Christiane; Thürmer, Andrea; Meinicke, Peter; Daniel, Rolf

2014-05-01

Soil microorganisms play an essential role in sustaining biogeochemical processes and cycling of nutrients across different land use types. To gain insights into microbial gene transcription in forest and grassland soil, we isolated mRNA from 32 sampling sites. After sequencing of generated complementary DNA (cDNA), a total of 5,824,229 sequences could be further analyzed. We were able to assign nonribosomal cDNA sequences to all three domains of life. A dominance of bacterial sequences, which were affiliated to 25 different phyla, was found. Bacterial groups capable of aromatic compound degradation such as Phenylobacterium and Burkholderia were detected in significantly higher relative abundance in forest soil than in grassland soil. Accordingly, KEGG pathway categories related to degradation of aromatic ring-containing molecules (e.g., benzoate degradation) were identified in high abundance within forest soil-derived metatranscriptomic datasets. The impact of land use type forest on community composition and activity is evidently to a high degree caused by the presence of wood breakdown products. Correspondingly, bacterial groups known to be involved in lignin degradation and containing ligninolytic genes such as Burkholderia, Bradyrhizobium, and Azospirillum exhibited increased transcriptional activity in forest soil. Higher solar radiation in grassland presumably induced increased transcription of photosynthesis-related genes within this land use type. This is in accordance with high abundance of photosynthetic organisms and plant-infecting viruses in grassland.

Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis.

Science.gov (United States)

Nikolakakis, K; Lehnert, E; McFall-Ngai, M J; Ruby, E G

2015-07-01

The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis.

Science.gov (United States)

Mueller, R F; Characklis, W G; Jones, W L; Sears, J T

1992-05-01

The processes leading to bacterial colonization on solid-water interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless-steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m(-2). The surface roughness varied among the substrata from 0.002 microm (for silicon) to 0.015 microm (for copper). Surface free energies varied from 25.1 dynes cm(-1) for silicon to 31.2 dynes cm(-1) for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.The adsorption rate coefficient varied by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell-containing bulk water. A mathematical model describing cell accumulation on a substratum is presented.

Dissecting specific and global transcriptional regulation of bacterial gene expression

NARCIS (Netherlands)

Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe

Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an

The Transcriptional Landscape of the Production Organism Pseudomonas putida

DEFF Research Database (Denmark)

D'Arrigo, Isotta

Bacterial cell factories represent a valid alternative to fossil fuel-based production. A promising bacterium that can be optimized as cell factory is Pseudomonas putida. However, its development in bioproduction applications poses some challenges including a clear understanding of the bacterial...... system biology. This thesis has the aim of facilitating the development of P. putida KT2440 as a bacterial cell factory by investigating the transcriptome of the bacterium under different conditions (e.g. growth and stress). The main goals are the identification of differentially expressed genes, which...... provide information on bacterial adaptation to different environments, and the identification of non-coding RNAs, which regulate gene expression. This work focuses on several aspects of P. putida highlighting genomic features such as transcription start sites (TSSs), RNA regulatory elements...

Transcription regulation by the Mediator complex.

Science.gov (United States)

Soutourina, Julie

2018-04-01

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators

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Teresa Milano

2016-12-01

Full Text Available Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro–Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for β-turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties.

Bacterially-Associated Transcriptional Remodelling in a Distinct Genomic Subtype of Colorectal Cancer Provides a Plausible Molecular Basis for Disease Development.

Directory of Open Access Journals (Sweden)

Katie S Lennard

Full Text Available The relevance of specific microbial colonisation to colorectal cancer (CRC disease pathogenesis is increasingly recognised, but our understanding of possible underlying molecular mechanisms that may link colonisation to disease in vivo remains limited. Here, we investigate the relationships between the most commonly studied CRC-associated bacteria (Enterotoxigenic Bacteroides fragilis, pks+ Escherichia coli, Fusobacterium spp., afaC+ E. coli, Enterococcus faecalis & Enteropathogenic E. coli and altered transcriptomic and methylation profiles of CRC patients, in order to gain insight into the potential contribution of these bacteria in the aetiopathogenesis of CRC. We show that colonisation by E. faecalis and high levels of Fusobacterium is associated with a specific transcriptomic subtype of CRC that is characterised by CpG island methylation, microsatellite instability and a significant increase in inflammatory and DNA damage pathways. Analysis of the significant, bacterially-associated changes in host gene expression, both at the level of individual genes as well as pathways, revealed a transcriptional remodeling that provides a plausible mechanistic link between specific bacterial colonisation and colorectal cancer disease development and progression in this subtype; these included upregulation of REG3A, REG1A and REG1P in the case of high-level colonization by Fusobacterium, and CXCL10 and BMI1 in the case of colonisation by E. faecalis. The enrichment of both E. faecalis and Fusobacterium in this CRC subtype suggests that polymicrobial colonisation of the colonic epithelium may well be an important aspect of colonic tumourigenesis.

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

Science.gov (United States)

Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

2016-01-15

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses.

Science.gov (United States)

Gundogdu, Ozan; da Silva, Daiani T; Mohammad, Banaz; Elmi, Abdi; Mills, Dominic C; Wren, Brendan W; Dorrell, Nick

2015-01-01

The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid.

Science.gov (United States)

Zhai, Zhengyuan; An, Haoran; Wang, Guohong; Luo, Yunbo; Hao, Yanling

2015-11-19

Lactobacillus delbrueckii subsp. bulgaricus develops acid tolerance response when subjected to acid stress conditions, such as the induction of enzymes associated with carbohydrate metabolism. In this study, pyk gene encoding pyruvate kinase was over-expressed in heterologous host Lactococcus lactis NZ9000, and SDS-PAGE analysis revealed the successful expression of this gene in NZ9000. The survival rate of Pyk-overproducing strain was 45-fold higher than the control under acid stress condition (pH 4.0). In order to determine the transcription factor (TF) which regulates the expression of pyk by bacterial one-hybrid, we constructed a TF library including 65 TFs of L. bulgaricus. Western blotting indicated that TFs in this library could be successfully expressed in host strains. Subsequently, the promoter of pfk-pyk operon in L. bulgaricus was identified by 5'-RACE PCR. The bait plasmid pH3U3-p01 carrying the deletion fragment of pfk-pyk promoter captured catabolite control protein A (CcpA) which could regulate the expression of pyk by binding to a putative catabolite-responsive element (5'-TGTAAGCCCTAACA-3') upstream the -35 region. Real-time qPCR analysis revealed the transcription of pyk was positively regulated by CcpA. This is the first report about identifying the TF of pyk in L. bulgaricus, which will provide new insight into the regulatory network.

Transcription elongation factor GreA has functional chaperone activity.

Science.gov (United States)

Li, Kun; Jiang, Tianyi; Yu, Bo; Wang, Limin; Gao, Chao; Ma, Cuiqing; Xu, Ping; Ma, Yanhe

2012-01-01

Bacterial GreA is an indispensable factor in the RNA polymerase elongation complex. It plays multiple roles in transcriptional elongation, and may be implicated in resistance to various stresses. In this study, we show that Escherichia coli GreA inhibits aggregation of several substrate proteins under heat shock condition. GreA can also effectively promote the refolding of denatured proteins. These facts reveal that GreA has chaperone activity. Distinct from many molecular chaperones, GreA does not form stable complexes with unfolded substrates. GreA overexpression confers the host cells with enhanced resistance to heat shock and oxidative stress. Moreover, GreA expression in the greA/greB double mutant could suppress the temperature-sensitive phenotype, and dramatically alleviate the in vivo protein aggregation. The results suggest that bacterial GreA may act as chaperone in vivo. These results suggest that GreA, in addition to its function as a transcription factor, is involved in protection of cellular proteins against aggregation.

RNA-Seq-Based Transcript Structure Analysis with TrBorderExt.

Science.gov (United States)

Wang, Yejun; Sun, Ming-An; White, Aaron P

2018-01-01

RNA-Seq has become a routine strategy for genome-wide gene expression comparisons in bacteria. Despite lower resolution in transcript border parsing compared with dRNA-Seq, TSS-EMOTE, Cappable-seq, Term-seq, and others, directional RNA-Seq still illustrates its advantages: low cost, quantification and transcript border analysis with a medium resolution (±10-20 nt). To facilitate mining of directional RNA-Seq datasets especially with respect to transcript structure analysis, we developed a tool, TrBorderExt, which can parse transcript start sites and termination sites accurately in bacteria. A detailed protocol is described in this chapter for how to use the software package step by step to identify bacterial transcript borders from raw RNA-Seq data. The package was developed with Perl and R programming languages, and is accessible freely through the website: http://www.szu-bioinf.org/TrBorderExt .

TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

KAUST Repository

Schaefer, Ulf; Schmeier, Sebastian; Bajic, Vladimir B.

2010-01-01

The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

KAUST Repository

Schaefer, Ulf

2010-10-21

The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

Early canine plaque biofilms: characterization of key bacterial interactions involved in initial colonization of enamel.

Directory of Open Access Journals (Sweden)

Lucy J Holcombe

Full Text Available Periodontal disease (PD is a significant problem in dogs affecting between 44% and 63.6% of the population. The main etiological agent for PD is plaque, a microbial biofilm that colonizes teeth and causes inflammation of the gingiva. Understanding how this biofilm initiates on the tooth surface is of central importance in developing interventions against PD. Although the stages of plaque development on human teeth have been well characterized little is known about how canine plaque develops. Recent studies of the canine oral microbiome have revealed distinct differences between the canine and human oral environments and the bacterial communities they support, particularly with respect to healthy plaque. These differences mean knowledge about the nature of plaque formation in humans may not be directly translatable to dogs. The aim of this study was to identify the bacterial species important in the early stages of canine plaque formation in vivo and then use isolates of these species in a laboratory biofilm model to develop an understanding of the sequential processes which take place during the initial colonization of enamel. Supra-gingival plaque samples were collected from 12 dogs at 24 and 48 hour time points following a full mouth descale and polish. Pyrosequencing of the 16S rDNA identified 134 operational taxonomic units after statistical analysis. The species with the highest relative abundance were Bergeyella zoohelcum, Neisseria shayeganii and a Moraxella species. Streptococcal species, which tend to dominate early human plaque biofilms, had very low relative abundance. In vitro testing of biofilm formation identified five primary colonizer species, three of which belonged to the genus Neisseria. Using these pioneer bacteria as a starting point, viable two and three species communities were developed. Combining in vivo and in vitro data has led us to construct novel models of how the early canine plaque biofilm develops.

A host basal transcription factor is a key component for infection of rice by TALE-carrying bacteria.

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Yuan, Meng; Ke, Yinggen; Huang, Renyan; Ma, Ling; Yang, Zeyu; Chu, Zhaohui; Xiao, Jinghua; Li, Xianghua; Wang, Shiping

2016-07-29

Transcription activator-like effectors (TALEs) are sequence-specific DNA binding proteins found in a range of plant pathogenic bacteria, where they play important roles in host-pathogen interactions. However, it has been unclear how TALEs, after they have been injected into the host cells, activate transcription of host genes required for infection success. Here, we show that the basal transcription factor IIA gamma subunit TFIIAγ5 from rice is a key component for infection by the TALE-carrying bacterium Xanthomonas oryzae pv. oryzae, the causal agent for bacterial blight. Direct interaction of several TALEs with TFIIAγ5 is required for activation of disease susceptibility genes. Conversely, reduced expression of the TFIIAγ5 host gene limits the induction of susceptibility genes and thus decreases bacterial blight symptoms. Suppression or mutation of TFIIAγ5 can also reduce bacterial streak, another devastating disease of rice caused by TALE-carrying X. oryzae pv. oryzicola. These results have important implications for formulating a widely applicable strategy with which to improve resistance of plants to TALE-carrying pathogens.

Transcriptional response of honey bee larvae infected with the bacterial pathogen Paenibacillus larvae.

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Cornman, Robert Scott; Lopez, Dawn; Evans, Jay D

2013-01-01

American foulbrood disease of honey bees is caused by the bacterium Paenibacillus larvae. Infection occurs per os in larvae and systemic infection requires a breaching of the host peritrophic matrix and midgut epithelium. Genetic variation exists for both bacterial virulence and host resistance, and a general immunity is achieved by larvae as they age, the basis of which has not been identified. To quickly identify a pool of candidate genes responsive to P. larvae infection, we sequenced transcripts from larvae inoculated with P. larvae at 12 hours post-emergence and incubated for 72 hours, and compared expression levels to a control cohort. We identified 75 genes with significantly higher expression and six genes with significantly lower expression. In addition to several antimicrobial peptides, two genes encoding peritrophic-matrix domains were also up-regulated. Extracellular matrix proteins, proteases/protease inhibitors, and members of the Osiris gene family were prevalent among differentially regulated genes. However, analysis of Drosophila homologs of differentially expressed genes revealed spatial and temporal patterns consistent with developmental asynchrony as a likely confounder of our results. We therefore used qPCR to measure the consistency of gene expression changes for a subset of differentially expressed genes. A replicate experiment sampled at both 48 and 72 hours post infection allowed further discrimination of genes likely to be involved in host response. The consistently responsive genes in our test set included a hymenopteran-specific protein tyrosine kinase, a hymenopteran specific serine endopeptidase, a cytochrome P450 (CYP9Q1), and a homolog of trynity, a zona pellucida domain protein. Of the known honey bee antimicrobial peptides, apidaecin was responsive at both time-points studied whereas hymenoptaecin was more consistent in its level of change between biological replicates and had the greatest increase in expression by RNA-seq analysis.

Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

DEFF Research Database (Denmark)

Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.

2015-01-01

organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10ºC. Multivariate statistical analysis of the bacterial diversity data (DNA......The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78º......N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable...

Immunomodulators targeting MARCO expression improve resistance to postinfluenza bacterial pneumonia.

Science.gov (United States)

Wu, Muzo; Gibbons, John G; DeLoid, Glen M; Bedugnis, Alice S; Thimmulappa, Rajesh K; Biswal, Shyam; Kobzik, Lester

2017-07-01

Downregulation of the alveolar macrophage (AM) receptor with collagenous structure (MARCO) leads to susceptibility to postinfluenza bacterial pneumonia, a major cause of morbidity and mortality. We sought to determine whether immunomodulation of MARCO could improve host defense and resistance to secondary bacterial pneumonia. RNAseq analysis identified a striking increase in MARCO expression between days 9 and 11 after influenza infection and indicated important roles for Akt and Nrf2 in MARCO recovery. In vitro, primary human AM-like monocyte-derived macrophages (AM-MDMs) and THP-1 macrophages were treated with IFNγ to model influenza effects. Activators of Nrf2 (sulforaphane) or Akt (SC79) caused increased MARCO expression and a MARCO-dependent improvement in phagocytosis in IFNγ-treated cells and improved survival in mice with postinfluenza pneumococcal pneumonia. Transcription factor analysis also indicated a role for transcription factor E-box (TFEB) in MARCO recovery. Overexpression of TFEB in THP-1 cells led to marked increases in MARCO. The ability of Akt activation to increase MARCO expression in IFNγ-treated AM-MDMs was abrogated in TFEB-knockdown cells, indicating Akt increases MARCO expression through TFEB. Increasing MARCO expression by targeting Nrf2 signaling or the Akt-TFEB-MARCO pathway are promising strategies to improve bacterial clearance and survival in postinfluenza bacterial pneumonia. Copyright © 2017 the American Physiological Society.

A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins

Directory of Open Access Journals (Sweden)

Teresa Milano

2016-01-01

Full Text Available The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average that is homologous to fold type-I pyridoxal 5′-phosphate (PLP dependent enzymes like aspartate aminotransferase (AAT. These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs. Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups.

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

Science.gov (United States)

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

2017-09-29

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association.

Science.gov (United States)

Chun, Carlene K; Troll, Joshua V; Koroleva, Irina; Brown, Bartley; Manzella, Liliana; Snir, Einat; Almabrazi, Hakeem; Scheetz, Todd E; Bonaldo, Maria de Fatima; Casavant, Thomas L; Soares, M Bento; Ruby, Edward G; McFall-Ngai, Margaret J

2008-08-12

The light-organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution.

RNA transcriptional biosignature analysis for identifying febrile infants with serious bacterial infections in the emergency department: a feasibility study.

Science.gov (United States)

Mahajan, Prashant; Kuppermann, Nathan; Suarez, Nicolas; Mejias, Asuncion; Casper, Charlie; Dean, J Michael; Ramilo, Octavio

2015-01-01

To develop the infrastructure and demonstrate the feasibility of conducting microarray-based RNA transcriptional profile analyses for the diagnosis of serious bacterial infections in febrile infants 60 days and younger in a multicenter pediatric emergency research network. We designed a prospective multicenter cohort study with the aim of enrolling more than 4000 febrile infants 60 days and younger. To ensure success of conducting complex genomic studies in emergency department (ED) settings, we established an infrastructure within the Pediatric Emergency Care Applied Research Network, including 21 sites, to evaluate RNA transcriptional profiles in young febrile infants. We developed a comprehensive manual of operations and trained site investigators to obtain and process blood samples for RNA extraction and genomic analyses. We created standard operating procedures for blood sample collection, processing, storage, shipping, and analyses. We planned to prospectively identify, enroll, and collect 1 mL blood samples for genomic analyses from eligible patients to identify logistical issues with study procedures. Finally, we planned to batch blood samples and determined RNA quantity and quality at the central microarray laboratory and organized data analysis with the Pediatric Emergency Care Applied Research Network data coordinating center. Below we report on establishment of the infrastructure and the feasibility success in the first year based on the enrollment of a limited number of patients. We successfully established the infrastructure at 21 EDs. Over the first 5 months we enrolled 79% (74 of 94) of eligible febrile infants. We were able to obtain and ship 1 mL of blood from 74% (55 of 74) of enrolled participants, with at least 1 sample per participating ED. The 55 samples were shipped and evaluated at the microarray laboratory, and 95% (52 of 55) of blood samples were of adequate quality and contained sufficient RNA for expression analysis. It is possible to

Analysis of convergent gene transcripts in the obligate intracellular bacterium Rickettsia prowazekii.

Directory of Open Access Journals (Sweden)

Andrew Woodard

2011-01-01

Full Text Available Termination of transcription is an important component of bacterial gene expression. However, little is known concerning this process in the obligate intracellular pathogen and model for reductive evolution, Rickettsia prowazekii. To assess transcriptional termination in this bacterium, transcripts of convergent gene pairs, some containing predicted intrinsic terminators, were analyzed. These analyses revealed that, rather than terminating at a specific site within the intervening region between the convergent genes, most of the transcripts demonstrated either a lack of termination within this region, which generated antisense RNA, or a putative non-site-specific termination that occurred throughout the intervening sequence. Transcripts terminating at predicted intrinsic terminators, as well as at a putative Rho-dependant terminator, were also examined and found to vary based on the rickettsial host environment. These results suggest that transcriptional termination, or lack thereof, plays a role in rickettsial gene regulation.

TAF(II)250: a transcription toolbox.

Science.gov (United States)

Wassarman, D A; Sauer, F

2001-08-01

Activation of RNA-polymerase-II-dependent transcription involves conversion of signals provided by gene-specific activator proteins into the synthesis of messenger RNA. This conversion requires dynamic structural changes in chromatin and assembly of general transcription factors (GTFs) and RNA polymerase II at core promoter sequence elements surrounding the transcription start site of genes. One hallmark of transcriptional activation is the interaction of DNA-bound activators with coactivators such as the TATA-box binding protein (TBP)-associated factors (TAF(II)s) within the GTF TFIID. TAF(II)250 possesses a variety of activities that are likely to contribute to the initial steps of RNA polymerase II transcription. TAF(II)250 is a scaffold for assembly of other TAF(II)s and TBP into TFIID, TAF(II)250 binds activators to recruit TFIID to particular promoters, TAF(II)250 regulates binding of TBP to DNA, TAF(II)250 binds core promoter initiator elements, TAF(II)250 binds acetylated lysine residues in core histones, and TAF(II)250 possesses protein kinase, ubiquitin-activating/conjugating and acetylase activities that modify histones and GTFs. We speculate that these activities achieve two goals--(1) they aid in positioning and stabilizing TFIID at particular promoters, and (2) they alter chromatin structure at the promoter to allow assembly of GTFs--and we propose a model for how TAF(II)250 converts activation signals into active transcription.

Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

Energy Technology Data Exchange (ETDEWEB)

Tennyson, C.N.; Worton, R.G. [Univ. of Toronto and the Hospital for Sick Children, Ontario (Canada)

1994-09-01

The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

Childhood asthma after bacterial colonization of the airway in neonates

DEFF Research Database (Denmark)

Bisgaard, Hans; Hermansen, Mette Northman; Buchvald, Frederik

2007-01-01

Pathological features of the airway in young children with severe recurrent wheeze suggest an association between bacterial colonization and the initiating events of early asthma. We conducted a study to investigate a possible association between bacterial colonization of the hypopharynx in asymp......Pathological features of the airway in young children with severe recurrent wheeze suggest an association between bacterial colonization and the initiating events of early asthma. We conducted a study to investigate a possible association between bacterial colonization of the hypopharynx...... in asymptomatic neonates and later development of recurrent wheeze, asthma, and allergy during the first 5 years of life....

PRODORIC2: the bacterial gene regulation database in 2018

Science.gov (United States)

Dudek, Christian-Alexander; Hartlich, Juliane; Brötje, David; Jahn, Dieter

2018-01-01

Abstract Bacteria adapt to changes in their environment via differential gene expression mediated by DNA binding transcriptional regulators. The PRODORIC2 database hosts one of the largest collections of DNA binding sites for prokaryotic transcription factors. It is the result of the thoroughly redesigned PRODORIC database. PRODORIC2 is more intuitive and user-friendly. Besides significant technical improvements, the new update offers more than 1000 new transcription factor binding sites and 110 new position weight matrices for genome-wide pattern searches with the Virtual Footprint tool. Moreover, binding sites deduced from high-throughput experiments were included. Data for 6 new bacterial species including bacteria of the Rhodobacteraceae family were added. Finally, a comprehensive collection of sigma- and transcription factor data for the nosocomial pathogen Clostridium difficile is now part of the database. PRODORIC2 is publicly available at http://www.prodoric2.de. PMID:29136200

Deciphering Transcriptional Regulation

DEFF Research Database (Denmark)

Valen, Eivind

The myriad of cells in the human body are all made from the same blueprint: the human genome. At the heart of this diversity lies the concept of gene regulation, the process in which it is decided which genes are used where and when. Genes do not function as on/off buttons, but more like a volume...... mostly near the start of the gene known as the promoter. This region contains patterns scattered in the DNA that the TFs can recognize and bind to. Such binding can prompt the assembly of the pre-initiation complex which ultimately leads to transcription of the gene. In order to achieve the regulation...... on what characterizes a hippocampus promoter. Pairing CAGE with TF binding site prediction we identi¿ed a likely key regulator of hippocampus. Finally, we developed a method for CAGE exploration. While the DeepCAGE library characterized a full 1.4 million transcription initiation events it did not capture...

Effects of chemical and biological pesticides on plant growth parameters and rhizospheric bacterial community structure in Vigna radiata

Energy Technology Data Exchange (ETDEWEB)

Singh, Sunil; Gupta, Rashi; Sharma, Shilpi, E-mail: shilpi@dbeb.iitd.ac.in

2015-06-30

Highlights: • Non-target effects of pesticides employing qualitative and quantitative approaches. • Qualitative shifts in resident and active bacterial community structure. • Abundance of 16S rRNA gene and transcripts were reduced significantly. • Effects of biological pesticide similar to chemical pesticides on rhizospheric bacteria. - Abstract: With increasing application of pesticides in agriculture, their non-target effects on soil microbial communities are critical to soil health maintenance. The present study aimed to evaluate the effects of chemical pesticides (chlorpyrifos and cypermethrin) and a biological pesticide (azadirachtin) on growth parameters and the rhizospheric bacterial community of Vigna radiata. Qualitative and quantitative analysis by PCR-denaturing gradient gel electrophoresis (DGGE) and q-PCR, respectively, of the 16S rRNA gene and transcript were performed to study the impact of these pesticides on the resident and active rhizospheric bacterial community. While plant parameters were not affected significantly by the pesticides, a shift in the bacterial community structure was observed with an adverse effect on the abundance of 16S rRNA gene and transcripts. Chlorpyrifos showed almost complete degradation toward the end of the experiment. These non-target impacts on soil ecosystems and the fact that the effects of the biopesticide mimic those of chemical pesticides raise serious concerns regarding their application in agriculture.

Effects of chemical and biological pesticides on plant growth parameters and rhizospheric bacterial community structure in Vigna radiata

International Nuclear Information System (INIS)

Singh, Sunil; Gupta, Rashi; Sharma, Shilpi

2015-01-01

Highlights: • Non-target effects of pesticides employing qualitative and quantitative approaches. • Qualitative shifts in resident and active bacterial community structure. • Abundance of 16S rRNA gene and transcripts were reduced significantly. • Effects of biological pesticide similar to chemical pesticides on rhizospheric bacteria. - Abstract: With increasing application of pesticides in agriculture, their non-target effects on soil microbial communities are critical to soil health maintenance. The present study aimed to evaluate the effects of chemical pesticides (chlorpyrifos and cypermethrin) and a biological pesticide (azadirachtin) on growth parameters and the rhizospheric bacterial community of Vigna radiata. Qualitative and quantitative analysis by PCR-denaturing gradient gel electrophoresis (DGGE) and q-PCR, respectively, of the 16S rRNA gene and transcript were performed to study the impact of these pesticides on the resident and active rhizospheric bacterial community. While plant parameters were not affected significantly by the pesticides, a shift in the bacterial community structure was observed with an adverse effect on the abundance of 16S rRNA gene and transcripts. Chlorpyrifos showed almost complete degradation toward the end of the experiment. These non-target impacts on soil ecosystems and the fact that the effects of the biopesticide mimic those of chemical pesticides raise serious concerns regarding their application in agriculture

Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

NARCIS (Netherlands)

Kort, R.; Keijser, B.J.; Caspers, M.P.M.; Schuren, F.H.; Montijn, R.

2008-01-01

Background: In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific

Basal transcription machinery

Indian Academy of Sciences (India)

2007-03-29

Mar 29, 2007 ... The holoenzyme of prokaryotic RNA polymerase consists of the core enzyme, made of two , , ' and subunits, which lacks promoter selectivity and a sigma () subunit which enables the core enzyme to initiate transcription in a promoter dependent fashion. A stress sigma factor s, in prokaryotes ...

CoryneRegNet: an ontology-based data warehouse of corynebacterial transcription factors and regulatory networks.

Science.gov (United States)

Baumbach, Jan; Brinkrolf, Karina; Czaja, Lisa F; Rahmann, Sven; Tauch, Andreas

2006-02-14

The application of DNA microarray technology in post-genomic analysis of bacterial genome sequences has allowed the generation of huge amounts of data related to regulatory networks. This data along with literature-derived knowledge on regulation of gene expression has opened the way for genome-wide reconstruction of transcriptional regulatory networks. These large-scale reconstructions can be converted into in silico models of bacterial cells that allow a systematic analysis of network behavior in response to changing environmental conditions. CoryneRegNet was designed to facilitate the genome-wide reconstruction of transcriptional regulatory networks of corynebacteria relevant in biotechnology and human medicine. During the import and integration process of data derived from experimental studies or literature knowledge CoryneRegNet generates links to genome annotations, to identified transcription factors and to the corresponding cis-regulatory elements. CoryneRegNet is based on a multi-layered, hierarchical and modular concept of transcriptional regulation and was implemented by using the relational database management system MySQL and an ontology-based data structure. Reconstructed regulatory networks can be visualized by using the yFiles JAVA graph library. As an application example of CoryneRegNet, we have reconstructed the global transcriptional regulation of a cellular module involved in SOS and stress response of corynebacteria. CoryneRegNet is an ontology-based data warehouse that allows a pertinent data management of regulatory interactions along with the genome-scale reconstruction of transcriptional regulatory networks. These models can further be combined with metabolic networks to build integrated models of cellular function including both metabolism and its transcriptional regulation.

Metronidazole combined with nystatin (vagitories) in the prevention of bacterial vaginosis after initial treatment with oral metronidazole.

Science.gov (United States)

Pulkkinen, P; Saranen, M; Kaaja, R

1993-01-01

In a double-blind trial comprising 66 patients we assessed the effect of metronidazole-nystatin vagitories on the prevention of bacterial vaginosis (BV) in women using IUD as a contraceptive method after an initial oral single dose of 2.0 g metronidazole and 7 days of intravaginal metronidazole-nystatin or placebo treatment. The prophylactic treatment consisted of metronidazole-nystatin or placebo vagitories applied at bedtime for 3 days after menstruation over 6 consecutive menstrual periods. The patients were randomized in two study groups: a treatment group of 32 patients (group A) and a placebo group of 34 patients (group B). The overall objective cure rate after the initial treatment was 97% in group A and 91% in group B. After 6 months of follow-up, the overall cumulative objective cure rate in group A was 100%, and 76% in group B. The single-dose oral treatment was well tolerated and no notable side effects were recorded.

Insulated transcriptional elements enable precise design of genetic circuits.

Science.gov (United States)

Zong, Yeqing; Zhang, Haoqian M; Lyu, Cheng; Ji, Xiangyu; Hou, Junran; Guo, Xian; Ouyang, Qi; Lou, Chunbo

2017-07-03

Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels. Here we address this issue at the level of prokaryotic transcription by insulating minimal promoters and operators to prevent their interaction and enable the biophysical modeling of synthetic transcription without free parameters. This approach allows genetic circuit design with extraordinary precision and diversity, and consequently simplifies the design-build-test-learn cycle of circuit engineering to a mix-and-match workflow. As a demonstration, combinatorial promoters encoding NOT-gate functions were designed from scratch with mean errors of 96% using our insulated transcription elements. Furthermore, four-node transcriptional networks with incoherent feed-forward loops that execute stripe-forming functions were obtained without any trial-and-error work. This insulation-based engineering strategy improves the resolution of genetic circuit technology and provides a simple approach for designing genetic circuits for systems and synthetic biology.Unwanted interactions between cellular components can complicate rational engineering of biological systems. Here the authors design insulated minimal promoters and operators that enable biophysical modeling of bacterial transcription without free parameters for precise circuit design.

Shaping the growth behaviour of biofilms initiated from bacterial aggregates

DEFF Research Database (Denmark)

Melaugh, Gavin; Hutchison, Jaime; Kragh, Kasper Nørskov

2016-01-01

Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell ag...

Bacterial biofilms: prokaryotic adventures in multicellularity

DEFF Research Database (Denmark)

Webb, J.S.; Givskov, Michael Christian; Kjelleberg, S.

2003-01-01

The development of bacterial biofilms includes both the initial social behavior of undifferentiated cells, as well as cell death and differentiation in the mature biofilm, and displays several striking similarities with higher organisms. Recent advances in the field provide new insight...... into differentiation and cell death events in bacterial biofilm development and propose that biofilms have an unexpected level of multicellularity....

Characterization of a novel radiation-inducible transcript, uscA, and analysis of its transcriptional regulation

Energy Technology Data Exchange (ETDEWEB)

Lim, Sang Yong; Kim, Dong Ho; Joe, Min Ho

2010-03-15

The transcriptional expression of the uscA promote (P{sub uscA}) only occurred under aerobic conditions and a dose of 2Gy maximally activated transcription of P{sub uscA}. However, various environmental stress including physical shocks (pH, temperature, osmotic shock), DNA damaging agents (UV and MMC) or oxidative stressagents (paraquat, menadione, and H{sub 2}O{sub 2}) didn't cause the transcriptional activationof P{sub uscA}. The transcription of uscA was initiated at 170 bp upstream of the cyoA start codon, and ended around the ampG stop codon. The size of uscA was determined through reverse transcription assay, approximately 250 bp. The deletion analysis of uscA promoter demonstrates that radiation inducibility of P{sub uscA} is mediated by sequences present between -20 and +111 relativeto +1 of P{sub uscA} and radiation causes P{sub uscA} activation thorough permitting the expression that is repressed under non-irradiated conditions

Characterization of a novel radiation-inducible transcript, uscA, and analysis of its transcriptional regulation

International Nuclear Information System (INIS)

Lim, Sang Yong; Kim, Dong Ho; Joe, Min Ho

2010-03-01

The transcriptional expression of the uscA promote (P uscA ) only occurred under aerobic conditions and a dose of 2Gy maximally activated transcription of P uscA . However, various environmental stress including physical shocks (pH, temperature, osmotic shock), DNA damaging agents (UV and MMC) or oxidative stressagents (paraquat, menadione, and H 2 O 2 ) didn't cause the transcriptional activationof P uscA . The transcription of uscA was initiated at 170 bp upstream of the cyoA start codon, and ended around the ampG stop codon. The size of uscA was determined through reverse transcription assay, approximately 250 bp. The deletion analysis of uscA promoter demonstrates that radiation inducibility of P uscA is mediated by sequences present between -20 and +111 relativeto +1 of P uscA and radiation causes P uscA activation thorough permitting the expression that is repressed under non-irradiated conditions

Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

International Nuclear Information System (INIS)

Sahu, Geetaram; Farley, Kalamo; El-Hage, Nazira; Aiamkitsumrit, Benjamas; Fassnacht, Ryan; Kashanchi, Fatah; Ochem, Alex; Simon, Gary L.; Karn, Jonathan; Hauser, Kurt F.; Tyagi, Mudit

2015-01-01

Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. - Highlights: • Cocaine induces the initiation phase of HIV transcription by activating NF-ĸB. • Cocaine induced NF-ĸB phosphorylation promotes its interaction with P300. • Cocaine enhances the elongation phase of HIV transcription by stimulating MSK1. • Cocaine activated MSK1 catalyzes the phosphorylation of histone H3 at its Ser10. • Cocaine induced H3S10 phosphorylation facilitates the recruitment of P-TEFb at LTR

Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

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Sahu, Geetaram; Farley, Kalamo [Division of Infectious Diseases, Department of Medicine, George Washington University, Washington, DC (United States); El-Hage, Nazira [Virginia Commonwealth University, Richmond, VA (United States); Aiamkitsumrit, Benjamas; Fassnacht, Ryan [Division of Infectious Diseases, Department of Medicine, George Washington University, Washington, DC (United States); Kashanchi, Fatah [George Mason University, Manassas, VA (United States); Ochem, Alex [ICGEB, Wernher and Beit Building, Anzio Road, Observatory, 7925 Cape Town (South Africa); Simon, Gary L. [Division of Infectious Diseases, Department of Medicine, George Washington University, Washington, DC (United States); Karn, Jonathan [Case Western Reserve University, Cleveland, OH (United States); Hauser, Kurt F. [Virginia Commonwealth University, Richmond, VA (United States); Tyagi, Mudit, E-mail: tmudit@email.gwu.edu [Division of Infectious Diseases, Department of Medicine, George Washington University, Washington, DC (United States); Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC 20037 (United States)

2015-09-15

Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. - Highlights: • Cocaine induces the initiation phase of HIV transcription by activating NF-ĸB. • Cocaine induced NF-ĸB phosphorylation promotes its interaction with P300. • Cocaine enhances the elongation phase of HIV transcription by stimulating MSK1. • Cocaine activated MSK1 catalyzes the phosphorylation of histone H3 at its Ser10. • Cocaine induced H3S10 phosphorylation facilitates the recruitment of P-TEFb at LTR.

Epigenetics regulates transcription and pathogenesis in the parasite Trichomonas vaginalis.

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Pachano, Tomas; Nievas, Yesica R; Lizarraga, Ayelen; Johnson, Patricia J; Strobl-Mazzulla, Pablo H; de Miguel, Natalia

2017-06-01

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Different T. vaginalis strains vary greatly in their adherence and cytolytic capacities. These phenotypic differences might be attributed to differentially expressed genes as a consequence of extra-genetic variation, such as epigenetic modifications. In this study, we explored the role of histone acetylation in regulating gene transcription and pathogenesis in T. vaginalis. Here, we show that histone 3 lysine acetylation (H3KAc) is enriched in nucleosomes positioned around the transcription start site of active genes (BAP1 and BAP2) in a highly adherent parasite strain; compared with the low acetylation abundance in contrast to that observed in a less-adherent strain that expresses these genes at low levels. Additionally, exposition of less-adherent strain with a specific histone deacetylases inhibitor, trichostatin A, upregulated the transcription of BAP1 and BAP2 genes in concomitance with an increase in H3KAc abundance and chromatin accessibility around their transcription start sites. Moreover, we demonstrated that the binding of initiator binding protein, the transcription factor responsible for the initiation of transcription of ~75% of known T. vaginalis genes, depends on the histone acetylation state around the metazoan-like initiator to which initiator binding protein binds. Finally, we found that trichostatin A treatment increased parasite aggregation and adherence to host cells. Our data demonstrated for the first time that H3KAc is a permissive histone modification that functions to mediate both transcription and pathogenesis of the parasite T. vaginalis. © 2017 John Wiley & Sons Ltd.

Core Promoter Plasticity Between Maize Tissues and Genotypes Contrasts with Predominance of Sharp Transcription Initiation Sites.

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Mejía-Guerra, María Katherine; Li, Wei; Galeano, Narmer F; Vidal, Mabel; Gray, John; Doseff, Andrea I; Grotewold, Erich

2015-12-01

Core promoters are crucial for gene regulation, providing blueprints for the assembly of transcriptional machinery at transcription start sites (TSSs). Empirically, TSSs define the coordinates of core promoters and other regulatory sequences. Thus, experimental TSS identification provides an essential step in the characterization of promoters and their features. Here, we describe the application of CAGE (cap analysis of gene expression) to identify genome-wide TSSs used in root and shoot tissues of two maize (Zea mays) inbred lines (B73 and Mo17). Our studies indicate that most TSS clusters are sharp in maize, similar to mice, but distinct from Arabidopsis thaliana, Drosophila melanogaster, or zebra fish, in which a majority of genes have broad-shaped TSS clusters. We established that ∼38% of maize promoters are characterized by a broader TATA-motif consensus, and this motif is significantly enriched in genes with sharp TSSs. A noteworthy plasticity in TSS usage between tissues and inbreds was uncovered, with ∼1500 genes showing significantly different dominant TSSs, sometimes affecting protein sequence by providing alternate translation initiation codons. We experimentally characterized instances in which this differential TSS utilization results in protein isoforms with additional domains or targeted to distinct subcellular compartments. These results provide important insights into TSS selection and gene expression in an agronomically important crop. © 2015 American Society of Plant Biologists. All rights reserved.

CoryneRegNet: An ontology-based data warehouse of corynebacterial transcription factors and regulatory networks

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Czaja Lisa F

2006-02-01

Full Text Available Abstract Background The application of DNA microarray technology in post-genomic analysis of bacterial genome sequences has allowed the generation of huge amounts of data related to regulatory networks. This data along with literature-derived knowledge on regulation of gene expression has opened the way for genome-wide reconstruction of transcriptional regulatory networks. These large-scale reconstructions can be converted into in silico models of bacterial cells that allow a systematic analysis of network behavior in response to changing environmental conditions. Description CoryneRegNet was designed to facilitate the genome-wide reconstruction of transcriptional regulatory networks of corynebacteria relevant in biotechnology and human medicine. During the import and integration process of data derived from experimental studies or literature knowledge CoryneRegNet generates links to genome annotations, to identified transcription factors and to the corresponding cis-regulatory elements. CoryneRegNet is based on a multi-layered, hierarchical and modular concept of transcriptional regulation and was implemented by using the relational database management system MySQL and an ontology-based data structure. Reconstructed regulatory networks can be visualized by using the yFiles JAVA graph library. As an application example of CoryneRegNet, we have reconstructed the global transcriptional regulation of a cellular module involved in SOS and stress response of corynebacteria. Conclusion CoryneRegNet is an ontology-based data warehouse that allows a pertinent data management of regulatory interactions along with the genome-scale reconstruction of transcriptional regulatory networks. These models can further be combined with metabolic networks to build integrated models of cellular function including both metabolism and its transcriptional regulation.

Molecular cloning of the human gene for von Willebrand factor and identification of the transcription initiation site

International Nuclear Information System (INIS)

Collins, C.J.; Underdahl, J.P.; Levene, R.B.; Ravera, C.P.; Morin, M.J.; Dombalagian, M.J.; Ricca, G.; Livingston, D.M.; Lynch, D.C.

1987-01-01

A series of overlapping cosmid genomic clones have been isolated that contain the entire coding unit of the human gene for van Willebrand factor (vWf), a major component of the hemostatic system. The cloned segments span ≅ 175 kilobases of human DNA sequence, and hybridization analysis suggest that the vWf coding unit is ≅150 kilobases in length. Within one of these clones, the vWF transcription initiation site has been mapped and a portion of the vWf promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the transcription start site. Sequencing of a segment of another genomic clone has revealed the vWF translation termination codon. Where tested, comparative restriction analysis of cloned and chromosomal DNA segments strongly suggests that no major alterations occurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-producing endothelial cells and nonexpressing leukocytes suggest that vWf gene expression is not accompanied by gross genomic rearrangements. In addition, there is significant homology of C-terminal coding sequences among the vWf genes of several vertebrate species

Crystal structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

Energy Technology Data Exchange (ETDEWEB)

Zheng, Meiying; Cooper, David; Grossoehmerb, Nickolas; Yu, Minmin; Hung, Li-Wei; Cieslik, Murcin; Derewendaro, Urszula; Lesley, Scott; Wilson, Ian; Giedrocb, David; Derewenda, Zygmunt

2009-06-06

The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged helix (WH) DNA-binding domains and diverse C-terminal, regulatory domains, which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all a-helical regulatory domains classified into two related Pfam families: FadR{_}C and FCD. Only two crystal structures of the FadR family members, i.e. the E. coli FadR protein and the LldR from C. glutamicum, have been described to date in literature. Here we describe the crystal structure of TM0439, a GntR regulator with an FCD domain, found in the Thermotoga maritima genome. The FCD domain is similar to that of the LldR regulator, and contains a buried metal binding site. Using atomic absorption spectroscopy and Trp fluorescence, we show that the recombinant protein contains bound Ni{sup 2+} ions, but it is able to bind Zn{sup 2+} with K{sub D} < 70 nM . We conclude that Zn{sup 2+} is the likely physiological metal, where it may perform either or both structural and regulatory roles. Finally, we compare the TM0439 structure to two other FadR family structures recently deposited by Structural Genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.

The chemical structure of DNA sequence signals for RNA transcription

Science.gov (United States)

George, D. G.; Dayhoff, M. O.

1982-01-01

The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors.

Science.gov (United States)

Ishihama, Akira; Shimada, Tomohiro; Yamazaki, Yukiko

2016-03-18

Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which achieves gene selectivity through interaction with sigma factors that recognize promoters, and transcription factors (TFs) that control the activity and specificity of RNAP holoenzyme. To understand the molecular mechanisms of transcriptional regulation, the identification of regulatory targets is needed for all these factors. We then performed genomic SELEX screenings of targets under the control of each sigma factor and each TF. Here we describe the assembly of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence of effector ligands. The results reveal several novel concepts: (i) each TF regulates more targets than hitherto recognized; (ii) each promoter is regulated by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are located within operons and even inside open reading frames. The binding sites of a set of global regulators, including cAMP receptor protein, LeuO and Lrp, overlap with those of the silencer H-NS, suggesting that certain global regulators play an anti-silencing role. To facilitate sharing of these accumulated SELEX datasets with the research community, we compiled a database, 'Transcription Profile of Escherichia coli' (www.shigen.nig.ac.jp/ecoli/tec/). © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

International Nuclear Information System (INIS)

Zheng, Meiying; Cooper, David R.; Grossoehme, Nickolas E.; Yu, Minmin; Hung, Li-Wei; Cieslik, Marcin; Derewenda, Urszula; Lesley, Scott A.; Wilson, Ian A.; Giedroc, David P.; Derewenda, Zygmunt S.

2009-01-01

Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR-C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni 2+ ions but that it is able to bind Zn 2+ with K d < 70 nM. It is concluded that Zn 2+ is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors

Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

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Christopher A Lavender

2016-08-01

Full Text Available Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.

Identification of regulatory targets for the bacterial Nus factor complex.

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Baniulyte, Gabriele; Singh, Navjot; Benoit, Courtney; Johnson, Richard; Ferguson, Robert; Paramo, Mauricio; Stringer, Anne M; Scott, Ashley; Lapierre, Pascal; Wade, Joseph T

2017-12-11

Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. Here, we identify an additional cellular function for the Nus factor complex in Escherichia coli: repression of the Nus factor-encoding gene, suhB. This repression occurs primarily by translation inhibition, followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Conservation of putative NusB/E binding sites upstream of Nus factor genes suggests that Nus factor autoregulation occurs in many bacterial species. Additionally, many putative NusB/E binding sites are also found upstream of other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory.

Transcriptional Response in Mouse Thyroid Tissue after 211At Administration: Effects of Absorbed Dose, Initial Dose-Rate and Time after Administration.

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Nils Rudqvist

Full Text Available 211At-labeled radiopharmaceuticals are potentially useful for tumor therapy. However, a limitation has been the preferential accumulation of released 211At in the thyroid gland, which is a critical organ for such therapy. The aim of this study was to determine the effect of absorbed dose, dose-rate, and time after 211At exposure on genome-wide transcriptional expression in mouse thyroid gland.BALB/c mice were i.v. injected with 1.7, 7.5 or 100 kBq 211At. Animals injected with 1.7 kBq were killed after 1, 6, or 168 h with mean thyroid absorbed doses of 0.023, 0.32, and 1.8 Gy, respectively. Animals injected with 7.5 and 100 kBq were killed after 6 and 1 h, respectively; mean thyroid absorbed dose was 1.4 Gy. Total RNA was extracted from pooled thyroids and the Illumina RNA microarray platform was used to determine mRNA levels. Differentially expressed transcripts and enriched GO terms were determined with adjusted p-value 1.5, and p-value <0.05, respectively.In total, 1232 differentially expressed transcripts were detected after 211At administration, demonstrating a profound effect on gene regulation. The number of regulated transcripts increased with higher initial dose-rate/absorbed dose at 1 or 6 h. However, the number of regulated transcripts decreased with mean absorbed dose/time after 1.7 kBq 211At administration. Furthermore, similar regulation profiles were seen for groups administered 1.7 kBq. Interestingly, few previously proposed radiation responsive genes were detected in the present study. Regulation of immunological processes were prevalent at 1, 6, and 168 h after 1.7 kBq administration (0.023, 0.32, 1.8 Gy.

Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations.

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Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

2017-01-01

Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.

Genome-wide mapping of transcription start sites yields novel insights into the primary transcriptome of Pseudomonas putida

DEFF Research Database (Denmark)

D'Arrigo, Isotta; Bojanovic, Klara; Yang, Xiaochen

2016-01-01

was examined using an in vivo assay with GFP-fusion vectors and shown to function via a translational repression mechanism. Furthermore, 56 novel intergenic small RNAs and 8 putative actuaton transcripts were detected, as well as 8 novel open reading frames (ORFs). This study illustrates how global mapping...... of TSSs can yield novel insights into the transcriptional features and RNA output of bacterial genomes....

Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

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Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Taş, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

2015-01-01

The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below −10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

Shedding light on cell compartmentation in the candidate phylum Poribacteria by high resolution visualisation and transcriptional profiling

KAUST Repository

Jahn, Martin T.; Markert, Sebastian M.; Ryu, Tae Woo; Ravasi, Timothy; Stigloher, Christian; Hentschel, Ute; Moitinho-Silva, Lucas

2016-01-01

metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC

Transcriptional analysis of exopolysaccharides biosynthesis gene clusters in Lactobacillus plantarum.

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Vastano, Valeria; Perrone, Filomena; Marasco, Rosangela; Sacco, Margherita; Muscariello, Lidia

2016-04-01

Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and find applications also in non-dairy foods and in therapeutics. Recently, four clusters of genes (cps) associated with surface polysaccharide production have been identified in Lactobacillus plantarum WCFS1, a probiotic and food-associated lactobacillus. These clusters are involved in cell surface architecture and probably in release and/or exposure of immunomodulating bacterial molecules. Here we show a transcriptional analysis of these clusters. Indeed, RT-PCR experiments revealed that the cps loci are organized in five operons. Moreover, by reverse transcription-qPCR analysis performed on L. plantarum WCFS1 (wild type) and WCFS1-2 (ΔccpA), we demonstrated that expression of three cps clusters is under the control of the global regulator CcpA. These results, together with the identification of putative CcpA target sequences (catabolite responsive element CRE) in the regulatory region of four out of five transcriptional units, strongly suggest for the first time a role of the master regulator CcpA in EPS gene transcription among lactobacilli.

RegPrecise 3.0--a resource for genome-scale exploration of transcriptional regulation in bacteria.

Science.gov (United States)

Novichkov, Pavel S; Kazakov, Alexey E; Ravcheev, Dmitry A; Leyn, Semen A; Kovaleva, Galina Y; Sutormin, Roman A; Kazanov, Marat D; Riehl, William; Arkin, Adam P; Dubchak, Inna; Rodionov, Dmitry A

2013-11-01

Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in prokaryotes is one of the critical tasks of modern genomics. Bacteria from different taxonomic groups, whose lifestyles and natural environments are substantially different, possess highly diverged transcriptional regulatory networks. The comparative genomics approaches are useful for in silico reconstruction of bacterial regulons and networks operated by both transcription factors (TFs) and RNA regulatory elements (riboswitches). RegPrecise (http://regprecise.lbl.gov) is a web resource for collection, visualization and analysis of transcriptional regulons reconstructed by comparative genomics. We significantly expanded a reference collection of manually curated regulons we introduced earlier. RegPrecise 3.0 provides access to inferred regulatory interactions organized by phylogenetic, structural and functional properties. Taxonomy-specific collections include 781 TF regulogs inferred in more than 160 genomes representing 14 taxonomic groups of Bacteria. TF-specific collections include regulogs for a selected subset of 40 TFs reconstructed across more than 30 taxonomic lineages. Novel collections of regulons operated by RNA regulatory elements (riboswitches) include near 400 regulogs inferred in 24 bacterial lineages. RegPrecise 3.0 provides four classifications of the reference regulons implemented as controlled vocabularies: 55 TF protein families; 43 RNA motif families; ~150 biological processes or metabolic pathways; and ~200 effectors or environmental signals. Genome-wide visualization of regulatory networks and metabolic pathways covered by the reference regulons are available for all studied genomes. A separate section of RegPrecise 3.0 contains draft regulatory networks in 640 genomes obtained by an conservative propagation of the reference regulons to closely related genomes. RegPrecise 3.0 gives access to the transcriptional regulons reconstructed in

Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene.

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Raul A Cernadas

2014-02-01

Full Text Available Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo, which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.

Stimulation of host immune defenses by a small molecule protects C. elegans from bacterial infection.

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Pukkila-Worley, Read; Feinbaum, Rhonda; Kirienko, Natalia V; Larkins-Ford, Jonah; Conery, Annie L; Ausubel, Frederick M

2012-01-01

The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes. We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested. Here we show that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode. Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7. These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans-based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens.

Stimulation of host immune defenses by a small molecule protects C. elegans from bacterial infection.

Directory of Open Access Journals (Sweden)

Read Pukkila-Worley

Full Text Available The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes. We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested. Here we show that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode. Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7. These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans-based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens.

Concentration and length dependence of DNA looping in transcriptional regulation.

Directory of Open Access Journals (Sweden)

Lin Han

2009-05-01

Full Text Available In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage, to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

Elongation factor P mediates a novel post-transcriptional regulatory pathway critical for bacterial virulence

DEFF Research Database (Denmark)

Zou, S Betty; Roy, Hervé; Ibba, Michael

2012-01-01

Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability of the path......Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability...... our laboratory and others now suggests that EF-P, previously thought to be essential, instead plays an ancillary role in translation by regulating the synthesis of a relatively limited subset of proteins. Other observations suggest that the eukaryotic homolog of EF-P, eIF5A, may illicit similar...

Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors

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Estruch Francisco

2012-09-01

Full Text Available Abstract Background The various steps of mRNP biogenesis (transcription, processing and export are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation. Results We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of dbp5 and mex67 mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC formation. Conclusions Our results indicate that mere reduction in the amount of mRNP produced is not sufficient to suppress the defects caused by a defective mRNA export factor. Suppression occurs only with mutants affecting events within a narrow window of the mRNP biogenesis process. We propose that reducing the speed with which transcription converts from initiation and promoter clearance to elongation may have a positive effect on mRNP formation by permitting more effective recruitment of partially-functional mRNP proteins to the nascent mRNP.

Interplay of DNA repair with transcription: from structures to mechanisms.

Science.gov (United States)

Deaconescu, Alexandra M; Artsimovitch, Irina; Grigorieff, Nikolaus

2012-12-01

Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

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Anna Lena Jung

2016-04-01

Full Text Available The formation and release of outer membrane vesicles (OMVs is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila, a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.

Crystallization and preliminary crystal structure analysis of the ligand-binding domain of PqsR (MvfR), the Pseudomonas quinolone signal (PQS) responsive quorum-sensing transcription factor of Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Xu, Ningna; Yu, Shen; Moniot, Sébastien; Weyand, Michael; Blankenfeldt, Wulf

2012-01-01

The ligand-binding domain of the transcription factor PqsR from P. aeruginosa has been crystallized and initial phases have been obtained using SAD data from seleno-l-methionine-labelled crystals. The opportunistic bacterial pathogen Pseudomonas aeruginosa employs three transcriptional regulators, LasR, RhlR and PqsR, to control the transcription of a large subset of its genes in a cell-density-dependent process known as quorum sensing. Here, the recombinant production, crystallization and structure solution of the ligand-binding domain of PqsR (MvfR), the LysR-type transcription factor that responds to the Pseudomonas quinolone signal (PQS), a quinolone-based quorum-sensing signal that is unique to P. aeruginosa and possibly a small number of other bacteria, is reported. PqsR regulates the expression of many virulence genes and may therefore be an interesting drug target. The ligand-binding domain (residues 91–319) was produced as a fusion with SUMO, and hexagonal-shaped crystals of purified PqsR-91–319 were obtained using the vapour-diffusion method. Crystallization in the presence of a PQS precursor allowed data collection to 3.25 Å resolution on a synchrotron beamline, and initial phases have been obtained using single-wavelength anomalous diffraction data from seleno-l-methionine-labelled crystals, revealing the space group to be P6 5 22, with unit-cell parameters a = b = 116–120, c = 115–117 Å

CHD chromatin remodelers and the transcription cycle

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Murawska, Magdalena

2011-01-01

It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by “opening” or “closing” chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but also are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts. PMID:22223048

Francisella tularensis subsp. tularensis induces a unique pulmonary inflammatory response: role of bacterial gene expression in temporal regulation of host defense responses.

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Kathie-Anne Walters

Full Text Available Pulmonary exposure to Francisella tularensis is associated with severe lung pathology and a high mortality rate. The lack of induction of classical inflammatory mediators, including IL1-β and TNF-α, during early infection has led to the suggestion that F. tularensis evades detection by host innate immune surveillance and/or actively suppresses inflammation. To gain more insight into the host response to Francisella infection during the acute stage, transcriptomic analysis was performed on lung tissue from mice exposed to virulent (Francisella tularensis ssp tularensis SchuS4. Despite an extensive transcriptional response in the lungs of animals as early as 4 hrs post-exposure, Francisella tularensis was associated with an almost complete lack of induction of immune-related genes during the initial 24 hrs post-exposure. This broad subversion of innate immune responses was particularly evident when compared to the pulmonary inflammatory response induced by other lethal (Yersinia pestis and non-lethal (Legionella pneumophila, Pseudomonas aeruginosa pulmonary infections. However, the unique induction of a subset of inflammation-related genes suggests a role for dysregulation of lymphocyte function and anti-inflammatory pathways in the extreme virulence of Francisella. Subsequent activation of a classical inflammatory response 48 hrs post-exposure was associated with altered abundance of Francisella-specific transcripts, including those associated with bacterial surface components. In summary, virulent Francisella induces a unique pulmonary inflammatory response characterized by temporal regulation of innate immune pathways correlating with altered bacterial gene expression patterns. This study represents the first simultaneous measurement of both host and Francisella transcriptome changes that occur during in vivo infection and identifies potential bacterial virulence factors responsible for regulation of host inflammatory pathways.

Overexpression of miR169o, an Overlapping microRNA in Response to Both Nitrogen Limitation and Bacterial Infection, Promotes Nitrogen Use Efficiency and Susceptibility to Bacterial Blight in Rice.

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Chao, Yu; Chen, Yutong; Cao, Yaqian; Chen, Huamin; Wang, Jichun; Bi, Yong-Mei; Tian, Fang; Yang, Fenghuan; Rothstein, Steven J; Zhou, Xueping; He, Chenyang

2018-03-15

Limiting nitrogen (N) supply contributes to improved resistance to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) in susceptible rice (Oryza sativa). To understand the regulatory roles of microRNAs in this phenomenon, sixty-three differentially-expressed overlapping miRNAs in response to Xoo infection and N-limitation stress in rice were identified through deep RNA-sequence and stem loop qRT-PCR. Among these, miR169o was further assessed as a typical overlapping miRNA through the overexpression of the miR169o primary gene. Osa-miR169o-OX plants were taller, and had more biomass accumulation with significantly increased nitrate and total amino acid contents in roots than wild type (WT). Transcript level assays showed that under different N supply conditions miR169o opposite regulated NRT2 which is reduced under normal N supply condition but remarkably induced under N limiting stress. On the other hand, osa-miR169o-OX plants also displayed increased disease lesion lengths and reduced transcriptional levels of defense gene (PR1b, PR10a, PR10b and PAL) compared with WT after inoculation with Xoo. In addition, miR169o impeded Xoo-mediated NRT transcription. Therefore, the overlapping miR169o contributes to increase N use efficiency and negatively regulates the resistance to bacterial blight in rice. Consistently, transient expression of NF-YAs in rice protoplast promoted the transcripts of PR genes and NRT2 genes, while reduced the transcripts of NRT1 genes. Our results provide novel and additional insights into the coordinated regulatory mechanisms of crosstalk between Xoo infection and N-deficiency responses in rice.

Genomic and chromatin signals underlying transcription start-site selection

DEFF Research Database (Denmark)

Valen, Eivind; Sandelin, Albin Gustav

2011-01-01

A central question in cellular biology is how the cell regulates transcription and discerns when and where to initiate it. Locating transcription start sites (TSSs), the signals that specify them, and ultimately elucidating the mechanisms of regulated initiation has therefore been a recurrent theme....... In recent years substantial progress has been made towards this goal, spurred by the possibility of applying genome-wide, sequencing-based analysis. We now have a large collection of high-resolution datasets identifying locations of TSSs, protein-DNA interactions, and chromatin features over whole genomes...

Modelling reveals kinetic advantages of co-transcriptional splicing.

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Stuart Aitken

2011-10-01

Full Text Available Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

Modelling reveals kinetic advantages of co-transcriptional splicing.

Science.gov (United States)

Aitken, Stuart; Alexander, Ross D; Beggs, Jean D

2011-10-01

Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

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Groscurth Peter

2007-06-01

Full Text Available Abstract Background Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522 were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. Results In this study we investigated generation of granulysin in lymphokine activated killer (LAK cells and antigen (Listeria specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation. Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. Conclusion This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing.

A downstream CpG island controls transcript initiation and elongation and the methylation state of the imprinted Airn macro ncRNA promoter.

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Martha V Koerner

Full Text Available A CpG island (CGI lies at the 5' end of the Airn macro non-protein-coding (nc RNA that represses the flanking Igf2r promoter in cis on paternally inherited chromosomes. In addition to being modified on maternally inherited chromosomes by a DNA methylation imprint, the Airn CGI shows two unusual organization features: its position immediately downstream of the Airn promoter and transcription start site and a series of tandem direct repeats (TDRs occupying its second half. The physical separation of the Airn promoter from the CGI provides a model to investigate if the CGI plays distinct transcriptional and epigenetic roles. We used homologous recombination to generate embryonic stem cells carrying deletions at the endogenous locus of the entire CGI or just the TDRs. The deleted Airn alleles were analyzed by using an ES cell imprinting model that recapitulates the onset of Igf2r imprinted expression in embryonic development or by using knock-out mice. The results show that the CGI is required for efficient Airn initiation and to maintain the unmethylated state of the Airn promoter, which are both necessary for Igf2r repression on the paternal chromosome. The TDRs occupying the second half of the CGI play a minor role in Airn transcriptional elongation or processivity, but are essential for methylation on the maternal Airn promoter that is necessary for Igf2r to be expressed from this chromosome. Together the data indicate the existence of a class of regulatory CGIs in the mammalian genome that act downstream of the promoter and transcription start.

Dual Regulation of Bacillus subtilis kinB Gene Encoding a Sporulation Trigger by SinR through Transcription Repression and Positive Stringent Transcription Control.

Science.gov (United States)

Fujita, Yasutaro; Ogura, Mitsuo; Nii, Satomi; Hirooka, Kazutake

2017-01-01

It is known that transcription of kinB encoding a trigger for Bacillus subtilis sporulation is under repression by SinR, a master repressor of biofilm formation, and under positive stringent transcription control depending on the adenine species at the transcription initiation nucleotide (nt). Deletion and base substitution analyses of the kinB promoter (P kinB ) region using lacZ fusions indicated that either a 5-nt deletion (Δ5, nt -61/-57, +1 is the transcription initiation nt) or the substitution of G at nt -45 with A (G-45A) relieved kinB repression. Thus, we found a pair of SinR-binding consensus sequences (GTTCTYT; Y is T or C) in an inverted orientation (SinR-1) between nt -57/-42, which is most likely a SinR-binding site for kinB repression. This relief from SinR repression likely requires SinI, an antagonist of SinR. Surprisingly, we found that SinR is essential for positive stringent transcription control of P kinB . Electrophoretic mobility shift assay (EMSA) analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 (nt -29/-8) consisting of another pair of SinR consensus sequences in a tandem repeat arrangement; the two sequences partially overlap the '-35' and '-10' regions of P kinB . Introduction of base substitutions (T-27C C-26T) in the upstream consensus sequence of SinR-2 affected positive stringent transcription control of P kinB , suggesting that SinR binding to SinR-2 likely causes this positive control. EMSA also implied that RNA polymerase and SinR are possibly bound together to SinR-2 to form a transcription initiation complex for kinB transcription. Thus, it was suggested in this work that derepression of kinB from SinR repression by SinI induced by Spo0A∼P and occurrence of SinR-dependent positive stringent transcription control of kinB might induce effective sporulation cooperatively, implying an intimate interplay by stringent response, sporulation, and biofilm formation.

Harnessing transcription for bioproduction in cyanobacteria

DEFF Research Database (Denmark)

Stensjö, Karin; Vavitsas, Konstantinos; Tyystjärvi, Taina

2018-01-01

Sustainable production of biofuels and other valuable compounds is one of our future challenges. One tempting possibility is to use photosynthetic cyanobacteria as production factories. Currently, tools for genetic engineering of cyanobacteria are yet not good enough to exploit the full potential...... of cyanobacteria. A wide variety of expression systems will be required to adjust both the expression of heterologous enzyme(s) and metabolic routes to the best possible balance, allowing the optimal production of a particular substance. In bacteria, transcription, especially the initiation of transcription, has...

Structural insights into transcription complexes

NARCIS (Netherlands)

Berger, I.; Blanco, A.G.; Boelens, R.; Cavarelli, J.; Coll, M.; Folkers, G.E.; Nie, Y.; Pogenberg, V.; Schultz, P.; Wilmanns, M.; Moras, D.; Poterszman, A.

2011-01-01

Control of transcription allows the regulation of cell activity in response to external stimuli and research in the field has greatly benefited from efforts in structural biology. In this review, based on specific examples from the European SPINE2-COMPLEXES initiative, we illustrate the impact of

Linking Core Promoter Classes to Circadian Transcription.

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Pål O Westermark

2016-08-01

Full Text Available Circadian rhythms in transcription are generated by rhythmic abundances and DNA binding activities of transcription factors. Propagation of rhythms to transcriptional initiation involves the core promoter, its chromatin state, and the basal transcription machinery. Here, I characterize core promoters and chromatin states of genes transcribed in a circadian manner in mouse liver and in Drosophila. It is shown that the core promoter is a critical determinant of circadian mRNA expression in both species. A distinct core promoter class, strong circadian promoters (SCPs, is identified in mouse liver but not Drosophila. SCPs are defined by specific core promoter features, and are shown to drive circadian transcriptional activities with both high averages and high amplitudes. Data analysis and mathematical modeling further provided evidence for rhythmic regulation of both polymerase II recruitment and pause release at SCPs. The analysis provides a comprehensive and systematic view of core promoters and their link to circadian mRNA expression in mouse and Drosophila, and thus reveals a crucial role for the core promoter in regulated, dynamic transcription.

Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes.

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Teichmann, Martin; Dumay-Odelot, Hélène; Fribourg, Sébastien

2012-01-01

The winged helix (WH) domain is found in core components of transcription systems in eukaryotes and prokaryotes. It represents a sub-class of the helix-turn-helix motif. The WH domain participates in establishing protein-DNA and protein-protein-interactions. Here, we discuss possible explanations for the enrichment of this motif in transcription systems.

Bacterial Community Succession in Pine-Wood Decomposition.

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Kielak, Anna M; Scheublin, Tanja R; Mendes, Lucas W; van Veen, Johannes A; Kuramae, Eiko E

2016-01-01

Though bacteria and fungi are common inhabitants of decaying wood, little is known about the relationship between bacterial and fungal community dynamics during natural wood decay. Based on previous studies involving inoculated wood blocks, strong fungal selection on bacteria abundance and community composition was expected to occur during natural wood decay. Here, we focused on bacterial and fungal community compositions in pine wood samples collected from dead trees in different stages of decomposition. We showed that bacterial communities undergo less drastic changes than fungal communities during wood decay. Furthermore, we found that bacterial community assembly was a stochastic process at initial stage of wood decay and became more deterministic in later stages, likely due to environmental factors. Moreover, composition of bacterial communities did not respond to the changes in the major fungal species present in the wood but rather to the stage of decay reflected by the wood density. We concluded that the shifts in the bacterial communities were a result of the changes in wood properties during decomposition and largely independent of the composition of the wood-decaying fungal communities.

[Characterization and transcriptional analysis of a new CC chemokine associated with innate imimune response in cobia (Rachycentron canadum)].

Science.gov (United States)

Su, Y; Feng, J; Sun, X; Guo, Z; Xu, L; Jiang, J

2013-01-01

Chemokines are small, secreted cytokine peptides, known principally for their ability to induce migration and activation of leukocyte populations under both pathological and physiological conditions. On the basis of previously constructed express sequence tags (ESTs) of the head kidney and spleen cDNA library of the perciform marine fish Rachycentron canadum (common name cobia). We used bi-directional rapid amplification of cDNA ends (RACE) and obtained a full-length cDNA of a new CC chemokine gene (designated RcCC3). The RcCC3 putative peptide exhibits sequence similarity to the group of CCL19/21/25 CC chemokines. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used in transcript expression studies of RcCC3. We examined the constitutive expression of the transcripts in 12 tissues of non-stressed cobia; RcCC3 transcripts were detected in all tissues examined, with the highest expression in gill and liver, following by head kidney, kidney, spleen, skin, intestine, muscle, stomach, heart, blood and brain. Transcript expression of RcCC3 was examined in immune-related organs, including head kidney, spleen and liver, following intraperitoneal injection of phosphate-buffered saline control, polyriboinosinic polyribocytidylic acid (poly(I:C)) and formalin-killed Vibrio carchariae (bacterial vaccine). The transcripts in these tissues were quickly up-regulated by the injection of poly(I:C) and bacterial vaccine at early time points, although with different expression profiles. These results indicate RcCC3 represents an important component of innate immunity in cobia.

Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma

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Dinesh K. Singh

2017-01-01

Full Text Available Efforts to identify and target glioblastoma (GBM drivers have primarily focused on receptor tyrosine kinases (RTKs. Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2 transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2 and zinc-finger E-box binding homeobox 1 (ZEB1, which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

Regulation of host-pathogen interactions via the post-transcriptional Csr/Rsm system.

Science.gov (United States)

Kusmierek, Maria; Dersch, Petra

2018-02-01

A successful colonization of specific hosts requires a rapid and efficient adaptation of the virulence-relevant gene expression program by bacterial pathogens. An important element in this endeavor is the Csr/Rsm system. This multi-component, post-transcriptional control system forms a central hub within complex regulatory networks and coordinately adjusts virulence properties with metabolic and physiological attributes of the pathogen. A key function is elicited by the RNA-binding protein CsrA/RsmA. CsrA/RsmA interacts with numerous target mRNAs, many of which encode crucial virulence factors, and alters their translation, stability or elongation of transcription. Recent studies highlighted that important colonization factors, toxins, and bacterial secretion systems are under CsrA/RsmA control. CsrA/RsmA deficiency impairs host colonization and attenuates virulence, making this post-transcriptional regulator a suitable drug target. The CsrA/RsmA protein can be inactivated through sequestration by non-coding RNAs, or via binding to specific highly abundant mRNAs and interacting proteins. The wide range of interaction partners and RNA targets, as well as the overarching, interlinked genetic control circuits illustrate the complexity of this regulatory system in the different pathogens. Future work addressing spatio-temporal changes of Csr/Rsm-mediated control during the course of an infection will help us to understand how bacteria reprogram their expression profile to cope with continuous changes experienced in colonized niches. Copyright © 2017 Elsevier Ltd. All rights reserved.

Intrinsic terminators in Mycoplasma hyopneumoniae transcription.

Science.gov (United States)

Fritsch, Tiago Ebert; Siqueira, Franciele Maboni; Schrank, Irene Silveira

2015-04-08

Mycoplasma hyopneumoniae, an important pathogen of swine, exhibits a low guanine and cytosine (GC) content genome. M. hyopneumoniae genome is organised in long transcriptional units and promoter sequences have been mapped upstream of all transcription units. These analysis provided insights into the gene organisation and transcription initiation at the genome scale. However, the presence of transcriptional terminator sequences in the M. hyopneumoniae genome is poorly understood. In silico analyses demonstrated the presence of putative terminators in 82% of the 33 monocistronic units (mCs) and in 74% of the 116 polycistronic units (pCs) considering different classes of terminators. The functional activity of 23 intrinsic terminators was confirmed by RT-PCR and qPCR. Analysis of all terminators found by three software algorithms, combined with experimental results, allowed us to propose a pattern of RNA hairpin formation during the termination process and to predict the location of terminators in the M. hyopneumoniae genome sequence. The stem-loop structures of intrinsic terminators of mycoplasma diverge from the pattern of terminators found in other bacteria due the low content of guanine and cytosine. In M. hyopneumoniae, transcription can end after a transcriptional unit and before its terminator sequence and can also continue past the terminator sequence with RNA polymerases gradually releasing the RNA.

Effect of crushed mussel shell addition on bacterial growth in acid polluted soils

DEFF Research Database (Denmark)

Fernandez Calviño, David; Garrido-Rodríguez, B.; Arias-Estévez, M.

2015-01-01

We applied three different doses of crushed mussel shell (CMS) on two Cu-polluted acid soils to study the effect of these amendments on the growth of the bacterial community during 730 days. Soil pH increased in the short and medium term due to CMS addition. In a first stage, bacterial growth...... was lower in the CMS-amended than in the un-amended samples. Thereafter, bacterial growth increased slowly. The soil having the highest initial pH value (4.5) showed the first significant increase in bacterial growth 95 days after the CMS amendment. However, in the soil with the lowest initial pH value (3...... as an agronomic sound practice for strongly acid soils (pH

Transcription of minute virus of mice, an autonomous parvovirus, may be regulated by attenuation

International Nuclear Information System (INIS)

Ben-Asher, E.; Aloni, Y.

1984-01-01

To characterize the transcriptional organization and regulation of minute virus of mice, an autonomous parvovirus, viral transcriptional complexes were isolated and cleaved with restriction enzymes. The in vivo preinitiated nascent RNA was elongated in vitro in the presence of [alpha- 32 P]UTP to generate runoff transcripts. The lengths of the runoff transcripts were analyzed by gel electrophoresis under denaturing conditions. On the basis of the map locations of the restriction sites and the lengths of the runoff transcripts, the in vivo initiation sites were determined. Two major initiation sites having similar activities were thus identified at residues 201 +/- 5 and 2005 +/- 5; both of them were preceded by a TATAA sequence. When uncleaved viral transcriptional complexes or isolated nuclei were incubated in vitro in the presence of [alpha- 32 P]UTP or [alpha- 32 P]CTP, they synthesized labeled RNA that, as determined by polyacrylamide gel electrophoresis, contained a major band of 142 nucleotides. The RNA of the major band was mapped between the initiation site at residue 201 +/- 5 and residue 342. We noticed the potential of forming two mutually exclusive stem-and-loop structures in the 142-nucleotide RNA; one of them is followed by a string of uridylic acid residues typical of a procaryotic transcription termination signal. We propose that, as in the transcription of simian virus 40, RNA transcription in minute virus of mice may be regulated by attenuation and may involve eucaryotic polymerase B, which can respond to a transcription termination signal similar to that of the procaryotic polymerase

Initiation of protein synthesis in bacteria

DEFF Research Database (Denmark)

Laursen, Brian Søgaard; Sørensen, Hans Peter; Mortensen, Kim Kusk

2005-01-01

Valuable information on translation initiation is available from biochemical data and recently solved structures. We present a detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation. The f...

Differential Rickettsial Transcription in Bloodfeeding and Non-Bloodfeeding Arthropod Hosts.

Directory of Open Access Journals (Sweden)

Victoria I Verhoeve

Full Text Available Crucial factors influencing the epidemiology of Rickettsia felis rickettsiosis include pathogenesis and transmission. Detection of R. felis DNA in a number of arthropod species has been reported, with characterized isolates, R. felis strain LSU and strain LSU-Lb, generated from the cat flea, Ctenocephalides felis, and the non-hematophagous booklouse, Liposcelis bostrychophila, respectively. While it is realized that strain influence on host biology varies, the rickettsial response to these distinct host environments remained undefined. To identify a panel of potential rickettsial transmission determinants in the cat flea, the transcriptional profile for these two strains of R. felis were compared in their arthropod hosts using RNAseq. Rickettsial genes with increased transcription in the flea as compared to the booklouse were identified. Genes previously associated with bacterial virulence including LPS biosynthesis, Type IV secretion system, ABC transporters, and a toxin-antitoxin system were selected for further study. Transcription of putative virulence-associated genes was determined in a flea infection bioassay for both strains of R. felis. A host-dependent transcriptional profile during bloodfeeding, specifically, an increased expression of selected transcripts in newly infected cat fleas and flea feces was detected when compared to arthropod cell culture and incubation in vertebrate blood. Together, these studies have identified novel, host-dependent rickettsial factors that likely contribute to successful horizontal transmission by bloodfeeding arthropods.

A transcription activator-like effector (TALE) induction system mediated by proteolysis.

Science.gov (United States)

Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

2016-04-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

Method for determining transcriptional linkage by means of inhibition of deoxyribonucleic acid transcription by ultraviolet irradiation: evaluation in application to the investigation of in vivo transcription in bacteriophage T7

International Nuclear Information System (INIS)

Brautigam, A.R.

1975-01-01

A technique is presented for mapping promotor sites that utilizes the introduction of transcription-terminating lesions in DNA through uv irradiation which prevents transcription of genes in proportion to their distance from the promotor. This technique was applied to and evaluated in investigations of the transcriptional linkage of bacteriophage T7. All results substantiate the hypothesis that transcription in vivo does not proceed beyond the first uv lesion encountered in the template DNA and that such premature termination of transcription is the principal effect of the uv irradiation on the transcriptional template function of DNA. UV-induced inhibition of the initiation of transcription is insignificant by comparison. Uv inactivation of expression of individual T7 genes was found to follow pseudo first-order kinetics, allowing a gene-specific uv inactivation cross section to be evaluated for each gene. Promotor locations were inferred from the discontinuity in the numerical values of inactivation cross sections arising at the start of each new unit. By such analysis the bacteriophage T7 genome was found to consist of seven transcription units. In vivo E. coli RNA polymerase transcribes the T7 early region as a single unit from a pomotor region located at the left end of the genome. The T7 late region was found to consist of six transcription units, with promotors located just ahead of genes 1.7, 7, 9, 11, 13 and 17

VirF-Independent Regulation of Shigella virB Transcription is Mediated by the Small RNA RyhB

Science.gov (United States)

Broach, William H.; Egan, Nicholas; Wing, Helen J.; Payne, Shelley M.; Murphy, Erin R.

2012-01-01

Infection of the human host by Shigella species requires the coordinated production of specific Shigella virulence factors, a process mediated largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional activator of several virulence-associated genes. This study reveals that transcription of virB is also regulated by the small RNA RyhB, and importantly, that this regulation is not achieved indirectly via modulation of VirF activity. These data are the first to demonstrate that the regulation of virB transcription can be uncoupled from the master regulator VirF. It is also established that efficient RyhB-dependent regulation of transcription is facilitated by specific nucleic acid sequences within virB. This study not only reveals RyhB-dependent regulation of virB transcription as a novel point of control in the central regulatory circuit modulating Shigella virulence, but also highlights the versatility of RyhB in controlling bacterial gene expression. PMID:22701677

Requirements for DNA strand transfer during reverse transcription in mutant HIV-1 virions

NARCIS (Netherlands)

Berkhout, B.; van Wamel, J.; Klaver, B.

1995-01-01

Retroviruses convert their RNA genome into a DNA form by means of reverse transcription. According to the current model of reverse transcription, two strand transfer reactions are needed to synthesize a full-length DNA genome. Because reverse transcription is initiated close to the 5' end of the RNA

Higher-Order Structure in Bacterial VapBC Toxin-Antitoxin Complexes

DEFF Research Database (Denmark)

Bendtsen, Kirstine L; Brodersen, Ditlev E

2017-01-01

Toxin-antitoxin systems are widespread in the bacterial kingdom, including in pathogenic species, where they allow rapid adaptation to changing environmental conditions through selective inhibition of key cellular processes, such as DNA replication or protein translation. Under normal growth...... that allow auto-regulation of transcription by direct binding to promoter DNA. In this chapter, we review our current understanding of the structural characteristics of type II toxin-antitoxin complexes in bacterial cells, with a special emphasis on the staggering variety of higher-order architecture...... conditions, type II toxins are inhibited through tight protein-protein interaction with a cognate antitoxin protein. This toxin-antitoxin complex associates into a higher-order macromolecular structure, typically heterotetrameric or heterooctameric, exposing two DNA binding domains on the antitoxin...

Effects of chemical and biological pesticides on plant growth parameters and rhizospheric bacterial community structure in Vigna radiata.

Science.gov (United States)

Singh, Sunil; Gupta, Rashi; Sharma, Shilpi

2015-06-30

With increasing application of pesticides in agriculture, their non-target effects on soil microbial communities are critical to soil health maintenance. The present study aimed to evaluate the effects of chemical pesticides (chlorpyrifos and cypermethrin) and a biological pesticide (azadirachtin) on growth parameters and the rhizospheric bacterial community of Vigna radiata. Qualitative and quantitative analysis by PCR-denaturing gradient gel electrophoresis (DGGE) and q-PCR, respectively, of the 16S rRNA gene and transcript were performed to study the impact of these pesticides on the resident and active rhizospheric bacterial community. While plant parameters were not affected significantly by the pesticides, a shift in the bacterial community structure was observed with an adverse effect on the abundance of 16S rRNA gene and transcripts. Chlorpyrifos showed almost complete degradation toward the end of the experiment. These non-target impacts on soil ecosystems and the fact that the effects of the biopesticide mimic those of chemical pesticides raise serious concerns regarding their application in agriculture. Copyright © 2015 Elsevier B.V. All rights reserved.

A unified architecture of transcriptional regulatory elements

DEFF Research Database (Denmark)

Andersson, Robin; Sandelin, Albin Gustav; Danko, Charles G.

2015-01-01

Gene expression is precisely controlled in time and space through the integration of signals that act at gene promoters and gene-distal enhancers. Classically, promoters and enhancers are considered separate classes of regulatory elements, often distinguished by histone modifications. However...... and enhancers are considered a single class of functional element, with a unified architecture for transcription initiation. The context of interacting regulatory elements and the surrounding sequences determine local transcriptional output as well as the enhancer and promoter activities of individual elements....

Copine-I: Modulator of NF-kappa B Transcription and Prostate Cancer Survival

National Research Council Canada - National Science Library

Mayo, Marty W; Creutz, Carl

2008-01-01

The purpose of our studies is to elucidate how Copine-I antagonizes NF-.B transcription. Nuclear factor-.B (NF-.B) is a dynamic transcription factor that regulates important biological processes involved in cancer initiation and progression...

Bacterial community succession in pine-wood decomposition

Directory of Open Access Journals (Sweden)

Anna eKielak

2016-03-01

Full Text Available Though bacteria and fungi are common inhabitants of decaying wood, little is known about the relationship between bacterial and fungal community dynamics during natural wood decay. Based on previous studies involving inoculated wood blocks, strong fungal selection on bacteria abundance and community composition was expected to occur during natural wood decay. Here we focused on bacterial and fungal community compositions in pine wood samples collected from dead trees in different stages of decomposition. We showed that bacterial communities undergo less drastic changes than fungal communities during wood decay. Furthermore, we found that bacterial community assembly was a stochastic process at initial stage of wood decay and became more deterministic in later stages, likely due to environmental factors. Moreover, composition of bacterial communities did not respond to the changes in the major fungal species present in the wood but rather to the stage of decay reflected by the wood density. We concluded that the shifts in the bacterial communities were a result of the changes in wood properties during decomposition and largely independent of the composition of the wood-decaying fungal communities.

Transcriptional and posttranscriptional regulation of cyanobacterial photosynthesis.

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Wilde, Annegret; Hihara, Yukako

2016-03-01

Cyanobacteria are well established model organisms for the study of oxygenic photosynthesis, nitrogen metabolism, toxin biosynthesis, and salt acclimation. However, in comparison to other model bacteria little is known about regulatory networks, which allow cyanobacteria to acclimate to changing environmental conditions. The current work has begun to illuminate how transcription factors modulate expression of different photosynthetic regulons. During the past few years, the research on other regulatory principles like RNA-based regulation showed the importance of non-protein regulators for bacterial lifestyle. Investigations on modulation of photosynthetic components should elucidate the contributions of all factors within the context of a larger regulatory network. Here, we focus on regulation of photosynthetic processes including transcriptional and posttranscriptional mechanisms, citing examples from a limited number of cyanobacterial species. Though, the general idea holds true for most species, important differences exist between various organisms, illustrating diversity of acclimation strategies in the very heterogeneous cyanobacterial clade. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of uv irradiation on lambda DNA transcription

Energy Technology Data Exchange (ETDEWEB)

Ranade, S S [Cancer Research Inst., Bombay (India)

1977-05-01

The effect of uv irradiation of template DNA has been studied in vitro in the E.coli RNA polymerase system with native and uv treated lambda DNA. Lambda DNA was more susceptible to uv than was calf-thymus DNA, yet a residual activity was observed at a uv dose of 0.5 x 10/sup 4/ erg/mm/sup 2/. From the kinetic analysis of the reaction and the incorporation of lambda /sup 32/P-labelled nucleoside triphosphates, it seems reasonable to conclude that uv irradiation probably did not affect the DNA initiation sites, recognizable by RNA polymerase. The transcription products made with uv irradiated lambda DNA were asymmetrical, and hybridized to the right half (R) and the left half (L) of lambda DNA with the ratio of R/L=4/1, and they showed a lower hybridizability than the transcripts with native lambda DNA. The initiation sites recognizable by RNA polymerase seemed to be the same on both native and uv irradiated lambda DNA, though the transcription of uv treated lambda DNA appeared to terminate with rather short RNA chains.

Inter-replicon Gene Flow Contributes to Transcriptional Integration in the Sinorhizobium meliloti Multipartite Genome.

Science.gov (United States)

diCenzo, George C; Wellappili, Deelaka; Golding, G Brian; Finan, Turlough M

2018-05-04

Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT). Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT. The Sinorhizobium meliloti genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid consisting primarily of recently acquired genes. Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid. Removal of the pSymA megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ∼8% of chromosomal genes and ∼4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome. No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched. Down-regulated genes were enriched in motility and sensory transduction pathways. Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions. In particularly, a chromosomal transporter was induced due to deletion of bdhA likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome. Copyright © 2018 diCenzo, et al.

Inter-replicon Gene Flow Contributes to Transcriptional Integration in the Sinorhizobium meliloti Multipartite Genome

Directory of Open Access Journals (Sweden)

George C. diCenzo

2018-05-01

Full Text Available Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT. Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT. The Sinorhizobium meliloti genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid consisting primarily of recently acquired genes. Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid. Removal of the pSymA megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ∼8% of chromosomal genes and ∼4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome. No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched. Down-regulated genes were enriched in motility and sensory transduction pathways. Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions. In particularly, a chromosomal transporter was induced due to deletion of bdhA likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome.

Genomic structure and cloning of two transcript isoforms of human Sp8.

NARCIS (Netherlands)

M.A. Milona (Maria-athina); J.E. Gough (Julie); A.J. Edgar (Alasdair)

2004-01-01

textabstractBACKGROUND: The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

Science.gov (United States)

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

A Laboratory Assessment of Factors That Affect Bacterial Adhesion to Contact Lenses

Science.gov (United States)

Dutta, Debarun; Willcox, Mark DP

2013-01-01

Adhesion of pathogenic microbes, particularly bacteria, to contact lenses is implicated in contact lens related microbial adverse events. Various in vitro conditions such as type of bacteria, the size of initial inoculum, contact lens material, nutritional content of media, and incubation period can influence bacterial adhesion to contact lenses and the current study investigated the effect of these conditions on bacterial adhesion to contact lenses. There was no significant difference in numbers of bacteria that adhered to hydrogel etafilcon A or silicone hydrogel senofilcon A contact lenses. Pseudomonas aeruginosa adhered in higher numbers compared to Staphylococcus aureus. Within a genera/species, adhesion of different bacterial strains did not differ appreciably. The size of initial inoculum, nutritional content of media, and incubation period played significant roles in bacterial adhesion to lenses. A set of in vitro assay conditions to help standardize adhesion between studies have been recommended. PMID:24833224

A Laboratory Assessment of Factors That Affect Bacterial Adhesion to Contact Lenses

Directory of Open Access Journals (Sweden)

Debarun Dutta

2013-11-01

Full Text Available Adhesion of pathogenic microbes, particularly bacteria, to contact lenses is implicated in contact lens related microbial adverse events. Various in vitro conditions such as type of bacteria, the size of initial inoculum, contact lens material, nutritional content of media, and incubation period can influence bacterial adhesion to contact lenses and the current study investigated the effect of these conditions on bacterial adhesion to contact lenses. There was no significant difference in numbers of bacteria that adhered to hydrogel etafilcon A or silicone hydrogel senofilcon A contact lenses. Pseudomonas aeruginosa adhered in higher numbers compared to Staphylococcus aureus. Within a genera/species, adhesion of different bacterial strains did not differ appreciably. The size of initial inoculum, nutritional content of media, and incubation period played significant roles in bacterial adhesion to lenses. A set of in vitro assay conditions to help standardize adhesion between studies have been recommended.

Molecular signatures in Arabidopsis thaliana in response to insect attack and bacterial infection.

Science.gov (United States)

Barah, Pankaj; Winge, Per; Kusnierczyk, Anna; Tran, Diem Hong; Bones, Atle M

2013-01-01

Under the threat of global climatic change and food shortages, it is essential to take the initiative to obtain a comprehensive understanding of common and specific defence mechanisms existing in plant systems for protection against different types of biotic invaders. We have implemented an integrated approach to analyse the overall transcriptomic reprogramming and systems-level defence responses in the model plant species Arabidopsis thaliana (A. thaliana henceforth) during insect Brevicoryne brassicae (B. brassicae henceforth) and bacterial Pseudomonas syringae pv. tomato strain DC3000 (P. syringae henceforth) attacks. The main aim of this study was to identify the attacker-specific and general defence response signatures in A. thaliana when attacked by phloem-feeding aphids or pathogenic bacteria. The obtained annotated networks of differentially expressed transcripts indicated that members of transcription factor families, such as WRKY, MYB, ERF, BHLH and bZIP, could be crucial for stress-specific defence regulation in Arabidopsis during aphid and P. syringae attack. The defence response pathways, signalling pathways and metabolic processes associated with aphid attack and P. syringae infection partially overlapped. Components of several important biosynthesis and signalling pathways, such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and glucosinolates, were differentially affected during the two the treatments. Several stress-regulated transcription factors were known to be associated with stress-inducible microRNAs. The differentially regulated gene sets included many signature transcription factors, and our co-expression analysis showed that they were also strongly co-expressed during 69 other biotic stress experiments. Defence responses and functional networks that were unique and specific to aphid or P. syringae stresses were identified. Furthermore, our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and

Molecular signatures in Arabidopsis thaliana in response to insect attack and bacterial infection.

Directory of Open Access Journals (Sweden)

Pankaj Barah

Full Text Available BACKGROUND: Under the threat of global climatic change and food shortages, it is essential to take the initiative to obtain a comprehensive understanding of common and specific defence mechanisms existing in plant systems for protection against different types of biotic invaders. We have implemented an integrated approach to analyse the overall transcriptomic reprogramming and systems-level defence responses in the model plant species Arabidopsis thaliana (A. thaliana henceforth during insect Brevicoryne brassicae (B. brassicae henceforth and bacterial Pseudomonas syringae pv. tomato strain DC3000 (P. syringae henceforth attacks. The main aim of this study was to identify the attacker-specific and general defence response signatures in A. thaliana when attacked by phloem-feeding aphids or pathogenic bacteria. RESULTS: The obtained annotated networks of differentially expressed transcripts indicated that members of transcription factor families, such as WRKY, MYB, ERF, BHLH and bZIP, could be crucial for stress-specific defence regulation in Arabidopsis during aphid and P. syringae attack. The defence response pathways, signalling pathways and metabolic processes associated with aphid attack and P. syringae infection partially overlapped. Components of several important biosynthesis and signalling pathways, such as salicylic acid (SA, jasmonic acid (JA, ethylene (ET and glucosinolates, were differentially affected during the two the treatments. Several stress-regulated transcription factors were known to be associated with stress-inducible microRNAs. The differentially regulated gene sets included many signature transcription factors, and our co-expression analysis showed that they were also strongly co-expressed during 69 other biotic stress experiments. CONCLUSIONS: Defence responses and functional networks that were unique and specific to aphid or P. syringae stresses were identified. Furthermore, our analysis revealed a probable link between

Childhood asthma after bacterial colonization of the airway in neonates

DEFF Research Database (Denmark)

Bisgaard, H.; Hermansen, M.N.; Buchvald, F.

2007-01-01

Pathological features of the airway in young children with severe recurrent wheeze suggest an association between bacterial colonization and the initiating events of early asthma. We conducted a study to investigate a possible association between bacterial colonization of the hypopharynx in asymp...... in asymptomatic neonates and later development of recurrent wheeze, asthma, and allergy during the first 5 years of life....

A Transcription and Translation Protocol for Sensitive Cross-Cultural Team Research.

Science.gov (United States)

Clark, Lauren; Birkhead, Ana Sanchez; Fernandez, Cecilia; Egger, Marlene J

2017-10-01

Assurance of transcript accuracy and quality in interview-based qualitative research is foundational for data accuracy and study validity. Based on our experience in a cross-cultural ethnographic study of women's pelvic organ prolapse, we provide practical guidance to set up step-by-step interview transcription and translation protocols for team-based research on sensitive topics. Beginning with team decisions about level of detail in transcription, completeness, and accuracy, we operationalize the process of securing vendors to deliver the required quality of transcription and translation. We also share rubrics for assessing transcript quality and the team protocol for managing transcripts (assuring consistency of format, insertion of metadata, anonymization, and file labeling conventions) and procuring an acceptable initial translation of Spanish-language interviews. Accurate, complete, and systematically constructed transcripts in both source and target languages respond to the call for more transparency and reproducibility of scientific methods.

Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

Science.gov (United States)

Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

2015-12-01

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape.

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Scarlet S Shell

2015-11-01

Full Text Available RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.

Archaeal RNA polymerase arrests transcription at DNA lesions.

Science.gov (United States)

Gehring, Alexandra M; Santangelo, Thomas J

2017-01-01

Transcription elongation is not uniform and transcription is often hindered by protein-bound factors or DNA lesions that limit translocation and impair catalysis. Despite the high degree of sequence and structural homology of the multi-subunit RNA polymerases (RNAP), substantial differences in response to DNA lesions have been reported. Archaea encode only a single RNAP with striking structural conservation with eukaryotic RNAP II (Pol II). Here, we demonstrate that the archaeal RNAP from Thermococcus kodakarensis is sensitive to a variety of DNA lesions that pause and arrest RNAP at or adjacent to the site of DNA damage. DNA damage only halts elongation when present in the template strand, and the damage often results in RNAP arresting such that the lesion would be encapsulated with the transcription elongation complex. The strand-specific halt to archaeal transcription elongation on modified templates is supportive of RNAP recognizing DNA damage and potentially initiating DNA repair through a process akin to the well-described transcription-coupled DNA repair (TCR) pathways in Bacteria and Eukarya.

Transcriptional profiling of Actinobacillus pleuropneumoniae during the acute phase of a natural infection in pigs

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Harel Josée

2010-02-01

Full Text Available Abstract Background Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. Many virulence factors are involved in the pathogenesis, namely capsular polysaccharides, RTX toxins, LPS and many iron acquisition systems. In order to identify genes that are expressed in vivo during a natural infection, we undertook transcript profiling experiments with an A. pleuropneumoniae DNA microarray, after recovery of bacterial mRNAs from serotype 5b-infected porcine lungs. AppChip2 contains 2033 PCR amplicons based on the genomic sequence of App serotype 5b strain L20, representing more than 95% of ORFs greater than 160 bp in length. Results Transcriptional profiling of A. pleuropneumoniae recovered from the lung of a pig suffering from a natural infection or following growth of the bacterial isolate in BHI medium was performed. An RNA extraction protocol combining beadbeating and hot-acid-phenol was developed in order to maximize bacterial mRNA yields and quality following total RNA extraction from lung lesions. Nearly all A. pleuropneumoniae transcripts could be detected on our microarrays, and 150 genes were deemed differentially expressed in vivo during the acute phase of the infection. Our results indicate that, for example, gene apxIVA from an operon coding for RTX toxin ApxIV is highly up-regulated in vivo, and that two genes from the operon coding for type IV fimbriae (APL_0878 and APL_0879 were also up-regulated. These transcriptional profiling data, combined with previous comparative genomic hybridizations performed by our group, revealed that 66 out of the 72 up-regulated genes are conserved amongst all serotypes and that 3 of them code for products that are predicted outer membrane proteins (genes irp and APL_0959, predicted to code for a TonB-dependent receptor and a filamentous hemagglutinin/adhesin respectively or lipoproteins (gene APL_0920. Only 4

A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

Science.gov (United States)

Calderwood, Stuart K

2016-05-26

Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

RNA search engines empower the bacterial intranet.

Science.gov (United States)

Dendooven, Tom; Luisi, Ben F

2017-08-15

RNA acts not only as an information bearer in the biogenesis of proteins from genes, but also as a regulator that participates in the control of gene expression. In bacteria, small RNA molecules (sRNAs) play controlling roles in numerous processes and help to orchestrate complex regulatory networks. Such processes include cell growth and development, response to stress and metabolic change, transcription termination, cell-to-cell communication, and the launching of programmes for host invasion. All these processes require recognition of target messenger RNAs by the sRNAs. This review summarizes recent results that have provided insights into how bacterial sRNAs are recruited into effector ribonucleoprotein complexes that can seek out and act upon target transcripts. The results hint at how sRNAs and their protein partners act as pattern-matching search engines that efficaciously regulate gene expression, by performing with specificity and speed while avoiding off-target effects. The requirements for efficient searches of RNA patterns appear to be common to all domains of life. © 2017 The Author(s).

Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB.

Science.gov (United States)

Magill, C P; Jackson, S P; Bell, S D

2001-12-14

Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

Prevalence of transcription promoters within archaeal operons and coding sequences.

Science.gov (United States)

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

Dissimilatory Metabolism of Nitrogen Oxides in Bacteria:Comparative Reconstruction of Transcriptional Networks

Energy Technology Data Exchange (ETDEWEB)

Rodionov, Dmitry A.; Dubchak, Inna L.; Arkin, Adam P.; Alm, EricJ.; Gelfand, Mikhail S.

2005-09-01

Bacterial response to nitric oxide (NO) is of major importance since NO is an obligatory intermediate of the nitrogen cycle. Transcriptional regulation of the dissimilatory nitric oxides metabolism in bacteria is diverse and involves FNR-like transcription factors HcpR, DNR and NnrR, two-component systems NarXL and NarQP, NO-responsive activator NorR, and nitrite sensitive repressor NsrR. Using comparative genomics approaches we predict DNA-binding signals for these transcriptional factors and describe corresponding regulons in available bacterial genomes. Within the FNR family of regulators, we observed a correlation of two specificity-determining amino acids and contacting bases in corresponding DNA signal. Highly conserved regulon HcpR for the hybrid cluster protein and some other redox enzymes is present in diverse anaerobic bacteria including Clostridia, Thermotogales and delta-proteobacteria. NnrR and DNR control denitrification in alpha- and beta-proteobacteria, respectively. Sigma-54-dependent NorR regulon found in some gamma- and beta-proteobacteria contains various enzymes involved in the NO detoxification. Repressor NsrR, which was previously known to control only nitrite reductase operon in Nitrosomonas spp., appears to be the master regulator of the nitric oxides metabolism not only in most gamma- and beta-proteobacteria (including well-studied species like Escherichia coli), but also in Gram-positive Bacillus and Streptomyces species. Positional analysis and comparison of regulatory regions of NO detoxification genes allows us to propose the candidate NsrR-binding signal. The most conserved member of the predicted NsrR regulon is the NO-detoxifying flavohemoglobin Hmp. In enterobacteria, the regulon includes also two nitrite-responsive loci, nipAB (hcp-hcr) and nipC(dnrN), thus confirming the identity of the effector, i.e., nitrite. The proposed NsrR regulons in Neisseria and some other species are extended to include denitrification genes. As the

Dissimilatory metabolism of nitrogen oxides in bacteria: comparative reconstruction of transcriptional networks.

Directory of Open Access Journals (Sweden)

2005-10-01

Full Text Available Bacterial response to nitric oxide (NO is of major importance since NO is an obligatory intermediate of the nitrogen cycle. Transcriptional regulation of the dissimilatory nitric oxides metabolism in bacteria is diverse and involves FNR-like transcription factors HcpR, DNR, and NnrR; two-component systems NarXL and NarQP; NO-responsive activator NorR; and nitrite-sensitive repressor NsrR. Using comparative genomics approaches, we predict DNA-binding motifs for these transcriptional factors and describe corresponding regulons in available bacterial genomes. Within the FNR family of regulators, we observed a correlation of two specificity-determining amino acids and contacting bases in corresponding DNA recognition motif. Highly conserved regulon HcpR for the hybrid cluster protein and some other redox enzymes is present in diverse anaerobic bacteria, including Clostridia, Thermotogales, and delta-proteobacteria. NnrR and DNR control denitrification in alpha- and beta-proteobacteria, respectively. Sigma-54-dependent NorR regulon found in some gamma- and beta-proteobacteria contains various enzymes involved in the NO detoxification. Repressor NsrR, which was previously known to control only nitrite reductase operon in Nitrosomonas spp., appears to be the master regulator of the nitric oxides' metabolism, not only in most gamma- and beta-proteobacteria (including well-studied species such as Escherichia coli, but also in Gram-positive Bacillus and Streptomyces species. Positional analysis and comparison of regulatory regions of NO detoxification genes allows us to propose the candidate NsrR-binding motif. The most conserved member of the predicted NsrR regulon is the NO-detoxifying flavohemoglobin Hmp. In enterobacteria, the regulon also includes two nitrite-responsive loci, nipAB (hcp-hcr and nipC (dnrN, thus confirming the identity of the effector, i.e. nitrite. The proposed NsrR regulons in Neisseria and some other species are extended to include

Transcriptome of American oysters, Crassostrea virginica, in response to bacterial challenge: insights into potential mechanisms of disease resistance.

Science.gov (United States)

McDowell, Ian C; Nikapitiya, Chamilani; Aguiar, Derek; Lane, Christopher E; Istrail, Sorin; Gomez-Chiarri, Marta

2014-01-01

The American oyster Crassostrea virginica, an ecologically and economically important estuarine organism, can suffer high mortalities in areas in the Northeast United States due to Roseovarius Oyster Disease (ROD), caused by the gram-negative bacterial pathogen Roseovarius crassostreae. The goals of this research were to provide insights into: 1) the responses of American oysters to R. crassostreae, and 2) potential mechanisms of resistance or susceptibility to ROD. The responses of oysters to bacterial challenge were characterized by exposing oysters from ROD-resistant and susceptible families to R. crassostreae, followed by high-throughput sequencing of cDNA samples from various timepoints after disease challenge. Sequence data was assembled into a reference transcriptome and analyzed through differential gene expression and functional enrichment to uncover genes and processes potentially involved in responses to ROD in the American oyster. While susceptible oysters experienced constant levels of mortality when challenged with R. crassostreae, resistant oysters showed levels of mortality similar to non-challenged oysters. Oysters exposed to R. crassostreae showed differential expression of transcripts involved in immune recognition, signaling, protease inhibition, detoxification, and apoptosis. Transcripts involved in metabolism were enriched in susceptible oysters, suggesting that bacterial infection places a large metabolic demand on these oysters. Transcripts differentially expressed in resistant oysters in response to infection included the immune modulators IL-17 and arginase, as well as several genes involved in extracellular matrix remodeling. The identification of potential genes and processes responsible for defense against R. crassostreae in the American oyster provides insights into potential mechanisms of disease resistance.

Temperate bacterial viruses as double-edged swords in bacterial warfare.

Science.gov (United States)

Gama, João Alves; Reis, Ana Maria; Domingues, Iolanda; Mendes-Soares, Helena; Matos, Ana Margarida; Dionisio, Francisco

2013-01-01

It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a "replicating toxin". However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails.

Temperate bacterial viruses as double-edged swords in bacterial warfare.

Directory of Open Access Journals (Sweden)

João Alves Gama

Full Text Available It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a "replicating toxin". However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails.

A comprehensive analysis of gene expression changes provoked by bacterial and fungal infection in C. elegans.

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Ilka Engelmann

Full Text Available While Caenorhabditis elegans specifically responds to infection by the up-regulation of certain genes, distinct pathogens trigger the expression of a common set of genes. We applied new methods to conduct a comprehensive and comparative study of the transcriptional response of C. elegans to bacterial and fungal infection. Using tiling arrays and/or RNA-sequencing, we have characterized the genome-wide transcriptional changes that underlie the host's response to infection by three bacterial (Serratia marcescens, Enterococcus faecalis and otorhabdus luminescens and two fungal pathogens (Drechmeria coniospora and Harposporium sp.. We developed a flexible tool, the WormBase Converter (available at http://wormbasemanager.sourceforge.net/, to allow cross-study comparisons. The new data sets provided more extensive lists of differentially regulated genes than previous studies. Annotation analysis confirmed that genes commonly up-regulated by bacterial infections are related to stress responses. We found substantial overlaps between the genes regulated upon intestinal infection by the bacterial pathogens and Harposporium, and between those regulated by Harposporium and D. coniospora, which infects the epidermis. Among the fungus-regulated genes, there was a significant bias towards genes that are evolving rapidly and potentially encode small proteins. The results obtained using new methods reveal that the response to infection in C. elegans is determined by the nature of the pathogen, the site of infection and the physiological imbalance provoked by infection. They form the basis for future functional dissection of innate immune signaling. Finally, we also propose alternative methods to identify differentially regulated genes that take into account the greater variability in lowly expressed genes.

HIF-1α is essential for effective PMN bacterial killing, antimicrobial peptide production and apoptosis in Pseudomonas aeruginosa keratitis.

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Elizabeth A Berger

Full Text Available Hypoxia-inducible factor (HIF-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals mice after ocular infection with Pseudomonas (P. aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN, the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG, the role of HIF-1α was assessed in P. aeruginosa-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.

The Mediator complex and transcription regulation

Science.gov (United States)

Poss, Zachary C.; Ebmeier, Christopher C.

2013-01-01

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress.

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Li Wang

2016-12-01

Full Text Available Both iron starvation and excess are detrimental to cellular life, especially for animal and plant pathogens since they always live in iron-limited environments produced by host immune responses. However, how organisms sense and respond to iron is incompletely understood. Herein, we reveal that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, VgrS (also named ColS is a membrane-bound receptor histidine kinase that senses extracytoplasmic iron limitation in the periplasm, while its cognate response regulator, VgrR (ColR, detects intracellular iron excess. Under iron-depleted conditions, dissociation of Fe3+ from the periplasmic sensor region of VgrS activates the VgrS autophosphorylation and subsequent phosphotransfer to VgrR, an OmpR-family transcription factor that regulates bacterial responses to take up iron. VgrR-VgrS regulon and the consensus DNA binding motif of the transcription factor VgrR were dissected by comparative proteomic and ChIP-seq analyses, which revealed that in reacting to iron-depleted environments, VgrR directly or indirectly controls the expressions of hundreds of genes that are involved in various physiological cascades, especially those associated with iron-uptake. Among them, we demonstrated that the phosphorylated VgrR tightly represses the transcription of a special TonB-dependent receptor gene, tdvA. This regulation is a critical prerequisite for efficient iron uptake and bacterial virulence since activation of tdvA transcription is detrimental to these processes. When the intracellular iron accumulates, the VgrR-Fe2+ interaction dissociates not only the binding between VgrR and the tdvA promoter, but also the interaction between VgrR and VgrS. This relieves the repression in tdvA transcription to impede continuous iron uptake and avoids possible toxic effects of excessive iron accumulation. Our results revealed a signaling system that directly senses both extracytoplasmic and intracellular

Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

Science.gov (United States)

Sahu, Geetaram; Farley, Kalamo; El-Hage, Nazira; Aiamkitsumrit, Benjamas; Fassnacht, Ryan; Kashanchi, Fatah; Ochem, Alex; Simon, Gary L.; Karn, Jonathan; Hauser, Kurt F.; Tyagi, Mudit

2015-01-01

Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-κB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-κB at 276th serine residue. These modifications enhance the interaction of NF-κB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. PMID:25980739

Dynamic usage of transcription start sites within core promoters

DEFF Research Database (Denmark)

Kawaji, Hideya; Frith, Martin C; Katayama, Shintaro

2006-01-01

BACKGROUND: Mammalian promoters do not initiate transcription at single, well defined base pairs, but rather at multiple, alternative start sites spread across a region. We previously characterized the static structures of transcription start site usage within promoters at the base pair level......, based on large-scale sequencing of transcript 5' ends. RESULTS: In the present study we begin to explore the internal dynamics of mammalian promoters, and demonstrate that start site selection within many mouse core promoters varies among tissues. We also show that this dynamic usage of start sites...... is associated with CpG islands, broad and multimodal promoter structures, and imprinting. CONCLUSION: Our results reveal a new level of biologic complexity within promoters--fine-scale regulation of transcription starting events at the base pair level. These events are likely to be related to epigenetic...

Discriminative identification of transcriptional responses of promoters and enhancers after stimulus

KAUST Repository

Kleftogiannis, Dimitrios A.

2016-10-17

Promoters and enhancers regulate the initiation of gene expression and maintenance of expression levels in spatial and temporal manner. Recent findings stemming from the Cap Analysis of Gene Expression (CAGE) demonstrate that promoters and enhancers, based on their expression profiles after stimulus, belong to different transcription response subclasses. One of the most promising biological features that might explain the difference in transcriptional response between subclasses is the local chromatin environment. We introduce a novel computational framework, PEDAL, for distinguishing effectively transcriptional profiles of promoters and enhancers using solely histone modification marks, chromatin accessibility and binding sites of transcription factors and co-activators. A case study on data from MCF-7 cell-line reveals that PEDAL can identify successfully the transcription response subclasses of promoters and enhancers from two different stimulations. Moreover, we report subsets of input markers that discriminate with minimized classification error MCF-7 promoter and enhancer transcription response subclasses. Our work provides a general computational approach for identifying effectively cell-specific and stimulation-specific promoter and enhancer transcriptional profiles, and thus, contributes to improve our understanding of transcriptional activation in human.

Simplified Method for Predicting a Functional Class of Proteins in Transcription Factor Complexes

KAUST Repository

Piatek, Marek J.

2013-07-12

Background:Initiation of transcription is essential for most of the cellular responses to environmental conditions and for cell and tissue specificity. This process is regulated through numerous proteins, their ligands and mutual interactions, as well as interactions with DNA. The key such regulatory proteins are transcription factors (TFs) and transcription co-factors (TcoFs). TcoFs are important since they modulate the transcription initiation process through interaction with TFs. In eukaryotes, transcription requires that TFs form different protein complexes with various nuclear proteins. To better understand transcription regulation, it is important to know the functional class of proteins interacting with TFs during transcription initiation. Such information is not fully available, since not all proteins that act as TFs or TcoFs are yet annotated as such, due to generally partial functional annotation of proteins. In this study we have developed a method to predict, using only sequence composition of the interacting proteins, the functional class of human TF binding partners to be (i) TF, (ii) TcoF, or (iii) other nuclear protein. This allows for complementing the annotation of the currently known pool of nuclear proteins. Since only the knowledge of protein sequences is required in addition to protein interaction, the method should be easily applicable to many species.Results:Based on experimentally validated interactions between human TFs with different TFs, TcoFs and other nuclear proteins, our two classification systems (implemented as a web-based application) achieve high accuracies in distinguishing TFs and TcoFs from other nuclear proteins, and TFs from TcoFs respectively.Conclusion:As demonstrated, given the fact that two proteins are capable of forming direct physical interactions and using only information about their sequence composition, we have developed a completely new method for predicting a functional class of TF interacting protein partners

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

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Clément Monot

2013-05-01

Full Text Available L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP first uses its endonuclease (EN to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A tail, a process known as target-primed reverse transcription (TPRT. Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

Science.gov (United States)

Monot, Clément; Kuciak, Monika; Viollet, Sébastien; Mir, Ashfaq Ali; Gabus, Caroline; Darlix, Jean-Luc; Cristofari, Gaël

2013-05-01

L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

First Exon Length Controls Active Chromatin Signatures and Transcription

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Nicole I. Bieberstein

2012-07-01

Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

CRISPR/Cas systems: new players in gene regulation and bacterial physiology

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David eWeiss

2014-04-01

Full Text Available CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP. Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2, CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes.

TSSer: an automated method to identify transcription start sites in prokaryotic genomes from differential RNA sequencing data.

Science.gov (United States)

Jorjani, Hadi; Zavolan, Mihaela

2014-04-01

Accurate identification of transcription start sites (TSSs) is an essential step in the analysis of transcription regulatory networks. In higher eukaryotes, the capped analysis of gene expression technology enabled comprehensive annotation of TSSs in genomes such as those of mice and humans. In bacteria, an equivalent approach, termed differential RNA sequencing (dRNA-seq), has recently been proposed, but the application of this approach to a large number of genomes is hindered by the paucity of computational analysis methods. With few exceptions, when the method has been used, annotation of TSSs has been largely done manually. In this work, we present a computational method called 'TSSer' that enables the automatic inference of TSSs from dRNA-seq data. The method rests on a probabilistic framework for identifying both genomic positions that are preferentially enriched in the dRNA-seq data as well as preferentially captured relative to neighboring genomic regions. Evaluating our approach for TSS calling on several publicly available datasets, we find that TSSer achieves high consistency with the curated lists of annotated TSSs, but identifies many additional TSSs. Therefore, TSSer can accelerate genome-wide identification of TSSs in bacterial genomes and can aid in further characterization of bacterial transcription regulatory networks. TSSer is freely available under GPL license at http://www.clipz.unibas.ch/TSSer/index.php

The MogR Transcriptional Repressor Regulates Nonhierarchal Expression of Flagellar Motility Genes and Virulence in Listeria monocytogenes.

Directory of Open Access Journals (Sweden)

2006-04-01

Full Text Available Flagella are surface structures critical for motility and virulence of many bacterial species. In Listeria monocytogenes, MogR tightly represses expression of flagellin (FlaA during extracellular growth at 37 degrees C and during intracellular infection. MogR is also required for full virulence in a murine model of infection. Using in vitro and in vivo infection models, we determined that the severe virulence defect of MogR-negative bacteria is due to overexpression of FlaA. Specifically, overproduction of FlaA in MogR-negative bacteria caused pleiotropic defects in bacterial division (chaining phenotype, intracellular spread, and virulence in mice. DNA binding and microarray analyses revealed that MogR represses transcription of all known flagellar motility genes by binding directly to a minimum of two TTTT-N(5-AAAA recognition sites positioned within promoter regions such that RNA polymerase binding is occluded. Analysis of MogR protein levels demonstrated that modulation of MogR repression activity confers the temperature-specificity to flagellar motility gene expression. Epistasis analysis revealed that MogR repression of transcription is antagonized in a temperature-dependent manner by the DegU response regulator and that DegU further regulates FlaA levels through a posttranscriptional mechanism. These studies provide the first known example to our knowledge of a transcriptional repressor functioning as a master regulator controlling nonhierarchal expression of flagellar motility genes.

The logic of communication: roles for mobile transcription factors in plants.

Science.gov (United States)

Long, Yuchen; Scheres, Ben; Blilou, Ikram

2015-02-01

Mobile transcription factors play many roles in plant development. Here, we compare the use of mobile transcription factors as signals with some canonical signal transduction processes in prokaryotes and eukaryotes. After an initial survey, we focus on the SHORT-ROOT pathway in Arabidopsis roots to show that, despite the simplicity of the concept of mobile transcription factor signalling, many lines of evidence reveal a surprising complexity in control mechanisms linked to this process. We argue that these controls bestow precision, robustness, and versatility on mobile transcription factor signalling. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Synthesis of in vitro Co1E1 transcripts with 5'-terminal ribonucleotides that exhibit noncomplementarity with the DNA template

International Nuclear Information System (INIS)

Parker, R.C.

1986-01-01

A region that forms the S1 nuclease site in Co1E1 DNA is shown to code for an in vitro transcript, called S1 RNA-B, which contains a 5'-terminal GTP residue that exhibits noncomplementarity with the template's DNA sequence. The synthesis of S1 RNA-B initiates four bases upstream from the start point for S1 RNA-C. The initial four bases in S1 RNA-B and S1 RNA-C are identical. The relative synthesis of S1 RNA-B to S1 RNA-C is sensitive to the concentration of GTP, a substrate that is required for elongation past the +4 position in S1 RNA-C. Dinucleotides that are expected to only initiate synthesis of S1 RNA-C yield two transcripts that appear to initiate from the S1 RNA-C and S1 RNA-B start sites. In vitro studies involving other Co1E1 transcripts, RNA-B and RNA-C, provide similar observations concerning the noncomplementary initiation phenomenon. A model involving transcriptional slippage is suggested to explain the noncomplementary initiation phenomenon. The model proposes that the cycling reaction of Escherichia coli RNA polymerase produces tetranucleotides that are transposed to nearby upstream sequences for priming transcription

Dispersal networks for enhancing bacterial degradation in heterogeneous environments

International Nuclear Information System (INIS)

Banitz, Thomas; Wick, Lukas Y.; Fetzer, Ingo; Frank, Karin; Harms, Hauke; Johst, Karin

2011-01-01

Successful biodegradation of organic soil pollutants depends on their bioavailability to catabolically active microorganisms. In particular, environmental heterogeneities often limit bacterial access to pollutants. Experimental and modelling studies revealed that fungal networks can facilitate bacterial dispersal and may thereby improve pollutant bioavailability. Here, we investigate the influence of such bacterial dispersal networks on biodegradation performance under spatially heterogeneous abiotic conditions using a process-based simulation model. To match typical situations in polluted soils, two types of abiotic conditions are studied: heterogeneous bacterial dispersal conditions and heterogeneous initial resource distributions. The model predicts that networks facilitating bacterial dispersal can enhance biodegradation performance for a wide range of these conditions. Additionally, the time horizon over which this performance is assessed and the network's spatial configuration are key factors determining the degree of biodegradation improvement. Our results support the idea of stimulating the establishment of fungal mycelia for enhanced bioremediation of polluted soils. - Highlights: → Bacterial dispersal networks can considerably improve biodegradation performance. → They facilitate bacterial access to dispersal-limited areas and remote resources. → Abiotic conditions, time horizon and network structure govern the improvements. → Stimulating the establishment of fungal mycelia promises enhanced soil remediation. - Simulation modelling demonstrates that fungus-mediated bacterial dispersal can considerably improve the bioavailability of organic pollutants under spatially heterogeneous abiotic conditions typical for water-unsaturated soils.

Dispersal networks for enhancing bacterial degradation in heterogeneous environments

Energy Technology Data Exchange (ETDEWEB)

Banitz, Thomas, E-mail: thomas.banitz@ufz.de [Department of Ecological Modelling, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany); Wick, Lukas Y.; Fetzer, Ingo [Department of Environmental Microbiology, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany); Frank, Karin [Department of Ecological Modelling, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany); Harms, Hauke [Department of Environmental Microbiology, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany); Johst, Karin [Department of Ecological Modelling, UFZ - Helmholtz Centre for Environmental Research, Permoserstr. 15, 04318 Leipzig (Germany)

2011-10-15

Successful biodegradation of organic soil pollutants depends on their bioavailability to catabolically active microorganisms. In particular, environmental heterogeneities often limit bacterial access to pollutants. Experimental and modelling studies revealed that fungal networks can facilitate bacterial dispersal and may thereby improve pollutant bioavailability. Here, we investigate the influence of such bacterial dispersal networks on biodegradation performance under spatially heterogeneous abiotic conditions using a process-based simulation model. To match typical situations in polluted soils, two types of abiotic conditions are studied: heterogeneous bacterial dispersal conditions and heterogeneous initial resource distributions. The model predicts that networks facilitating bacterial dispersal can enhance biodegradation performance for a wide range of these conditions. Additionally, the time horizon over which this performance is assessed and the network's spatial configuration are key factors determining the degree of biodegradation improvement. Our results support the idea of stimulating the establishment of fungal mycelia for enhanced bioremediation of polluted soils. - Highlights: > Bacterial dispersal networks can considerably improve biodegradation performance. > They facilitate bacterial access to dispersal-limited areas and remote resources. > Abiotic conditions, time horizon and network structure govern the improvements. > Stimulating the establishment of fungal mycelia promises enhanced soil remediation. - Simulation modelling demonstrates that fungus-mediated bacterial dispersal can considerably improve the bioavailability of organic pollutants under spatially heterogeneous abiotic conditions typical for water-unsaturated soils.

Roles of Arabidopsis WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens

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Fan Baofang

2008-06-01

Full Text Available Abstract Background Plant WRKY DNA-binding transcription factors are involved in plant responses to biotic and abiotic responses. It has been previously shown that Arabidopsis WRKY3 and WRKY4, which encode two structurally similar WRKY transcription factors, are induced by pathogen infection and salicylic acid (SA. However, the role of the two WRKY transcription factors in plant disease resistance has not been directly analyzed. Results Both WRKY3 and WRKY4 are nuclear-localized and specifically recognize the TTGACC W-box sequences in vitro. Expression of WRKY3 and WRKY4 was induced rapidly by stress conditions generated by liquid infiltration or spraying. Stress-induced expression of WRKY4 was further elevated by pathogen infection and SA treatment. To determine directly their role in plant disease resistance, we have isolated T-DNA insertion mutants and generated transgenic overexpression lines for WRKY3 and WRKY4. Both the loss-of-function mutants and transgenic overexpression lines were examined for responses to the biotrophic bacterial pathogen Pseudomonas syringae and the necrotrophic fungal pathogen Botrytis cinerea. The wrky3 and wrky4 single and double mutants exhibited more severe disease symptoms and support higher fungal growth than wild-type plants after Botrytis infection. Although disruption of WRKY3 and WRKY4 did not have a major effect on plant response to P. syringae, overexpression of WRKY4 greatly enhanced plant susceptibility to the bacterial pathogen and suppressed pathogen-induced PR1 gene expression. Conclusion The nuclear localization and sequence-specific DNA-binding activity support that WRKY3 and WRKY4 function as transcription factors. Functional analysis based on T-DNA insertion mutants and transgenic overexpression lines indicates that WRKY3 and WRKY4 have a positive role in plant resistance to necrotrophic pathogens and WRKY4 has a negative effect on plant resistance to biotrophic pathogens.

Biomarker-based classification of bacterial and fungal whole-blood infections in a genome-wide expression study

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Andreas eDix

2015-03-01

Full Text Available Sepsis is a clinical syndrome that can be caused by bacteria or fungi. Early knowledge on the nature of the causative agent is a prerequisite for targeted anti-microbial therapy. Besides currently used detection methods like blood culture and PCR-based assays, the analysis of the transcriptional response of the host to infecting organisms holds great promise. In this study, we aim to examine the transcriptional footprint of infections caused by the bacterial pathogens Staphylococcus aureus and Escherichia coli and the fungal pathogens Candida albicans and Aspergillus fumigatus in a human whole-blood model. Moreover, we use the expression information to build a random forest classifier to classify if a sample contains a bacterial, fungal, or mock-infection. After normalizing the transcription intensities using stably expressed reference genes, we filtered the gene set for biomarkers of bacterial or fungal blood infections. This selection is based on differential expression and an additional gene relevance measure. In this way, we identified 38 biomarker genes, including IL6, SOCS3, and IRG1 which were already associated to sepsis by other studies. Using these genes, we trained the classifier and assessed its performance. It yielded a 96% accuracy (sensitivities >93%, specificities >97% for a 10-fold stratified cross-validation and a 92% accuracy (sensitivities and specificities >83% for an additional test dataset comprising Cryptococcus neoformans infections. Furthermore, the classifier is robust to Gaussian noise, indicating correct class predictions on datasets of new species. In conclusion, this genome-wide approach demonstrates an effective feature selection process in combination with the construction of a well-performing classification model. Further analyses of genes with pathogen-dependent expression patterns can provide insights into the systemic host responses, which may lead to new anti-microbial therapeutic advances.

TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions

KAUST Repository

Schmeier, Sebastian; Alam, Tanvir; Essack, Magbubah; Bajic, Vladimir B.

2016-01-01

Transcription factors (TFs) play a pivotal role in transcriptional regulation, making them crucial for cell survival and important biological functions. For the regulation of transcription, interactions of different regulatory proteins known as transcription co-factors (TcoFs) and TFs are essential in forming necessary protein complexes. Although TcoFs themselves do not bind DNA directly, their influence on transcriptional regulation and initiation, although indirect, has been shown to be significant, with the functionality of TFs strongly influenced by the presence of TcoFs. In the TcoF-DB v2 database, we collect information on TcoFs. In this article, we describe updates and improvements implemented in TcoF-DB v2. TcoF-DB v2 provides several new features that enables exploration of the roles of TcoFs. The content of the database has significantly expanded, and is enriched with information from Gene Ontology, biological pathways, diseases and molecular signatures. TcoF-DB v2 now includes many more TFs; has substantially increased the number of human TcoFs to 958, and now includes information on mouse (418 new TcoFs). TcoF-DB v2 enables the exploration of information on TcoFs and allows investigations into their influence on transcriptional regulation in humans and mice. TcoF-DB v2 can be accessed at http://tcofdb.org/.

TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions

KAUST Repository

Schmeier, Sebastian

2016-10-17

Transcription factors (TFs) play a pivotal role in transcriptional regulation, making them crucial for cell survival and important biological functions. For the regulation of transcription, interactions of different regulatory proteins known as transcription co-factors (TcoFs) and TFs are essential in forming necessary protein complexes. Although TcoFs themselves do not bind DNA directly, their influence on transcriptional regulation and initiation, although indirect, has been shown to be significant, with the functionality of TFs strongly influenced by the presence of TcoFs. In the TcoF-DB v2 database, we collect information on TcoFs. In this article, we describe updates and improvements implemented in TcoF-DB v2. TcoF-DB v2 provides several new features that enables exploration of the roles of TcoFs. The content of the database has significantly expanded, and is enriched with information from Gene Ontology, biological pathways, diseases and molecular signatures. TcoF-DB v2 now includes many more TFs; has substantially increased the number of human TcoFs to 958, and now includes information on mouse (418 new TcoFs). TcoF-DB v2 enables the exploration of information on TcoFs and allows investigations into their influence on transcriptional regulation in humans and mice. TcoF-DB v2 can be accessed at http://tcofdb.org/.

Transcriptional networks in epithelial-mesenchymal transition.

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Christo Venkov

Full Text Available Epithelial-mesenchymal transition (EMT changes polarized epithelial cells into migratory phenotypes associated with loss of cell-cell adhesion molecules and cytoskeletal rearrangements. This form of plasticity is seen in mesodermal development, fibroblast formation, and cancer metastasis.Here we identify prominent transcriptional networks active during three time points of this transitional process, as epithelial cells become fibroblasts. DNA microarray in cultured epithelia undergoing EMT, validated in vivo, were used to detect various patterns of gene expression. In particular, the promoter sequences of differentially expressed genes and their transcription factors were analyzed to identify potential binding sites and partners. The four most frequent cis-regulatory elements (CREs in up-regulated genes were SRY, FTS-1, Evi-1, and GC-Box, and RNA inhibition of the four transcription factors, Atf2, Klf10, Sox11, and SP1, most frequently binding these CREs, establish their importance in the initiation and propagation of EMT. Oligonucleotides that block the most frequent CREs restrain EMT at early and intermediate stages through apoptosis of the cells.Our results identify new transcriptional interactions with high frequency CREs that modulate the stability of cellular plasticity, and may serve as targets for modulating these transitional states in fibroblasts.

Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

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Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

2016-01-01

Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

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Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

2016-04-26

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

Bistable Bacterial Growth Rate in Response to Antibiotics with Low Membrane Permeability

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Elf, Johan; Nilsson, Karin; Tenson, Tanel; Ehrenberg, Måns

2006-12-01

We demonstrate that growth rate bistability for bacterial cells growing exponentially at a fixed external antibiotic concentration can emerge when the cell wall permeability for the drug is low and the growth rate sensitivity to the intracellular drug concentration is high. Under such conditions, an initially high growth rate can remain high, due to dilution of the intracellular drug concentration by rapid cell volume increase, while an initially low growth rate can remain low, due to slow cell volume increase and insignificant drug dilution. Our findings have implications for the testing of novel antibiotics on growing bacterial strains.

A third mode of surface‐associated growth: immobilization of Salmonella enterica serovar Typhimurium modulates the RpoS‐directed transcriptional programme

DEFF Research Database (Denmark)

Knudsen, Gitte Maegaard; Nielsen, Maj‐Britt; Grassby, Terri

2012-01-01

environments in both food products and the GI tract. This immobilized mode of growth has not been widely studied. To develop our understanding of the effects of immobilization upon a food‐borne bacterial pathogen, we used the IFR Gel Cassette model. The transcriptional programme and metabolomic profile...

Elucidation of Operon Structures across Closely Related Bacterial Genomes

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Li, Guojun

2014-01-01

About half of the protein-coding genes in prokaryotic genomes are organized into operons to facilitate co-regulation during transcription. With the evolution of genomes, operon structures are undergoing changes which could coordinate diverse gene expression patterns in response to various stimuli during the life cycle of a bacterial cell. Here we developed a graph-based model to elucidate the diversity of operon structures across a set of closely related bacterial genomes. In the constructed graph, each node represents one orthologous gene group (OGG) and a pair of nodes will be connected if any two genes, from the corresponding two OGGs respectively, are located in the same operon as immediate neighbors in any of the considered genomes. Through identifying the connected components in the above graph, we found that genes in a connected component are likely to be functionally related and these identified components tend to form treelike topology, such as paths and stars, corresponding to different biological mechanisms in transcriptional regulation as follows. Specifically, (i) a path-structure component integrates genes encoding a protein complex, such as ribosome; and (ii) a star-structure component not only groups related genes together, but also reflects the key functional roles of the central node of this component, such as the ABC transporter with a transporter permease and substrate-binding proteins surrounding it. Most interestingly, the genes from organisms with highly diverse living environments, i.e., biomass degraders and animal pathogens of clostridia in our study, can be clearly classified into different topological groups on some connected components. PMID:24959722

Persistence drives gene clustering in bacterial genomes

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Rocha Eduardo PC

2008-01-01

Full Text Available Abstract Background Gene clustering plays an important role in the organization of the bacterial chromosome and several mechanisms have been proposed to explain its extent. However, the controversies raised about the validity of each of these mechanisms remind us that the cause of this gene organization remains an open question. Models proposed to explain clustering did not take into account the function of the gene products nor the likely presence or absence of a given gene in a genome. However, genomes harbor two very different categories of genes: those genes present in a majority of organisms – persistent genes – and those present in very few organisms – rare genes. Results We show that two classes of genes are significantly clustered in bacterial genomes: the highly persistent and the rare genes. The clustering of rare genes is readily explained by the selfish operon theory. Yet, genes persistently present in bacterial genomes are also clustered and we try to understand why. We propose a model accounting specifically for such clustering, and show that indispensability in a genome with frequent gene deletion and insertion leads to the transient clustering of these genes. The model describes how clusters are created via the gene flux that continuously introduces new genes while deleting others. We then test if known selective processes, such as co-transcription, physical interaction or functional neighborhood, account for the stabilization of these clusters. Conclusion We show that the strong selective pressure acting on the function of persistent genes, in a permanent state of flux of genes in bacterial genomes, maintaining their size fairly constant, that drives persistent genes clustering. A further selective stabilization process might contribute to maintaining the clustering.

Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

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Reid-Bayliss, Kate S; Loeb, Lawrence A

2017-08-29

Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

Polyphenol Compound as a Transcription Factor Inhibitor

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Seyeon Park

2015-10-01

Full Text Available A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1, c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB and β-catenin/T cell factor (Tcf.

Polyphenol Compound as a Transcription Factor Inhibitor.

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Park, Seyeon

2015-10-30

A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)).

[Participation of the piRNA pathway in recruiting a component of RNA polymerase I transcription initiation complex to germline cell nucleoli].

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Fefelova, E A; Stolyarenko, A D; Yakushev, E Y; Gvozdev, V A; Klenov, M S

2017-01-01

Proteins of the Piwi family and short Piwi-interacting RNAs (piRNAs) ensure the protection of the genome from transposable elements. We have previously shown that nuclear Piwi protein tends to concentrate in the nucleoli of the cells of Drosophila melanogaster ovaries. It could be hypothesized that the function of Piwi in the nucleolus is associated with the repression of R1 and R2 retrotransposons inserted into the rDNA cluster. Here, we show that Piwi participates in recruiting Udd protein to nucleoli. Udd is a component of the conserved Selectivity Factor I-like (SL1-like) complex, which is required for transcription initiation by RNA polymerase I. We found that Udd localization depends on Piwi in germline cells, but not in somatic cells of the ovaries. In contrast, knockdowns of the SL1-like components (Udd or TAF1b) do not disrupt Piwi localization. We also observed that the absence of Udd or TAF1b in germline cells, as well as the impairment of Piwi nuclear localization lead to the accumulation of late stage egg chambers in the ovaries, which could be explained by reduced rRNA transcription. These results allow us to propose for the first time a role for Piwi in the nucleolus that is not directly associated with transposable element repression.

The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation.

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Henry, Romain; Bruneau, Emmanuelle; Gardan, Rozenn; Bertin, Stéphane; Fleuchot, Betty; Decaris, Bernard; Leblond-Bourget, Nathalie

2011-10-07

Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation

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Bertin Stéphane

2011-10-01

Full Text Available Abstract Background Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. Results In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. Conclusions These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

Characterization of herpes simplex virus 2 primary microRNA Transcript regulation.

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Tang, Shuang; Bosch-Marce, Marta; Patel, Amita; Margolis, Todd P; Krause, Philip R

2015-05-01

In order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans-activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation. The HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5' ends and evaluation of ICP4 response. These

Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA

International Nuclear Information System (INIS)

Vassylyeva, Marina N.; Svetlov, Vladimir; Klyuyev, Sergiy; Devedjiev, Yancho D.; Artsimovitch, Irina; Vassylyev, Dmitry G.

2006-01-01

The E. coli transcriptional regulator RfaH was cloned, expressed, purified and crystallized and the complex of RfaH with its target DNA oligonucleotide was cocrystallized. Complete diffraction data sets were collected for the apo protein and its nucleic acid complex at 2.4 and at 1.6 Å resolution, respectively. The bacterial transcriptional factor and virulence regulator RfaH binds to rapidly moving transcription elongation complexes through specific interactions with the exposed segment of the non-template DNA strand. To elucidate this unusual mechanism of recruitment, determination of the three-dimensional structure of RfaH and its complex with DNA was initiated. To this end, the Escherichia coli rfaH gene was cloned and expressed. The purified protein was crystallized by the sitting-drop vapor-diffusion technique. The space group was P6 1 22 or P6 5 22, with unit-cell parameters a = b = 45.46, c = 599.93 Å. A complex of RfaH and a nine-nucleotide oligodeoxyribonucleotide was crystallized by the same technique, but under different crystallization conditions, yielding crystals that belonged to space group P1 (unit-cell parameters a = 36.79, b = 44.01, c = 62.37 Å, α = 80.62, β = 75.37, γ = 75.41°). Complete diffraction data sets were collected for RfaH and its complex with DNA at 2.4 and 1.6 Å resolution, respectively. Crystals of selenomethionine-labeled proteins in both crystal forms were obtained by cross-microseeding using the native microcrystals. The structure determination of RfaH and its complex with DNA is in progress

Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP.

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Kim, Donghyuk; Seo, Sang Woo; Gao, Ye; Nam, Hojung; Guzman, Gabriela I; Cho, Byung-Kwan; Palsson, Bernhard O

2018-04-06

Two major transcriptional regulators of carbon metabolism in bacteria are Cra and CRP. CRP is considered to be the main mediator of catabolite repression. Unlike for CRP, in vivo DNA binding information of Cra is scarce. Here we generate and integrate ChIP-exo and RNA-seq data to identify 39 binding sites for Cra and 97 regulon genes that are regulated by Cra in Escherichia coli. An integrated metabolic-regulatory network was formed by including experimentally-derived regulatory information and a genome-scale metabolic network reconstruction. Applying analysis methods of systems biology to this integrated network showed that Cra enables optimal bacterial growth on poor carbon sources by redirecting and repressing glycolysis flux, by activating the glyoxylate shunt pathway, and by activating the respiratory pathway. In these regulatory mechanisms, the overriding regulatory activity of Cra over CRP is fundamental. Thus, elucidation of interacting transcriptional regulation of core carbon metabolism in bacteria by two key transcription factors was possible by combining genome-wide experimental measurement and simulation with a genome-scale metabolic model.

Influence of the nano-micro structure of the surface on bacterial adhesion

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Carolina Díaz

2007-03-01

Full Text Available Biomaterials failures are frequently associated to the formation of bacterial biofilms on the surface. The aim of this work is to study the adhesion of non motile bacteria streptococci consortium and motile Pseudomonas fluorescens. Substrates with micro and nanopatterned topography were used. The influence of surface characteristics on bacterial adhesion was investigated using optical and epifluorescence microscopy, scanning electron microscopy (SEM and atomic force microscopy (AFM. Results showed an important influence of the substratum nature. On microrough surfaces, initial bacterial adhesion was less significant than on smooth surfaces. In contrast, nanopatterned samples showed more bacterial attachment than the smooth control. It was also noted a remarkable difference in morphology, orientation and distribution of bacteria between the smooth and the nanostructured substrate. The results show the important effect of substratum nature and topography on bacterial adhesion which depended on the relation between roughness characteristics dimensions and bacterial size.

Acute Sleep Deprivation Enhances Post-Infection Sleep and Promotes Survival during Bacterial Infection in Drosophila

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Kuo, Tzu-Hsing; Williams, Julie A.

2014-01-01

Study Objectives: Sleep is known to increase as an acute response to infection. However, the function of this behavioral response in host defense is not well understood. To address this problem, we evaluated the effect of acute sleep deprivation on post-infection sleep and immune function in Drosophila. Setting: Laboratory. Participants: Drosophila melanogaster. Methods and Results: Flies were subjected to sleep deprivation before (early DEP) or after (late DEP) bacterial infection. Relative to a non-deprived control, flies subjected to early DEP had enhanced sleep after infection as well as increased bacterial clearance and survival outcome. Flies subjected to late DEP experienced enhanced sleep following the deprivation period, and showed a modest improvement in survival outcome. Continuous DEP (early and late DEP) throughout infection also enhanced sleep later during infection and improved survival. However, improved survival in flies subjected to late or continuous DEP did not occur until after flies had experienced sleep. During infection, both early and late DEP enhanced NFκB transcriptional activity as measured by a luciferase reporter (κB-luc) in living flies. Early DEP also increased NFκB activity prior to infection. Flies that were deficient in expression of either the Relish or Dif NFκB transcription factors showed normal responses to early DEP. However, the effect of early DEP on post-infection sleep and survival was abolished in double mutants, which indicates that Relish and Dif have redundant roles in this process. Conclusions: Acute sleep deprivation elevated NFκB-dependent activity, increased post-infection sleep, and improved survival during bacterial infection. Citation: Kuo TH, Williams JA. Acute sleep deprivation enhances post-infection sleep and promotes survival during bacterial infection in Drosophila. SLEEP 2014;37(5):859-869. PMID:24790264

Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida.

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Bojanovič, Klara; D'Arrigo, Isotta; Long, Katherine S

2017-04-01

Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one

The Mediator complex: a central integrator of transcription

Science.gov (United States)

Allen, Benjamin L.; Taatjes, Dylan J.

2016-01-01

The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

Prevalent Bacterial Species and Novel Phylotypes in Advanced Noma Lesions

OpenAIRE

Paster, B. J.; Falkler, Jr., W. A.; Enwonwu, C. O.; Idigbe, E. O.; Savage, K. O.; Levanos, V. A.; Tamer, M. A.; Ericson, R. L.; Lau, C. N.; Dewhirst, F. E.

2002-01-01

The purpose of this study was to determine the bacterial diversity in advanced noma lesions using culture-independent molecular methods. 16S ribosomal DNA bacterial genes from DNA isolated from advanced noma lesions of four Nigerian children were PCR amplified with universally conserved primers and spirochetal selective primers and cloned into Escherichia coli. Partial 16S rRNA sequences of approximately 500 bases from 212 cloned inserts were used initially to determine species identity or cl...

A linguistic representation of the regulation of transcription initiation. I. An ordered array of complex symbols with distinctive features.

Science.gov (United States)

Collado-Vides, J

1993-01-01

The inadequacy of context-free grammars in the description of regulatory information contained in DNA gave the formal justification for a linguistic approach to the study of gene regulation. Based on that result, we have initiated a linguistic formalization of the regulatory arrays of 107 sigma 70 E. coli promoters. The complete sequences of promoter (Pr), operator (Op) and activator binding sites (I) have previously been identified as the smallest elements, or categories, for a combinatorial analysis of the range of transcription initiation of sigma 70 promoters. These categories are conceptually equivalent to phonemes of natural language. Several features associated with these categories are required in a complete description of regulatory arrays of promoters. We have to select the best way to describe the properties that are pertinent for the description of such regulatory regions. In this paper we define distinctive features of regulatory regions based on the following criteria: identification of subclasses of substitutable elements, simplicity, selection of the most directly related information, and distinction of one array among the whole set of promoters. Alternative ways to represent distances in between regulatory sites are discussed, permitting, together with a principle of precedence, the identification of an ordered set of complex symbols as a unique representation for a promoter and its associated regulatory sites. In the accompanying paper additional distinctive features of promoters and regulatory sites are identified.

The DNA replication checkpoint directly regulates MBF-dependent G1/S transcription.

Science.gov (United States)

Dutta, Chaitali; Patel, Prasanta K; Rosebrock, Adam; Oliva, Anna; Leatherwood, Janet; Rhind, Nicholas

2008-10-01

The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G(1)/S transcriptional program by directly regulating MBF, the G(1)/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G(1)/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G(1)/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.

Abundance of bacterial and diatom fouling on various surfaces

Digital Repository Service at National Institute of Oceanography (India)

PrabhaDevi

Abundance of bacterial and diatom fouling on aluminium, fibreglass and stainless steel were studied from Dona Paula waters of the Zuari Estuary. Both these forms were reversibly attached in large numbers to surfaces during the initial 24 hr...

Persistent Hg contamination and occurrence of Hg-methylating transcript (hgcA) downstream of a chlor-alkali plant in the Olt River (Romania).

Science.gov (United States)

Bravo, Andrea G; Loizeau, Jean-Luc; Dranguet, Perrine; Makri, Stamatina; Björn, Erik; Ungureanu, Viorel Gh; Slaveykova, Vera I; Cosio, Claudia

2016-06-01

Chlor-alkali plants using mercury (Hg) cell technology are acute point sources of Hg pollution in the aquatic environment. While there have been recent efforts to reduce the use of Hg cells, some of the emitted Hg can be transformed to neurotoxic methylmercury (MeHg). Here, we aimed (i) to study the dispersion of Hg in four reservoirs located downstream of a chlor-alkali plant along the Olt River (Romania) and (ii) to track the activity of bacterial functional genes involved in Hg methylation. Total Hg (THg) concentrations in water and sediments decreased successively from the initial reservoir to downstream reservoirs. Suspended fine size particles and seston appeared to be responsible for the transport of THg into downstream reservoirs, while macrophytes reflected the local bioavailability of Hg. The concentration and proportion of MeHg were correlated with THg, but were not correlated with bacterial activity in sediments, while the abundance of hgcA transcript correlated with organic matter and Cl(-) concentration, indicating the importance of Hg bioavailability in sediments for Hg methylation. Our data clearly highlights the importance of considering Hg contamination as a legacy pollutant since there is a high risk of continued Hg accumulation in food webs long after Hg-cell phase out.

Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli.

Directory of Open Access Journals (Sweden)

Shan Goh

Full Text Available BACKGROUND: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development. METHODOLOGY/PRINCIPAL FINDINGS: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50. When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50 values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required. CONCLUSIONS: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

Transcriptional regulation by nonclassical action of thyroid hormone

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Moeller Lars C

2011-08-01

Full Text Available Abstract Thyroid hormone (TH is essential for normal development, growth and metabolism. Its effects were thought to be principally mediated through triiodothyronine (T3, acting as a ligand for the nuclear TH receptors (TRs α and β residing on thyroid hormone response elements (TREs in the promoter of TH target genes. In this classical model of TH action, T3 binding to TRs leads to recruitment of basal transcription factors and increased transcription of TH responsive genes. Recently, the concept of TH action on gene expression has become more diverse and now includes nonclassical actions of T3 and T4: T3 has been shown to activate PI3K via the TRs, which ultimately increases transcription of certain genes, e.g. HIF-1α. Additionally, both T3 and thyroxine (T4 can bind to a membrane integrin, αvβ3, which leads to activation of the PI3K and MAPK signal transduction pathways and finally also increases gene transcription, e.g. of the FGF2 gene. Therefore, these initially nongenomic, nonclassical actions seem to serve as additional interfaces for transcriptional regulation by TH. Aim of this perspective is to summarize the genes that are currently known to be induced by nonclassical TH action and the mechanisms involved.

Transcription-based model for the induction of chromosomal exchange events by ionising radiation

International Nuclear Information System (INIS)

Radford, I.A.

2003-01-01

The mechanistic basis for chromosomal aberration formation, following exposure of mammalian cells to ionising radiation, has long been debated. Although chromosomal aberrations are probably initiated by DNA double-strand breaks (DSB), little is understood about the mechanisms that generate and modulate DNA rearrangement. Based on results from our laboratory and data from the literature, a novel model of chromosomal aberration formation has been suggested (Radford 2002). The basic postulates of this model are that: (1) DSB, primarily those involving multiple individual damage sites (i.e. complex DSB), are the critical initiating lesion; (2) only those DSB occurring in transcription units that are associated with transcription 'factories' (complexes containing multiple transcription units) induce chromosomal exchange events; (3) such DSB are brought into contact with a DNA topoisomerase I molecule through RNA polymerase II catalysed transcription and give rise to trapped DNA-topo I cleavage complexes; and (4) trapped complexes interact with another topo I molecule on a temporarily inactive transcription unit at the same transcription factory leading to DNA cleavage and subsequent strand exchange between the cleavage complexes. We have developed a method using inverse PCR that allows the detection and sequencing of putative ionising radiation-induced DNA rearrangements involving different regions of the human genome (Forrester and Radford 1998). The sequences detected by inverse PCR can provide a test of the prediction of the transcription-based model that ionising radiation-induced DNA rearrangements occur between sequences in active transcription units. Accordingly, reverse transcriptase PCR was used to determine if sequences involved in rearrangements were transcribed in the test cells. Consistent with the transcription-based model, nearly all of the sequences examined gave a positive result to reverse transcriptase PCR (Forrester and Radford unpublished)

The transcriptional landscape

DEFF Research Database (Denmark)

Nielsen, Henrik

2011-01-01

The application of new and less biased methods to study the transcriptional output from genomes, such as tiling arrays and deep sequencing, has revealed that most of the genome is transcribed and that there is substantial overlap of transcripts derived from the two strands of DNA. In protein coding...... regions, the map of transcripts is very complex due to small transcripts from the flanking ends of the transcription unit, the use of multiple start and stop sites for the main transcript, production of multiple functional RNA molecules from the same primary transcript, and RNA molecules made...... by independent transcription from within the unit. In genomic regions separating those that encode proteins or highly abundant RNA molecules with known function, transcripts are generally of low abundance and short-lived. In most of these cases, it is unclear to what extent a function is related to transcription...

Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

Science.gov (United States)

Furuse, Yuki; Finethy, Ryan; Saka, Hector A; Xet-Mull, Ana M; Sisk, Dana M; Smith, Kristen L Jurcic; Lee, Sunhee; Coers, Jörn; Valdivia, Raphael H; Tobin, David M; Cullen, Bryan R

2014-01-01

MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

Directory of Open Access Journals (Sweden)

Yuki Furuse

Full Text Available MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

Danish initiatives to improve the safety of meat products

DEFF Research Database (Denmark)

Wegener, Henrik Caspar

2010-01-01

and Campylobacter, and to a lesser extent Yersinia, Escherichia coli O157 and Listeria. Danish initiatives to improve the safety of meat products have focused on the entire production chain from the farm to the consumer, with a special emphasis on the pre-harvest stage of production. The control of bacterial......During the last two decades the major food safety problems in Denmark, as determined by the number of human patients, has been associated with bacterial infections stemming from meat products and eggs. The bacterial pathogens causing the majority of human infections has been Salmonella...

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

Science.gov (United States)

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-07-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

Mapping the transcription termination region of the mouse immunoglobulin kappa gene

International Nuclear Information System (INIS)

Xu, M.; Garrard, W.T.

1986-01-01

To define the transcription termination region of the mouse immunoglobulin kappa gene, they have subcloned single copy DNA sequences corresponding to both the template and the non-template strands of this locus. In vitro nuclear transcription with isolated MPC-11 nuclei was performed and the resulting 32 P-labeled RNA was hybridized to slot-blotted, single-stranded M13 probes covering regions within and flanking the kappa gene. The hybridization pattern for the template-strand reveals that transcription terminates within the region between 1.1 to 2.3 kb downstream from the poly(A) site. Ten different short sequences (8-13 bp) reside within 460 bp of this region that exhibit homology with sequences found in the termination regions of mouse β-globin and chicken ovalbumin genes. Transcription of the non-template strand occurs on either side of this termination region. They note that no transcription is detectable on the non-template strand downstream of the enhancer, indicating that if RNA polymerase II enters at this site, it does not initiate transcription during transit to the promoter region. They conclude that transcription of the kappa gene passes the poly(A) addition site and terminates within 2.3 Kb downstream

Complete Genomes of Classical Swine Fever Virus Cloned into Bacterial Artificial Chromosomes

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Reimann, I.; Uttenthal, Åse

Complete genome amplification of viral RNA provides a new tool for the generation of modified pestiviruses. We have used our full-genome amplification strategy for generation of amplicons representing complete genomes of classical swine fever virus. The amplicons were cloned directly into a stabl...... single-copy bacterial artificial chromosome (BAC) generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived. Our strategy allows construction of stable infectious BAC DNAs from a single full-length PCR product....

RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

KAUST Repository

Baazim, Hatoon

2014-05-01

Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

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Benoît Couvigny

Full Text Available The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor, we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

Bacterial communities in the fruit bodies of ground basidiomycetes

Science.gov (United States)

Zagryadskaya, Yu. A.; Lysak, L. V.; Chernov, I. Yu.

2015-06-01

Fruit bodies of basidiomycetes at different stages of decomposition serve as specific habitats in forest biocenoses for bacteria and differ significantly with respect to the total bacterial population and abundance of particular bacterial genera. A significant increase in the total bacterial population estimated by the direct microscopic method with acridine orange staining and in the population of saprotrophic bacteria (inoculation of glucose peptone yeast agar) in fruit bodies of basidiomycetes Armillaria mellea and Coprinus comatus was recorded at the final stage of their decomposition in comparison with the initial stage. Gramnegative bacteria predominated in the tissues of fruit bodies at all the stages of decomposition and were represented at the final stage by the Aeromonas, Vibrio, and Pseudomonas genera (for fruit bodies of A. mellea) the Pseudomonas genus (for fruit bodies of C. comatus). The potential influence of bacterial communities in the fruit bodies of soil basidiomycetes on the formation of bacterial communities in the upper soil horizons in forest biocenoses is discussed. The loci connected with the development and decomposition of fruit bodies of basidiomycetes on the soil surface are promising for targeted search of Gram-negative bacteria, the important objects of biotechnology.

Bacterial Meningitis in the Absence of Cerebrospinal Fluid Pleocytosis: A Case Report and Review of the Literature

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Ryota Hase

2014-01-01

Full Text Available Elevation of cerebrospinal fluid (CSF cell count is a key sign in the diagnosis of bacterial meningitis. However, there have been reports of bacterial meningitis with no abnormalities in initial CSF testing. This type of presentation is extremely rare in adult patients. Here, a case involving an 83-year-old woman who was later diagnosed with bacterial meningitis caused by Neisseria meningitidis is described, in whom CSF at initial and second lumbar puncture did not show elevation of cell counts. Twenty-six non-neutropenic adult cases of bacterial meningitis in the absence of CSF pleocytosis were reviewed. The frequent causative organisms were Streptococcus pneumoniae and N meningitidis. Nineteen cases had bacteremia and seven died. The authors conclude that normal CSF at lumbar puncture at an early stage cannot rule out bacterial meningitis. Therefore, repeat CSF analysis should be considered, and antimicrobial therapy must be started immediately if there are any signs of sepsis or meningitis.

Transcriptional activation of the tad type IVb pilus operon by PypB in Yersinia enterocolitica.

Science.gov (United States)

Schilling, Jennifer; Wagner, Karin; Seekircher, Stephanie; Greune, Lilo; Humberg, Verena; Schmidt, M Alexander; Heusipp, Gerhard

2010-07-01

Type IV pili are virulence factors in various bacteria and mediate, among other functions, the colonization of diverse surfaces. Various subclasses of type IV pili have been identified, but information on pilus expression, biogenesis, and the associated phenotypes is sparse for the genus Yersinia. We recently described the identification of PypB as a transcriptional regulator in Yersinia enterocolitica. Here we show that the pypB gene is associated with the tad locus, a genomic island that is widespread among bacterial and archaeal species. The genetic linkage of pypB with the tad locus is conserved throughout the yersiniae but is not found among other bacteria carrying the tad locus. We show that the genes of the tad locus form an operon in Y. enterocolitica that is controlled by PypB and that pypB is part of this operon. The tad genes encode functions necessary for the biogenesis of the Flp subfamily of type IVb pili initially described for Aggregatibacter actinomycetemcomitans to mediate a tight-adherence phenotype. In Y. enterocolitica, the Flp pilin protein shows some peculiarities in its amino acid sequence that imply similarities as well as differences compared to typical motifs found in the Flp subtype of type IVb pili. Flp is expressed and processed after PypB overproduction, resulting in microcolony formation but not in increased adherence to biotic or abiotic surfaces. Our data describe the transcriptional regulation of the tad type IVb pilus operon by PypB in Y. enterocolitica but fail to show most previously described phenotypes associated with this type of pilus in other bacteria.

The DAF-16/FOXO transcription factor functions as a regulator of epidermal innate immunity.

Science.gov (United States)

Zou, Cheng-Gang; Tu, Qiu; Niu, Jie; Ji, Xing-Lai; Zhang, Ke-Qin

2013-01-01

The Caenorhabditis elegans DAF-16 transcription factor is critical for diverse biological processes, particularly longevity and stress resistance. Disruption of the DAF-2 signaling cascade promotes DAF-16 activation, and confers resistance to killing by pathogenic bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. However, daf-16 mutants exhibit similar sensitivity to these bacteria as wild-type animals, suggesting that DAF-16 is not normally activated by these bacterial pathogens. In this report, we demonstrate that DAF-16 can be directly activated by fungal infection and wounding in wild-type animals, which is independent of the DAF-2 pathway. Fungal infection and wounding initiate the Gαq signaling cascade, leading to Ca(2+) release. Ca(2+) mediates the activation of BLI-3, a dual-oxidase, resulting in the production of reactive oxygen species (ROS). ROS then activate DAF-16 through a Ste20-like kinase-1/CST-1. Our results indicate that DAF-16 in the epidermis is required for survival after fungal infection and wounding. Thus, the EGL-30-Ca(2+)-BLI-3-CST-1-DAF-16 signaling represents a previously unknown pathway to regulate epidermal damage response.

Simultaneous transcriptional profiling of bacteria and their host cells.

Directory of Open Access Journals (Sweden)

Michael S Humphrys

Full Text Available We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness. Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.

Pseudoalteromonas spp. serve as initial bacterial attractants in mesocosms of coastal waters but have subsequent antifouling capacity in mesocosms and when embedded in paint.

Science.gov (United States)

Bernbom, Nete; Ng, Yoke Yin; Olsen, Stefan Møller; Gram, Lone

2013-11-01

The purpose of the present study was to determine if the monoculture antifouling effect of several pigmented pseudoalteromonads was retained in in vitro mesocosm systems using natural coastal seawater and when the bacteria were embedded in paint used on surfaces submerged in coastal waters. Pseudoalteromonas piscicida survived on a steel surface and retained antifouling activity for at least 53 days in sterile seawater, whereas P. tunicata survived and had antifouling activity for only 1 week. However, during the first week, all Pseudoalteromonas strains facilitated rather than prevented bacterial attachment when used to coat stainless steel surfaces and submerged in mesocosms with natural seawater. The bacterial density on surfaces coated with sterile growth medium was 10(5) cells/cm(2) after 7 days, whereas counts on surfaces precoated with Pseudoalteromonas were significantly higher, at 10(6) to 10(8) cells/cm(2). However, after 53 days, seven of eight Pseudoalteromonas strains had reduced total bacterial adhesion compared to the control. P. piscicida, P. antarctica, and P. ulvae remained on the surface, at levels similar to those in the initial coating, whereas P. tunicata could not be detected. Larger fouling organisms were observed on all plates precoated with Pseudoalteromonas; however, plates coated only with sterile growth medium were dominated by a bacterial biofilm. Suspensions of a P. piscicida strain and a P. tunicata strain were incorporated into ship paints (Hempasil x3 87500 and Hempasil 77500) used on plates that were placed at the Hempel A/S test site in Jyllinge Harbor. For the first 4 months, no differences were observed between control plates and treated plates, but after 5 to 6 months, the control plates were more fouled than the plates with pseudoalteromonad-based paint. Our study demonstrates that no single laboratory assay can predict antifouling effects and that a combination of laboratory and real-life methods must be used to determine

Co-isolation of in vivo 32P-labeled specific transcripts and DNA without phenol extraction of nuclease digestion

International Nuclear Information System (INIS)

Hayes, S.; Hayes, C.; Brand, L.

1981-01-01

A method is described for isolation and quantitation of specific intact transcripts, for which a hybridization probe is available, from 32 P-labeled bacterial cells. The RNA is extracted in the absence of R Nase activity by incorporating an inert, physically removable R Nase inhibitor throughout the spheroplasting, cell lysis, and pronase digestion steps. [/sup 32/P]RNA is separated from [ 32 P]DNA, without recourse to phenol extraction of DNase treatment, on a Cs 2 SO/sub 4-/HCONH 2 step gradient in which the precipitated RNA forms a sharp band. Specific transcripts are purified from [ 32 P]RNA by physical separation of the transcript and hybridization probe using gel-exclusion chromatography. The gentleness of this technique enables the co-isolation of DNA and can facilitate the analysis of covalently joined RNA-DNA replication intermediates

The effect of U.V.-irradiation on lambda DNA transcription

International Nuclear Information System (INIS)

Ranade, S.S.

1977-01-01

The effect of U.V.-irradiation of template DNA has been studied in vitro in the E.coli RNA polymerase system with native and U.V.-treated lambda DNA. Lambda DNA was more susceptible to U.V. than was calf-thymus DNA, yet a residual activity was observed at a U.V. dose of 0.5 x 10 4 erg/mm 2 . From the kinetic analysis of the reaction and the incorporation of lambda 32 P-labelled nucleoside triphosphates, it seems reasonable to conclude that U.V.-irradiation probably did not affect the DNA initiation sites, recognizable by RNA polymerase. The transcription products made with U.V.-irradiated lambda DNA were asymmetrical, and hybridized to the right half (R) and the left half (L) of lambda DNA with the ratio of R/L=4/1, and they showed a lower hybridizability than the transcripts with native lambda DNA. The initiation sites recognizable by RNA polymerase seemed to be the same on both native and U.V.-irradiated lambda DNA, though the transcription of U.V.-treated lambda DNA appeared to terminate with rather short RNA chains. (author)

Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example

Science.gov (United States)

Lerner, Eitan; Ingargiola, Antonino; Weiss, Shimon

2018-03-01

Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few "structural snapshots" along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Förster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the technique's temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase-promoter open complex (RPo). We measured microsecond alternating laser excitation-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis, photon-by-photon hidden Markov modeling, and the FRET-restrained positioning and screening approach to identify two conformational states for RPo. The experimentally derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.

Acute sleep deprivation enhances post-infection sleep and promotes survival during bacterial infection in Drosophila.

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Kuo, Tzu-Hsing; Williams, Julie A

2014-05-01

Sleep is known to increase as an acute response to infection. However, the function of this behavioral response in host defense is not well understood. To address this problem, we evaluated the effect of acute sleep deprivation on post-infection sleep and immune function in Drosophila. Laboratory. Drosophila melanogaster. Flies were subjected to sleep deprivation before (early DEP) or after (late DEP) bacterial infection. Relative to a non-deprived control, flies subjected to early DEP had enhanced sleep after infection as well as increased bacterial clearance and survival outcome. Flies subjected to late DEP experienced enhanced sleep following the deprivation period, and showed a modest improvement in survival outcome. Continuous DEP (early and late DEP) throughout infection also enhanced sleep later during infection and improved survival. However, improved survival in flies subjected to late or continuous DEP did not occur until after flies had experienced sleep. During infection, both early and late DEP enhanced NFκB transcriptional activity as measured by a luciferase reporter (κB-luc) in living flies. Early DEP also increased NFκB activity prior to infection. Flies that were deficient in expression of either the Relish or Dif NFκB transcription factors showed normal responses to early DEP. However, the effect of early DEP on post-infection sleep and survival was abolished in double mutants, which indicates that Relish and Dif have redundant roles in this process. Acute sleep deprivation elevated NFκB-dependent activity, increased post-infection sleep, and improved survival during bacterial infection.

Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia

DEFF Research Database (Denmark)

Bjerrum, Jacob T; Nielsen, Ole H; Riis, Lene B

2014-01-01

to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. METHODS: The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription...... polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction...... and immunohistochemistry data by the Wilcoxon's rank-sum test. RESULTS: The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel...

Reagent-free bacterial identification using multivariate analysis of transmission spectra

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Smith, Jennifer M.; Huffman, Debra E.; Acosta, Dayanis; Serebrennikova, Yulia; García-Rubio, Luis; Leparc, German F.

2012-10-01

The identification of bacterial pathogens from culture is critical to the proper administration of antibiotics and patient treatment. Many of the tests currently used in the clinical microbiology laboratory for bacterial identification today can be highly sensitive and specific; however, they have the additional burdens of complexity, cost, and the need for specialized reagents. We present an innovative, reagent-free method for the identification of pathogens from culture. A clinical study has been initiated to evaluate the sensitivity and specificity of this approach. Multiwavelength transmission spectra were generated from a set of clinical isolates including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Spectra of an initial training set of these target organisms were used to create identification models representing the spectral variability of each species using multivariate statistical techniques. Next, the spectra of the blinded isolates of targeted species were identified using the model achieving >94% sensitivity and >98% specificity, with 100% accuracy for P. aeruginosa and S. aureus. The results from this on-going clinical study indicate this approach is a powerful and exciting technique for identification of pathogens. The menu of models is being expanded to include other bacterial genera and species of clinical significance.

Bacterial prostatitis.

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Gill, Bradley C; Shoskes, Daniel A

2016-02-01

The review provides the infectious disease community with a urologic perspective on bacterial prostatitis. Specifically, the article briefly reviews the categorization of prostatitis by type and provides a distillation of new findings published on bacterial prostatitis over the past year. It also highlights key points from the established literature. Cross-sectional prostate imaging is becoming more common and may lead to more incidental diagnoses of acute bacterial prostatitis. As drug resistance remains problematic in this condition, the reemergence of older antibiotics such as fosfomycin, has proven beneficial. With regard to chronic bacterial prostatitis, no clear clinical risk factors emerged in a large epidemiological study. However, bacterial biofilm formation has been associated with more severe cases. Surgery has a limited role in bacterial prostatitis and should be reserved for draining of a prostatic abscess or the removal of infected prostatic stones. Prostatitis remains a common and bothersome clinical condition. Antibiotic therapy remains the basis of treatment for both acute and chronic bacterial prostatitis. Further research into improving prostatitis treatment is indicated.

TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair

NARCIS (Netherlands)

G.S. Winkler (Sebastiaan); U. Fiedler; W. Vermeulen (Wim); F. Coin (Frédéric); R.D. Wood (Richard); H.T.M. Timmers (Marc); G. Weeda (Geert); J.H.J. Hoeijmakers (Jan); S.J. Araú jo; J-M. Egly (Jean-Marc)

2000-01-01

textabstractTFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and

Traveling Rocky Roads: The Consequences of Transcription-Blocking DNA Lesions on RNA Polymerase II.

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Steurer, Barbara; Marteijn, Jurgen A

2017-10-27

The faithful transcription of eukaryotic genes by RNA polymerase II (RNAP2) is crucial for proper cell function and tissue homeostasis. However, transcription-blocking DNA lesions of both endogenous and environmental origin continuously challenge the progression of elongating RNAP2. The stalling of RNAP2 on a transcription-blocking lesion triggers a series of highly regulated events, including RNAP2 processing to make the lesion accessible for DNA repair, R-loop-mediated DNA damage signaling, and the initiation of transcription-coupled DNA repair. The correct execution and coordination of these processes is vital for resuming transcription following the successful repair of transcription-blocking lesions. Here, we outline recent insights into the molecular consequences of RNAP2 stalling on transcription-blocking DNA lesions and how these lesions are resolved to restore mRNA synthesis. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Lipopolysaccharide (LPS Accumulates in Neocortical Neurons of Alzheimer’s Disease (AD Brain and Impairs Transcription in Human Neuronal-Glial Primary Co-cultures

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Yuhai Zhao

2017-12-01

Full Text Available Several independent laboratories have recently reported the detection of bacterial nucleic acid sequences or bacterial-derived neurotoxins, such as highly inflammatory lipopolysaccharide (LPS, within Alzheimer’s disease (AD affected brain tissues. Whether these bacterial neurotoxins originate from the gastrointestinal (GI tract microbiome, a possible brain microbiome or some dormant pathological microbiome is currently not well understood. Previous studies indicate that the co-localization of pro-inflammatory LPS with AD-affected brain cell nuclei suggests that there may be a contribution of this neurotoxin to genotoxic events that support inflammatory neurodegeneration and failure in homeostatic gene expression. In this report we provide evidence that in sporadic AD, LPS progressively accumulates in neuronal parenchyma and appears to preferentially associate with the periphery of neuronal nuclei. Run-on transcription studies utilizing [α-32P]-uridine triphosphate incorporation into newly synthesized total RNA further indicates that human neuronal-glial (HNG cells in primary co-culture incubated with LPS exhibit significantly reduced output of DNA transcription products. These studies suggest that in AD LPS may impair the efficient readout of neuronal genetic information normally required for the homeostatic operation of brain cell function and may contribute to a progressive disruption in the read-out of genetic information.

CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex.

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Bae, Brian; Chen, James; Davis, Elizabeth; Leon, Katherine; Darst, Seth A; Campbell, Elizabeth A

2015-09-08

A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

Characterization of the LysR-type transcriptional regulator YcjZ-like from Xylella fastidiosa overexpressed in Escherichia coli.

Science.gov (United States)

Santiago, André S; Santos, Clelton A; Mendes, Juliano S; Toledo, Marcelo A S; Beloti, Lilian L; Souza, Alessandra A; Souza, Anete P

2015-09-01

The Xylella fastidiosa 9a5c strain is a xylem-limited phytopathogen that is the causal agent of citrus variegated chlorosis (CVC). This bacterium is able to form a biofilm and occlude the xylem vessels of susceptible plants, which leads to significant agricultural and economic losses. Biofilms are associated with bacterial pathogenicity because they are very resistant to antibiotics and other metal-based chemicals that are used in agriculture. The X. fastidiosa YcjZ-like (XfYcjZ-like) protein belongs to the LysR-type transcriptional regulator (LTTR) family and is involved in various cellular functions that range from quorum sensing to bacterial survival. In the present study, we report the cloning, expression and purification of XfYcjZ-like, which was overexpressed in Escherichia coli. The secondary folding of the recombinant and purified protein was assessed by circular dichroism, which revealed that XfYcjZ-like contains a typical α/β fold. An initial hydrodynamic characterization showed that XfYcjZ-like is a globular tetramer in solution. In addition, using a polyclonal antibody against XfYcjZ-like, we assessed the expression profile of this protein during the different developmental phases of X. fastidiosa in in vitro cultivated biofilm cells and demonstrated that XfYcjZ-like is upregulated in planktonic cells in response to a copper shock treatment. Finally, the ability of XfYcjZ-like to interact with its own predicted promoter was confirmed in vitro, which is a typical feature of LysR. Taken together, our findings indicated that the XfYcjZ-like protein is involved in both the organization of the architecture and the maturation of the bacterial biofilm and that it is responsive to oxidative stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Bacterial Cellulose Production from Industrial Waste and by-Product Streams.

Science.gov (United States)

Tsouko, Erminda; Kourmentza, Constantina; Ladakis, Dimitrios; Kopsahelis, Nikolaos; Mandala, Ioanna; Papanikolaou, Seraphim; Paloukis, Fotis; Alves, Vitor; Koutinas, Apostolis

2015-07-01

The utilization of fermentation media derived from waste and by-product streams from biodiesel and confectionery industries could lead to highly efficient production of bacterial cellulose. Batch fermentations with the bacterial strain Komagataeibacter sucrofermentans DSM (Deutsche Sammlung von Mikroorganismen) 15973 were initially carried out in synthetic media using commercial sugars and crude glycerol. The highest bacterial cellulose concentration was achieved when crude glycerol (3.2 g/L) and commercial sucrose (4.9 g/L) were used. The combination of crude glycerol and sunflower meal hydrolysates as the sole fermentation media resulted in bacterial cellulose production of 13.3 g/L. Similar results (13 g/L) were obtained when flour-rich hydrolysates produced from confectionery industry waste streams were used. The properties of bacterial celluloses developed when different fermentation media were used showed water holding capacities of 102-138 g · water/g · dry bacterial cellulose, viscosities of 4.7-9.3 dL/g, degree of polymerization of 1889.1-2672.8, stress at break of 72.3-139.5 MPa and Young's modulus of 0.97-1.64 GPa. This study demonstrated that by-product streams from the biodiesel industry and waste streams from confectionery industries could be used as the sole sources of nutrients for the production of bacterial cellulose with similar properties as those produced with commercial sources of nutrients.

Clavibacter michiganensis ssp. michiganensis: bacterial canker of tomato, molecular interactions and disease management.

Science.gov (United States)

Nandi, Munmun; Macdonald, Jacqueline; Liu, Peng; Weselowski, Brian; Yuan, Ze-Chun

2018-03-12

Bacterial canker disease is considered to be one of the most destructive diseases of tomato (Solanum lycopersicum), and is caused by the seed-borne Gram-positive bacterium Clavibacter michiganensis ssp. michiganensis (Cmm). This vascular pathogen generally invades and proliferates in the xylem through natural openings or wounds, causing wilt and canker symptoms. The incidence of symptomless latent infections and the invasion of tomato seeds by Cmm are widespread. Pathogenicity is mediated by virulence factors and transcriptional regulators encoded by the chromosome and two natural plasmids. The virulence factors include serine proteases, cell wall-degrading enzymes (cellulases, xylanases, pectinases) and others. Mutational analyses of these genes and gene expression profiling (via quantitative reverse transcription-polymerase chain reaction, transcriptomics and proteomics) have begun to shed light on their roles in colonization and virulence, whereas the expression of tomato genes in response to Cmm infection suggests plant factors involved in the defence response. These findings may aid in the generation of target-specific bactericides or new resistant varieties of tomato. Meanwhile, various chemical and biological controls have been researched to control Cmm. This review presents a detailed investigation regarding the pathogen Cmm, bacterial canker infection, molecular interactions between Cmm and tomato, and current perspectives on improved disease management. © 2018 AGRICULTURE AND AGRI-FOOD CANADA. MOLECULAR PLANT PATHOLOGY © 2018 JOHN WILEY & SONS LTD.

Effect of Weak Magnetic Field on Bacterial Growth

Science.gov (United States)

Masood, Samina

Effects of weak magnetic fields are observed on the growth of various bacterial strains. Different sources of a constant magnetic field are used to demonstrate that ion transport in the nutrient broth and bacterial cellular dynamics is perturbed in the presence of weak magnetic field which affects the mobility and absorption of nutrients in cells and hence their doubling rate. The change is obvious after a few hours of exposure and keeps on increasing with time for all the observed species. The growth rate depends on the field strength and the nature of the magnetic field. The field effect varies with the shape and the structure of the bacterial cell wall as well as the concentration of nutrient broth. We closely study the growth of three species Escherichia coli, Pseudomonas aeruginosa and Staphylococcus epidermidis with the same initial concentrations at the same temperature in the same laboratory environment. Our results indicate that the weak static field of a few gauss after a few hours gives a measurable change in the growth rates of all bacterial species. This shows that the same magnetic field has different effects on different species in the same environment.

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

Science.gov (United States)

Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

2017-08-30

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (Prock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these

Discrete redox signaling pathways regulate photosynthetic light-harvesting and chloroplast gene transcription.

Directory of Open Access Journals (Sweden)

John F Allen

Full Text Available In photosynthesis in chloroplasts, two related regulatory processes balance the actions of photosystems I and II. These processes are short-term, post-translational redistribution of light-harvesting capacity, and long-term adjustment of photosystem stoichiometry initiated by control of chloroplast DNA transcription. Both responses are initiated by changes in the redox state of the electron carrier, plastoquinone, which connects the two photosystems. Chloroplast Sensor Kinase (CSK is a regulator of transcription of chloroplast genes for reaction centres of the two photosystems, and a sensor of plastoquinone redox state. We asked whether CSK is also involved in regulation of absorbed light energy distribution by phosphorylation of light-harvesting complex II (LHC II. Chloroplast thylakoid membranes isolated from a CSK T-DNA insertion mutant and from wild-type Arabidopsis thaliana exhibit similar light- and redox-induced (32P-labelling of LHC II and changes in 77 K chlorophyll fluorescence emission spectra, while room-temperature chlorophyll fluorescence emission transients from Arabidopsis leaves are perturbed by inactivation of CSK. The results indicate indirect, pleiotropic effects of reaction centre gene transcription on regulation of photosynthetic light-harvesting in vivo. A single, direct redox signal is transmitted separately to discrete transcriptional and post-translational branches of an integrated cytoplasmic regulatory system.

Synthesis and characterization of anti-bacterial and anti-fungal citrate-based mussel-inspired bioadhesives

Science.gov (United States)

Guo, Jinshan; Wang, Wei; Hu, Jianqing; Xie, Denghui; Gerhard, Ethan; Nisic, Merisa; Shan, Dingying; Qian, Guoying; Zheng, Siyang; Yang, Jian

2016-01-01

Bacterial and fungal infections in the use of surgical devices and medical implants remain a major concern. Traditional bioadhesives fail to incorporate anti-microbial properties, necessitating additional anti-microbial drug injection. Herein, by the introduction of the clinically used and inexpensive anti-fungal agent, 10-undecylenic acid (UA), into our recently developed injectable citrate-based mussel-inspired bioadhesives (iCMBAs), a new family of anti-bacterial and anti-fungal iCMBAs (AbAf iCs) was developed. AbAf iCs not only showed strong wet tissue adhesion strength, but also exhibited excellent in vitro cyto-compatibility, fast degradation, and strong initial and considerable long-term anti-bacterial and anti-fungal ability. For the first time, the biocompatibility and anti-microbial ability of sodium metaperiodate (PI), an oxidant used as a cross-linking initiator in the AbAf iCs system, was also thoroughly investigated. Our results suggest that the PI-based bioadhesives showed better anti-microbial properties compared to the unstable silver-based bioadhesive materials. In conclusion, AbAf iCs family can serve as excellent anti-bacterial and anti-fungal bioadhesive candidates for tissue/wound closure, wound dressing, and bone regeneration, especially when bacterial or fungal infections are a major concern. PMID:26874283

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

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Elena García-Cano

Full Text Available Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis.

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF

Science.gov (United States)

García-Cano, Elena; Magori, Shimpei; Sun, Qi; Ding, Zehong; Lazarowitz, Sondra G.; Citovsky, Vitaly

2015-01-01

Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS) to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq) revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis. PMID:26571494

Transcriptional regulation by competing transcription factor modules.

Directory of Open Access Journals (Sweden)

Rutger Hermsen

2006-12-01

Full Text Available Gene regulatory networks lie at the heart of cellular computation. In these networks, intracellular and extracellular signals are integrated by transcription factors, which control the expression of transcription units by binding to cis-regulatory regions on the DNA. The designs of both eukaryotic and prokaryotic cis-regulatory regions are usually highly complex. They frequently consist of both repetitive and overlapping transcription factor binding sites. To unravel the design principles of these promoter architectures, we have designed in silico prokaryotic transcriptional logic gates with predefined input-output relations using an evolutionary algorithm. The resulting cis-regulatory designs are often composed of modules that consist of tandem arrays of binding sites to which the transcription factors bind cooperatively. Moreover, these modules often overlap with each other, leading to competition between them. Our analysis thus identifies a new signal integration motif that is based upon the interplay between intramodular cooperativity and intermodular competition. We show that this signal integration mechanism drastically enhances the capacity of cis-regulatory domains to integrate signals. Our results provide a possible explanation for the complexity of promoter architectures and could be used for the rational design of synthetic gene circuits.

Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans

Directory of Open Access Journals (Sweden)

Sager Jennifer

2008-05-01

Full Text Available Abstract Background Dietary restriction (DR increases life span and delays age-associated disease in many organisms. The mechanism by which DR enhances longevity is not well understood. Results Using bacterial food deprivation as a means of DR in C. elegans, we show that transient DR confers long-term benefits including stress resistance and increased longevity. Consistent with studies in the fruit fly and in mice, we demonstrate that DR also enhances survival when initiated late in life. DR by bacterial food deprivation significantly increases life span in worms when initiated as late as 24 days of adulthood, an age at which greater than 50% of the cohort have died. These survival benefits are, at least partially, independent of food consumption, as control fed animals are no longer consuming bacterial food at this advanced age. Animals separated from the bacterial lawn by a barrier of solid agar have a life span intermediate between control fed and food restricted animals. Thus, we find that life span extension from bacterial deprivation can be partially suppressed by a diffusible component of the bacterial food source, suggesting a calorie-independent mechanism for life span extension by dietary restriction. Conclusion Based on these findings, we propose that dietary restriction by bacterial deprivation increases longevity in C. elegans by a combination of reduced food consumption and decreased food sensing.

Transcriptional responses of Italian ryegrass during interaction with Xanthomonas translucens pv. graminis reveal novel candidate genes for bacterial wilt resistance

DEFF Research Database (Denmark)

Wichmann, Fabienne; Asp, Torben; Widmer, Franko

2011-01-01

Xanthomonas translucens pv. graminis (Xtg) causes bacterial wilt, a severe disease of forage grasses such as Italian ryegrass (Lolium multiflorum Lam.). In order to gain a more detailed understanding of the genetic control of resistance mechanisms and to provide prerequisites for marker assisted...... selection, the partial transcriptomes of two Italian ryegrass genotypes, one resistant and one susceptible to bacterial wilt were compared at four time points after Xtg infection. A cDNA microarray developed from a perennial ryegrass (Lolium perenne) expressed sequence tag set consisting of 9,990 unique...

New insights in the bacterial spore resistance to extreme terrestrial and extraterrestrial factors

Science.gov (United States)

Moeller, Ralf; Horneck, Gerda; Reitz, Guenther

Based on their unique resistance to various space parameters, Bacillus endospores are one of the model systems used for astrobiological studies. The extremely high resistance of bacterial endospores to environmental stress factors has intrigued researchers since long time and many characteristic spore features, especially those involved in the protection of spore DNA, have already been uncovered. The disclosure of the complete genomic sequence of Bacillus subtilis 168, one of the often used astrobiological model system, and the rapid development of tran-scriptional microarray techniques have opened new opportunities of gaining further insights in the enigma of spore resistance. Spores of B. subtilis were exposed to various extreme ter-restrial and extraterrestrial stressors to reach a better understanding of the DNA protection and repair strategies, which them to cope with the induced DNA damage. Following physical stress factors of environmental importance -either on Earth or in space -were selected for this thesis: (i) mono-and polychromatic UV radiation, (ii) ionizing radiation, (iii) exposure to ultrahigh vacuum; and (iv) high shock pressures simulating meteorite impacts. To reach a most comprehensive understanding of spore resistance to those harsh terrestrial or simulated extraterrestrial conditions, a standardized experimental protocol of the preparation and ana-lyzing methods was established including the determination of the following spore responses: (i) survival, (ii) induced mutations, (iii) DNA damage, (iv) role of different repair pathways by use of a set of repair deficient mutants, and (v) transcriptional responses during spore germi-nation by use of genome-wide transcriptome analyses and confirmation by RT-PCR. From this comprehensive set of data on spore resistance to a variety of environmental stress parameters a model of a "built-in" transcriptional program of bacterial spores in response to DNA damaging treatments to ensure DNA restoration

Antimicrobial Nanoplexes meet Model Bacterial Membranes: the key role of Cardiolipin

Science.gov (United States)

Marín-Menéndez, Alejandro; Montis, Costanza; Díaz-Calvo, Teresa; Carta, Davide; Hatzixanthis, Kostas; Morris, Christopher J.; McArthur, Michael; Berti, Debora

2017-01-01

Antimicrobial resistance to traditional antibiotics is a crucial challenge of medical research. Oligonucleotide therapeutics, such as antisense or Transcription Factor Decoys (TFDs), have the potential to circumvent current resistance mechanisms by acting on novel targets. However, their full translation into clinical application requires efficient delivery strategies and fundamental comprehension of their interaction with target bacterial cells. To address these points, we employed a novel cationic bolaamphiphile that binds TFDs with high affinity to form self-assembled complexes (nanoplexes). Confocal microscopy revealed that nanoplexes efficiently transfect bacterial cells, consistently with biological efficacy on animal models. To understand the factors affecting the delivery process, liposomes with varying compositions, taken as model synthetic bilayers, were challenged with nanoplexes and investigated with Scattering and Fluorescence techniques. Thanks to the combination of results on bacteria and synthetic membrane models we demonstrate for the first time that the prokaryotic-enriched anionic lipid Cardiolipin (CL) plays a key-role in the TFDs delivery to bacteria. Moreover, we can hypothesize an overall TFD delivery mechanism, where bacterial membrane reorganization with permeability increase and release of the TFD from the nanoplexes are the main factors. These results will be of great benefit to boost the development of oligonucleotides-based antimicrobials of superior efficacy.

Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei)

NARCIS (Netherlands)

Stricker, A.R.; Mach, R.L.; Graaff, de L.H.

2008-01-01

The filamentous fungi Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) have been the subject of many studies investigating the mechanism of transcriptional regulation of hemicellulase- and cellulase-encoding genes. The transcriptional regulator XlnR that was initially identified in A.

Hyperosmotic stress regulates the distribution and stability of myocardin-related transcription factor, a key modulator of the cytoskeleton

DEFF Research Database (Denmark)

Ly, Donald L.; Waheed, Faiza; Lodyga, Monika

2013-01-01

Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin-regulated coactiv......Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin......-regulated coactivator of serum response factor, is a major link between the actin skeleton and transcriptional control. We therefore investigated whether MRTF is regulated by hyperosmotic stress. Here we show that hypertonicity induces robust, rapid, and transient translocation of MRTF from the cytosol to the nucleus...... in kidney tubular cells. We found that the hyperosmolarity-triggered MRTF translocation is mediated by the RhoA/Rho kinase (ROK) pathway. Moreover, the Rho guanine nucleotide exchange factor GEF-H1 is activated by hyperosmotic stress, and it is a key contributor to the ensuing RhoA activation and MRTF...

A Laboratory Assessment of Factors That Affect Bacterial Adhesion to Contact Lenses

OpenAIRE

Dutta, Debarun; Willcox, Mark DP

2013-01-01

Adhesion of pathogenic microbes, particularly bacteria, to contact lenses is implicated in contact lens related microbial adverse events. Various in vitro conditions such as type of bacteria, the size of initial inoculum, contact lens material, nutritional content of media, and incubation period can influence bacterial adhesion to contact lenses and the current study investigated the effect of these conditions on bacterial adhesion to contact lenses. There was no significant difference in num...

KLF4 Nuclear Export Requires ERK Activation and Initiates Exit from Naive Pluripotency.

Science.gov (United States)

Dhaliwal, Navroop K; Miri, Kamelia; Davidson, Scott; Tamim El Jarkass, Hala; Mitchell, Jennifer A

2018-04-10

Cooperative action of a transcription factor complex containing OCT4, SOX2, NANOG, and KLF4 maintains the naive pluripotent state; however, less is known about the mechanisms that disrupt this complex, initiating exit from pluripotency. We show that, as embryonic stem cells (ESCs) exit pluripotency, KLF4 protein is exported from the nucleus causing rapid decline in Nanog and Klf4 transcription; as a result, KLF4 is the first pluripotency transcription factor removed from transcription-associated complexes during differentiation. KLF4 nuclear export requires ERK activation, and phosphorylation of KLF4 by ERK initiates interaction of KLF4 with nuclear export factor XPO1, leading to KLF4 export. Mutation of the ERK phosphorylation site in KLF4 (S132) blocks KLF4 nuclear export, the decline in Nanog, Klf4, and Sox2 mRNA, and differentiation. These findings demonstrate that relocalization of KLF4 to the cytoplasm is a critical first step in exit from the naive pluripotent state and initiation of ESC differentiation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Initiating Service Encounter-based Innovation by Word-of-Business

DEFF Research Database (Denmark)

Mattsson, Jan

2015-01-01

Purpose – This paper aims to set up a natural experiment as action research and to develop a framework of cognitive distance of informants to improve the initiation of service encounter-based innovation. Design/methodology/approach – Natural experiment as action research in one Scandinavian case...... transcriptions of interviews and transcriptions. Research limitations/implications – Only one Scandinavian company and a limited number of informants were activated. Also, the time period only included the initiation phase of service encounter-based innovation. Practical implications – Three different strategies...... in an emerging innovation field, open/user-driven innovation. Theory from business marketing, service encounter and innovation is also used....

Transcript structure and domain display: a customizable transcript visualization tool.

Science.gov (United States)

Watanabe, Kenneth A; Ma, Kaiwang; Homayouni, Arielle; Rushton, Paul J; Shen, Qingxi J

2016-07-01

Transcript Structure and Domain Display (TSDD) is a publicly available, web-based program that provides publication quality images of transcript structures and domains. TSDD is capable of producing transcript structures from GFF/GFF3 and BED files. Alternatively, the GFF files of several model organisms have been pre-loaded so that users only needs to enter the locus IDs of the transcripts to be displayed. Visualization of transcripts provides many benefits to researchers, ranging from evolutionary analysis of DNA-binding domains to predictive function modeling. TSDD is freely available for non-commercial users at http://shenlab.sols.unlv.edu/shenlab/software/TSD/transcript_display.html : jeffery.shen@unlv.nevada.edu. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Bacterial Adhesion & Blocking Bacterial Adhesion

DEFF Research Database (Denmark)

Vejborg, Rebecca Munk

2008-01-01

, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...... tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters...

Regulation of cell proliferation by the E2F transcription factors

DEFF Research Database (Denmark)

Helin, K

1998-01-01

Experimental data generated in the past year have further emphasized the essential role for the E2F transcription factors in the regulation of cell proliferation. Genetic studies have shown that E2F activity is required for normal development in fruitflies, and the generation of E2F-1(-/-) mice h......Fs in the proteasomes. Novel target genes for the E2F transcription factors have been identified that link the E2Fs directly to the initiation of DNA replication.......Experimental data generated in the past year have further emphasized the essential role for the E2F transcription factors in the regulation of cell proliferation. Genetic studies have shown that E2F activity is required for normal development in fruitflies, and the generation of E2F-1(-/-) mice has...... demonstrated that individual members of the E2F transcription factor family are likely to have distinct roles in mammalian development and homeostasis. Additional mechanisms regulating the activity of the E2F transcription factors have been reported, including subcellular localization and proteolysis of the E2...

Nano and Microscale Topographies for the Prevention of Bacterial Surface Fouling

Directory of Open Access Journals (Sweden)

Mary V. Graham

2014-01-01

Full Text Available Bacterial surface fouling is problematic for a wide range of applications and industries, including, but not limited to medical devices (implants, replacement joints, stents, pacemakers, municipal infrastructure (pipes, wastewater treatment, food production (food processing surfaces, processing equipment, and transportation (ship hulls, aircraft fuel tanks. One method to combat bacterial biofouling is to modify the topographical structure of the surface in question, thereby limiting the ability of individual cells to attach to the surface, colonize, and form biofilms. Multiple research groups have demonstrated that micro and nanoscale topographies significantly reduce bacterial biofouling, for both individual cells and bacterial biofilms. Antifouling strategies that utilize engineered topographical surface features with well-defined dimensions and shapes have demonstrated a greater degree of controllable inhibition over initial cell attachment, in comparison to undefined, texturized, or porous surfaces. This review article will explore the various approaches and techniques used by researches, including work from our own group, and the underlying physical properties of these highly structured, engineered micro/nanoscale topographies that significantly impact bacterial surface attachment.

Rickettsia conorii transcriptional response within inoculation eschar.

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Patricia Renesto

Full Text Available BACKGROUND: Rickettsia conorii, the causative agent of the Mediterranean spotted fever, is transmitted to humans by the bite of infected ticks Rhipicephalus sanguineus. The skin thus constitutes an important barrier for the entry and propagation of R. conorii. Given this, analysis of the survival strategies used by the bacterium within infected skin is critical for our understanding of rickettsiosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the first genome-wide analysis of R. conorii gene expression from infected human skin biopsies. Our data showed that R. conorii exhibited a striking transcript signature that is remarkably conserved across patients, regardless of genotype. The expression profiles obtained using custom Agilent microarrays were validated by quantitative RT-PCR. Within eschars, the amount of detected R. conorii transcripts was of 55%, this value being of 74% for bacteria grown in Vero cells. In such infected host tissues, approximately 15% (n = 211 of the total predicted R. conorii ORFs appeared differentially expressed compared to bacteria grown in standard laboratory conditions. These genes are mostly down-regulated and encode proteins essential for bacterial replication. Some of the strategies displayed by rickettsiae to overcome the host defense barriers, thus avoiding killing, were also pointed out. The observed up-regulation of rickettsial genes associated with DNA repair is likely to correspond to a DNA-damaging agent enriched environment generated by the host cells to eradicate the pathogens. Survival of R. conorii within eschars also involves adaptation to osmotic stress, changes in cell surface proteins and up-regulation of some virulence factors. Interestingly, in contrast to down-regulated transcripts, we noticed that up-regulated ones rather exhibit a small nucleotide size, most of them being exclusive for the spotted fever group rickettsiae. CONCLUSION/SIGNIFICANCE: Because eschar is a site for rickettsial

Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues.

Science.gov (United States)

Peyer, Suzanne M; Pankey, M Sabrina; Oakley, Todd H; McFall-Ngai, Margaret J

2014-02-01

The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or 'light organ', which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ∼1/4 into embryogenesis (Naef stage 18) and the light organ ∼3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light

Temperature adaptation of bacterial communities in experimentally warmed forest soils.

Science.gov (United States)

Rousk, Johannes; Frey, Serita D; Bååth, Erland

2012-10-01

A detailed understanding of the influence of temperature on soil microbial activity is critical to predict future atmospheric CO 2 concentrations and feedbacks to anthropogenic warming. We investigated soils exposed to 3-4 years of continuous 5 °C-warming in a field experiment in a temperate forest. We found that an index for the temperature adaptation of the microbial community, T min for bacterial growth, increased by 0.19 °C per 1 °C rise in temperature, showing a community shift towards one adapted to higher temperature with a higher temperature sensitivity (Q 10(5-15 °C) increased by 0.08 units per 1 °C). Using continuously measured temperature data from the field experiment we modelled in situ bacterial growth. Assuming that warming did not affect resource availability, bacterial growth was modelled to become 60% higher in warmed compared to the control plots, with the effect of temperature adaptation of the community only having a small effect on overall bacterial growth (bacterial growth, most likely due to substrate depletion because of the initially higher growth in warmed plots. When this was factored in, the result was similar rates of modelled in situ bacterial growth in warmed and control plots after 3 years, despite the temperature difference. We conclude that although temperature adaptation for bacterial growth to higher temperatures was detectable, its influence on annual bacterial growth was minor, and overshadowed by the direct temperature effect on growth rates. © 2012 Blackwell Publishing Ltd.

DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Pedersen, Jakob Madsen; Fredsøe, Jacob Christian; Rødgaard, Morten Terpager

2012-01-01

To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down......-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence......-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation...

Transcription Factors Expressed in Lateral Organ Boundaries: Identification of Downstream Targets

Energy Technology Data Exchange (ETDEWEB)

Springer, Patricia S

2010-07-12

The processes of lateral organ initiation and patterning are central to the generation of mature plant form. Characterization of the molecular mechanisms underlying these processes is essential to our understanding of plant development. Communication between the shoot apical meristem and initiating organ primordia is important both for functioning of the meristem and for proper organ patterning, and very little is known about this process. In particular, the boundary between meristem and leaf is emerging as a critical region that is important for SAM maintenance and regulation of organogenesis. The goal of this project was to characterize three boundary-expressed genes that encode predicted transcription factors. Specifically, we have studied LATERAL ORGAN BOUNDARIES (LOB), LATERAL ORGAN FUSION1 (LOF1), and LATERAL ORGAN FUSION2 (LOF2). LOB encodes the founding member of the LOB-DOMAIN (LBD) plant-specific DNA binding transcription factor family and LOF1 and LOF2 encode paralogous MYB-domain transcription factors. We characterized the genetic relationship between these three genes and other boundary and meristem genes. We also used an ectopic inducible expression system to identify direct targets of LOB.

Transcription of the soybean leghemoglobin genes during nodule development

DEFF Research Database (Denmark)

Marcker, Anne; Ø Jensen, Erik; Marcker, Kjeld A

1984-01-01

During the early stages of soybean nodule development the leghemoglobin (Lb) genes are activated sequentially in the opposite order to which they are arranged in the soybean genome. At a specific stage after the initial activation of all the Lb genes, a large increment occurs in the transcription...... of the Lb(c1), Lb(c3) and Lb(a) genes while the transcription of the Lb(c2) gene is not amplified to a similar extent. All the Lb genes retain significant activity for a long period during the lifetime of a nodule. Consequently the soybean Lb genes are not regulated by a developmental gene switching...

Antiviral activity and specific modes of action of bacterial prodigiosin against Bombyx mori nucleopolyhedrovirus in vitro.

Science.gov (United States)

Zhou, Wei; Zeng, Cheng; Liu, RenHua; Chen, Jie; Li, Ru; Wang, XinYan; Bai, WenWen; Liu, XiaoYuan; Xiang, TingTing; Zhang, Lin; Wan, YongJi

2016-05-01

Prodigiosin, the tripyrrole red pigment, is a bacterial secondary metabolite with multiple bioactivities; however, the antiviral activity has not been reported yet. In the present study, we found the antiviral activity of bacterial prodigiosin on Bombyx mori nucleopolyhedrovirus (BmNPV)-infected cells in vitro, with specific modes of action. Prodigiosin at nontoxic concentrations selectively killed virus-infected cells, inhibited viral gene transcription, especially viral early gene ie-1, and prevented virus-mediated membrane fusion. Under prodigiosin treatment, both progeny virus production and viral DNA replication were significantly inhibited. Fluorescent assays showed that prodigiosin predominantly located in cytoplasm which suggested it might interact with cytoplasm factors to inhibit virus replication. In conclusion, the present study clearly indicates that prodigiosin possesses significant antiviral activity against BmNPV.

Bacterial Cellulose Production from Industrial Waste and by-Product Streams

Directory of Open Access Journals (Sweden)

Erminda Tsouko

2015-07-01

Full Text Available The utilization of fermentation media derived from waste and by-product streams from biodiesel and confectionery industries could lead to highly efficient production of bacterial cellulose. Batch fermentations with the bacterial strain Komagataeibacter sucrofermentans DSM (Deutsche Sammlung von Mikroorganismen 15973 were initially carried out in synthetic media using commercial sugars and crude glycerol. The highest bacterial cellulose concentration was achieved when crude glycerol (3.2 g/L and commercial sucrose (4.9 g/L were used. The combination of crude glycerol and sunflower meal hydrolysates as the sole fermentation media resulted in bacterial cellulose production of 13.3 g/L. Similar results (13 g/L were obtained when flour-rich hydrolysates produced from confectionery industry waste streams were used. The properties of bacterial celluloses developed when different fermentation media were used showed water holding capacities of 102–138 g·water/g·dry bacterial cellulose, viscosities of 4.7–9.3 dL/g, degree of polymerization of 1889.1–2672.8, stress at break of 72.3–139.5 MPa and Young’s modulus of 0.97–1.64 GPa. This study demonstrated that by-product streams from the biodiesel industry and waste streams from confectionery industries could be used as the sole sources of nutrients for the production of bacterial cellulose with similar properties as those produced with commercial sources of nutrients.

Modeling bacterial contamination of fuel ethanol fermentation.

Science.gov (United States)

Bischoff, Kenneth M; Liu, Siqing; Leathers, Timothy D; Worthington, Ronald E; Rich, Joseph O

2009-05-01

The emergence of antibiotic-resistant bacteria may limit the effectiveness of antibiotics to treat bacterial contamination in fuel ethanol plants, and therefore, new antibacterial intervention methods and tools to test their application are needed. Using shake-flask cultures of Saccharomyces cerevisiae grown on saccharified corn mash and strains of lactic acid bacteria isolated from a dry-grind ethanol facility, a simple model to simulate bacterial contamination and infection was developed. Challenging the model with 10(8) CFU/mL Lactobacillus fermentum decreased ethanol yield by 27% and increased residual glucose from 6.2 to 45.5 g/L. The magnitude of the effect was proportional to the initial bacterial load, with 10(5) CFU/mL L. fermentum still producing an 8% decrease in ethanol and a 3.2-fold increase in residual glucose. Infection was also dependent on the bacterial species used to challenge the fermentation, as neither L. delbrueckii ATCC 4797 nor L. amylovorus 0315-7B produced a significant decrease in ethanol when inoculated at a density of 10(8) CFU/mL. In the shake-flask model, treatment with 2 microg/mL virginiamycin mitigated the infection when challenged with a susceptible strain of L. fermentum (MIC for virginiamycin model may find application in developing new antibacterial agents and management practices for use in controlling contamination in the fuel ethanol industry. Copyright 2008 Wiley Periodicals, Inc.

Impact of heavy metal contamination on oxidative stress of Eisenia andrei and bacterial community structure in Tunisian mine soil.

Science.gov (United States)

Boughattas, Iteb; Hattab, Sabrine; Boussetta, Hamadi; Banni, Mohamed; Navarro, Elisabeth

2017-08-01

The aims of this work were firstly to study the effect of heavy metal-polluted soils from Tunisian mine on earthworm biochemical biomarkers and on bacterial communities and therefore to analyze the interaction between earth worms and bacterial communities in these contaminated soils. For this purpose, we had introduced earthworm Eisenia andrei in six soils: one from mine spoils and five from agricultural soils, establishing a gradient of contamination. The response of worms to the presence of heavy metal was analyzed at the biochemical and transcriptional levels. In a second time, the impact of worm on bacterial community structure was investigated using automated ribosomal intergenic spacer analysis (ARISA) fingerprinting. An impact of heavy metal-contaminated soils on the oxidative status of E. andrei was observed, but this effect was dependent of the level of heavy metal contamination. Moreover, our results demonstrate that the introduction of earthworms E. andrei has an impact on bacterial community; however, the major change was observed in the less contaminated site. Furthermore, a significant correlation between earthworm oxidative status biomarkers and bacterial community structure was observed, mainly in the mine spoils. Therefore, we contribute to a better understanding of the relationships between epigenic earthworms and bacterial communities in heavy metal-contaminated soils.

Transcriptional responses to sucrose mimic the plant-associated life style of the plant growth promoting endophyte Enterobacter sp. 638.

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Taghavi, Safiyh; Wu, Xiao; Ouyang, Liming; Zhang, Yian Biao; Stadler, Andrea; McCorkle, Sean; Zhu, Wei; Maslov, Sergei; van der Lelie, Daniel

2015-01-01

Growth in sucrose medium was previously found to trigger the expression of functions involved in the plant associated life style of the endophytic bacterium Enterobacter sp. 638. Therefore, comparative transcriptome analysis between cultures grown in sucrose or lactate medium was used to gain insights in the expression levels of bacterial functions involved in the endophytic life style of strain 638. Growth on sucrose as a carbon source resulted in major changes in cell physiology, including a shift from a planktonic life style to the formation of bacterial aggregates. This shift was accompanied by a decrease in transcription of genes involved in motility (e.g., flagella biosynthesis) and an increase in the transcription of genes involved in colonization, adhesion and biofilm formation. The transcription levels of functions previously suggested as being involved in endophytic behavior and functions responsible for plant growth promoting properties, including the synthesis of indole-acetic acid, acetoin and 2,3-butanediol, also increased significantly for cultures grown in sucrose medium. Interestingly, despite an abundance of essential nutrients transcription levels of functions related to uptake and processing of nitrogen and iron became increased for cultures grown on sucrose as sole carbon source. Transcriptome data were also used to analyze putative regulatory relationships. In addition to the small RNA csrABCD regulon, which seems to play a role in the physiological adaptation and possibly the shift between free-living and plant-associated endophytic life style of Enterobacter sp. 638, our results also pointed to the involvement of rcsAB in controlling responses by Enterobacter sp. 638 to a plant-associated life style. Targeted mutagenesis was used to confirm this role and showed that compared to wild-type Enterobacter sp. 638 a ΔrcsB mutant was affected in its plant growth promoting ability.

Transcriptional responses to sucrose mimic the plant-associated life style of the plant growth promoting endophyte Enterobacter sp. 638.

Directory of Open Access Journals (Sweden)

Safiyh Taghavi

Full Text Available Growth in sucrose medium was previously found to trigger the expression of functions involved in the plant associated life style of the endophytic bacterium Enterobacter sp. 638. Therefore, comparative transcriptome analysis between cultures grown in sucrose or lactate medium was used to gain insights in the expression levels of bacterial functions involved in the endophytic life style of strain 638. Growth on sucrose as a carbon source resulted in major changes in cell physiology, including a shift from a planktonic life style to the formation of bacterial aggregates. This shift was accompanied by a decrease in transcription of genes involved in motility (e.g., flagella biosynthesis and an increase in the transcription of genes involved in colonization, adhesion and biofilm formation. The transcription levels of functions previously suggested as being involved in endophytic behavior and functions responsible for plant growth promoting properties, including the synthesis of indole-acetic acid, acetoin and 2,3-butanediol, also increased significantly for cultures grown in sucrose medium. Interestingly, despite an abundance of essential nutrients transcription levels of functions related to uptake and processing of nitrogen and iron became increased for cultures grown on sucrose as sole carbon source. Transcriptome data were also used to analyze putative regulatory relationships. In addition to the small RNA csrABCD regulon, which seems to play a role in the physiological adaptation and possibly the shift between free-living and plant-associated endophytic life style of Enterobacter sp. 638, our results also pointed to the involvement of rcsAB in controlling responses by Enterobacter sp. 638 to a plant-associated life style. Targeted mutagenesis was used to confirm this role and showed that compared to wild-type Enterobacter sp. 638 a ΔrcsB mutant was affected in its plant growth promoting ability.

Brood size modifications affect plumage bacterial assemblages of European starlings.

Science.gov (United States)

Lucas, Françoise S; Moureau, Benoit; Jourdie, Violaine; Heeb, Philipp

2005-02-01

During reproduction, birds face trade-offs between time and energy devoted to parental effort and traits associated with self-maintenance. We manipulated brood sizes to investigate the effects of such trade-offs on feather bacterial densities and the structure of bacterial assemblages on feathers in adult European starlings, Sturnus vulgaris, and in vitro feather degradation. As predicted by a trade-off between parental effort and self-maintenance, we found that birds with enlarged broods had more free-living bacteria on their feathers than birds with reduced broods. Furthermore, we found a significant interaction between brood manipulation and original brood size on free-living bacterial densities suggesting that the trade-off is mediated by the adults' initial reproductive investment. In contrast, brood size manipulations had no significant effect on densities of attached bacteria. Using ribosomal intergenic spacer analysis (RISA), we demonstrated that brood manipulations significantly modified the structure (band pattern) of feather-degrading bacterial assemblages, but had no significant effect on their richness (number of bands) or the in vitro feather degradation. In vitro feather degradation varied in relation to the premanipulation brood size and positively with the richness of the feather degrading bacterial community. Besides brood manipulation effect, we found that ecological factors and individual traits, such as the age, the nest location or the capture date, shaped bacterial assemblages and feather degradation capacities.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

Science.gov (United States)

Traverse, Charles C; Ochman, Howard

2017-08-29

. Knowledge of the full extent of sequences subject to transcription indels supports a new model of bacterial transcription slippage, one that relies on the number of complementary bases between the transcript and the DNA template to which it slipped. Copyright © 2017 Traverse and Ochman.

RNA Pol II promotes transcription of centromeric satellite DNA in beetles.

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Zeljka Pezer

Full Text Available Transcripts of centromeric satellite DNAs are known to play a role in heterochromatin formation as well as in establishment of the kinetochore. However, little is known about basic mechanisms of satellite DNA expression within constitutive heterochromatin and its regulation. Here we present comprehensive analysis of transcription of abundant centromeric satellite DNA, PRAT from beetle Palorus ratzeburgii (Coleoptera. This satellite is characterized by preservation and extreme sequence conservation among evolutionarily distant insect species. PRAT is expressed in all three developmental stages: larvae, pupae and adults at similar level. Transcripts are abundant comprising 0.033% of total RNA and are heterogeneous in size ranging from 0.5 kb up to more than 5 kb. Transcription proceeds from both strands but with 10 fold different expression intensity and transcripts are not processed into siRNAs. Most of the transcripts (80% are not polyadenylated and remain in the nucleus while a small portion is exported to the cytoplasm. Multiple, irregularly distributed transcription initiation sites as well as termination sites have been mapped within the PRAT sequence using primer extension and RLM-RACE. The presence of cap structure as well as poly(A tails in a portion of the transcripts indicate RNA polymerase II-dependent transcription and a putative polymerase II promoter site overlaps the most conserved part of the PRAT sequence. The treatment of larvae with alpha-amanitin decreases the level of PRAT transcripts at concentrations that selectively inhibit pol II activity. In conclusion, stable, RNA polymerase II dependant transcripts of abundant centromeric satellite DNA, not regulated by RNAi, have been identified and characterized. This study offers a basic understanding of expression of highly abundant heterochromatic DNA which in beetle species constitutes up to 50% of the genome.

A mechanistic overview of herbal medicine and botanical compounds to target transcriptional factors in Breast cancer.

Science.gov (United States)

Zhao, Yingke; Liu, Yue

2018-04-01

The abnormalities of transcription factors, such as NF-κB, STAT, estrogen receptor, play a critical role in the initiation and progression of breast cancer. Due to the limitation of current treatment, transcription factors could be promising therapeutic targets, which have received close attention. In this review, we introduced herbal medicines, as well as botanical compounds that had been verified with anti-tumor properties via regulating transcription factors. Herbs, compounds, as well as formulae reported with various transcriptional targets, were summarized thoroughly, to provide implication for the future research on basic experiment and clinical application. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteins mediating DNA loops effectively block transcription.

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Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

2017-07-01

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

Science.gov (United States)

Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

2016-01-01

The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

Requirement of the Epithelium-specific Ets Transcription Factor Spdef for Mucous Gland Cell Function in the Gastric Antrum*

OpenAIRE

Horst, David; Gu, Xuesong; Bhasin, Manoj; Yang, Quanli; Verzi, Michael; Lin, Dongxu; Joseph, Marie; Zhang, Xiaobo; Chen, Wei; Li, Yi-Ping; Shivdasani, Ramesh A.; Libermann, Towia A.

2010-01-01

Mucus-secreting cells of the stomach epithelium provide a protective barrier against damage that might result from bacterial colonization or other stimuli. Impaired barrier function contributes to chronic inflammation and cancer. Knock-out mice for the epithelium-specific transcription factor Spdef (also called Pdef) have defects in terminal differentiation of intestinal and bronchial secretory cells. We sought to determine the physiologic function of Spdef in the stomach, another site of sig...

Transcriptional responses of Treponema denticola to other oral bacterial species.

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Juni Sarkar

presented here provide an in-depth understanding of the transcriptional responses triggered by contact-dependent interactions between microorganisms inhabiting the periodontal pocket.

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

Science.gov (United States)

Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

2017-08-31

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

Microbial activity and bacterial community structure during degradation of microcystins

DEFF Research Database (Denmark)

Christoffersen, K.; Lyck, Susanne; Winding, A.

2002-01-01

experiments were analysed by polymerase chain reaction-density gradient gel electrophoresis (PCR-DGGE) of 16S rDNA, which showed that the indigenous bacterial community responded quickly to the addition of lysates. Our study confirms that bacteria can efficiently degrade microcystins in natural waters....... It was hypothesised that the bacterial community from a lake with frequent occurrence of toxic cyanobacteria can degrade microcystin along with other organic compounds. The initial dissolved microcystin concentrations ranged between 10 and 136 mug 1(-1) (microcystin-LR equivalents) in the laboratory experiment, using...... experiment to evaluate the effects of organic lysates on bacterial proliferation in the absence of microcystin. An exponential decline of the dissolved toxins was observed in all cases with toxins present, and the degradation rates ranged between 0.5 and 1.0 d(-1). No lag phases were observed but slow...

Interference of transcription across H-NS binding sites and repression by H-NS.

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Rangarajan, Aathmaja Anandhi; Schnetz, Karin

2018-05-01

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression. © 2018 John Wiley & Sons Ltd.

Co-transcriptomic Analysis by RNA Sequencing to Simultaneously Measure Regulated Gene Expression in Host and Bacterial Pathogen

KAUST Repository

Ravasi, Timothy; Mavromatis, Charalampos Harris; Bokil, Nilesh J.; Schembri, Mark A.; Sweet, Matthew J.

2016-01-01

Intramacrophage pathogens subvert antimicrobial defence pathways using various mechanisms, including the targeting of host TLR-mediated transcriptional responses. Conversely, TLR-inducible host defence mechanisms subject intramacrophage pathogens to stress, thus altering pathogen gene expression programs. Important biological insights can thus be gained through the analysis of gene expression changes in both the host and the pathogen during an infection. Traditionally, research methods have involved the use of qPCR, microarrays and/or RNA sequencing to identify transcriptional changes in either the host or the pathogen. Here we describe the application of RNA sequencing using samples obtained from in vitro infection assays to simultaneously quantify both host and bacterial pathogen gene expression changes, as well as general approaches that can be undertaken to interpret the RNA sequencing data that is generated. These methods can be used to provide insights into host TLR-regulated transcriptional responses to microbial challenge, as well as pathogen subversion mechanisms against such responses.

Co-transcriptomic Analysis by RNA Sequencing to Simultaneously Measure Regulated Gene Expression in Host and Bacterial Pathogen

KAUST Repository

Ravasi, Timothy

2016-01-24

Intramacrophage pathogens subvert antimicrobial defence pathways using various mechanisms, including the targeting of host TLR-mediated transcriptional responses. Conversely, TLR-inducible host defence mechanisms subject intramacrophage pathogens to stress, thus altering pathogen gene expression programs. Important biological insights can thus be gained through the analysis of gene expression changes in both the host and the pathogen during an infection. Traditionally, research methods have involved the use of qPCR, microarrays and/or RNA sequencing to identify transcriptional changes in either the host or the pathogen. Here we describe the application of RNA sequencing using samples obtained from in vitro infection assays to simultaneously quantify both host and bacterial pathogen gene expression changes, as well as general approaches that can be undertaken to interpret the RNA sequencing data that is generated. These methods can be used to provide insights into host TLR-regulated transcriptional responses to microbial challenge, as well as pathogen subversion mechanisms against such responses.

Non-Bacterial Chronic Recurrent Osteomyelitis of the Clavicle

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KL Pan

2012-03-01

Full Text Available This report details the case of a 12-year-old girl with a painful, progressive swelling of the medial portion of the clavicle with no history of trauma or other constitutional symptoms. All laboratory investigations were normal except for an elevated erythrocyte sedimentation rate (ESR. Initial plain radiographs showed a destructive lesion with magnetic resonance imaging showing features of malignancy. Biopsies revealed osteomyelitis, but with negative bacterial cultures and no evidence of malignancy. Treatment with antibiotics did not result in a favourable response. Over time, the swelling increased in size with episodic exacerbations of pain. Follow-up radiographs showed sclerosis and hyperostosis. After five years, this was recognized as non-bacterial chronic recurrent osteomyelitis of the clavicle.

Effect of CPP/ACP on Initial Bioadhesion to Enamel and Dentin In Situ

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Susann Grychtol

2014-01-01

Full Text Available The present in situ study investigated the influence of a preparation containing CPP/ACP (caseinphosphopeptide-amorphous calcium phosphate (GC Tooth mousse on initial bacterial colonization of enamel and dentin. Therefore, pellicle formation was performed in situ on bovine enamel and dentin specimens fixed to individual upper jaw splints worn by 8 subjects. After 1 min of pellicle formation GC Tooth mousse was used according to manufacturer’s recommendations. Rinses with chlorhexidine served as positive controls. Specimens carried without any rinse served as negative controls. After 8 h overnight exposure of the splints, bacterial colonization was quantified by fluorescence microscopy (DAPI and BacLight live/dead staining. Additionally, the colony forming units (CFU were determined after desorption. Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight. There was no significant impact of CPP/ACP on initial bacterial colonization proved with DAPI and BacLight. Determination of CFU showed statistical significance for CPP/ACP to reduce bacterial adherence on enamel. The in vitro investigation indicated no antimicrobial effects for CPP/ACP on Streptococcus mutans suspension. Under the chosen conditions, CPP/ACP (GC Tooth mousse had no significant impact on initial biofilm formation on dental hard tissues. The tested preparation cannot be recommended for biofilm management.

Novel core promoter elements and a cognate transcription factor in the divergent unicellular eukaryote Trichomonas vaginalis.

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Smith, Alias J; Chudnovsky, Lorissa; Simoes-Barbosa, Augusto; Delgadillo-Correa, Maria G; Jonsson, Zophonias O; Wohlschlegel, James A; Johnson, Patricia J

2011-04-01

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5' untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif.

Novel Core Promoter Elements and a Cognate Transcription Factor in the Divergent Unicellular Eukaryote Trichomonas vaginalis▿

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Smith, Alias J.; Chudnovsky, Lorissa; Simoes-Barbosa, Augusto; Delgadillo-Correa, Maria G.; Jonsson, Zophonias O.; Wohlschlegel, James A.; Johnson, Patricia J.

2011-01-01

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5′ untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif. PMID:21245378

Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs.

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Kutejova, Eva; Sasai, Noriaki; Shah, Ankita; Gouti, Mina; Briscoe, James

2016-03-21

In the vertebrate neural tube, a morphogen-induced transcriptional network produces multiple molecularly distinct progenitor domains, each generating different neuronal subtypes. Using an in vitro differentiation system, we defined gene expression signatures of distinct progenitor populations and identified direct gene-regulatory inputs corresponding to locations of specific transcription factor binding. Combined with targeted perturbations of the network, this revealed a mechanism in which a progenitor identity is installed by active repression of the entire transcriptional programs of other neural progenitor fates. In the ventral neural tube, sonic hedgehog (Shh) signaling, together with broadly expressed transcriptional activators, concurrently activates the gene expression programs of several domains. The specific outcome is selected by repressive input provided by Shh-induced transcription factors that act as the key nodes in the network, enabling progenitors to adopt a single definitive identity from several initially permitted options. Together, the data suggest design principles relevant to many developing tissues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Dynamics of immune system gene expression upon bacterial challenge and wounding in a social insect (Bombus terrestris.

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Silvio Erler

2011-03-01

Full Text Available The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT. There is a lack of immune genes in social insects (e.g. honeybees when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals. The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge.Antimicrobial peptides (AMP (abaecin, defensin 1, hymenoptaecin were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish and JNK pathway (basket. Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment.Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the

Molecular mechanisms of cell-cell spread of intracellular bacterial pathogens.

Science.gov (United States)

Ireton, Keith

2013-07-17

Several bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and Rickettsia spp., have evolved mechanisms to actively spread within human tissues. Spreading is initiated by the pathogen-induced recruitment of host filamentous (F)-actin. F-actin forms a tail behind the microbe, propelling it through the cytoplasm. The motile pathogen then encounters the host plasma membrane, forming a bacterium-containing protrusion that is engulfed by an adjacent cell. Over the past two decades, much progress has been made in elucidating mechanisms of F-actin tail formation. Listeria and Shigella produce tails of branched actin filaments by subverting the host Arp2/3 complex. By contrast, Rickettsia forms tails with linear actin filaments through a bacterial mimic of eukaryotic formins. Compared with F-actin tail formation, mechanisms controlling bacterial protrusions are less well understood. However, recent findings have highlighted the importance of pathogen manipulation of host cell-cell junctions in spread. Listeria produces a soluble protein that enhances bacterial protrusions by perturbing tight junctions. Shigella protrusions are engulfed through a clathrin-mediated pathway at 'tricellular junctions'--specialized membrane regions at the intersection of three epithelial cells. This review summarizes key past findings in pathogen spread, and focuses on recent developments in actin-based motility and the formation and internalization of bacterial protrusions.

Effects of Fe nanoparticles on bacterial growth and biosurfactant production

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Liu, Jia; Vipulanandan, Cumaraswamy; Cooper, Tim F.; Vipulanandan, Geethanjali

2013-01-01

Environmental conditions can have a major impact on bacterial growth and production of secondary products. In this study, the effect of different concentrations of Fe nanoparticles on the growth of Serratia sp. and on its production of a specific biosurfactant was investigated. The Fe nanoparticles were produced using the foam method, and the needle-shaped nanoparticles were about 30 nm in diameter. It was found that Fe nanoparticles can have either a positive or a negative impact on the bacterial growth and biosurfactant production, depending on their concentration. At 1 mg/L of Fe nanoparticle concentration the bacterial growth increased by 57 % and biosurfactant production increased by 63 %. When the Fe nanoparticle concentration was increased to 1 g/L, the bacterial growth decreased by 77 % and biosurfactant activity was undetectable. The biosurfactant itself was not directly affected by Fe nanoparticles over the range of concentrations studied, indicating that the observed changes in biosurfactant activity resulted indirectly from the effect of nanoparticles on the bacteria. These negative effects with nanoparticle exposures were temporary, demonstrated by the restoration of biosurfactant activity when the bacteria initially exposed to Fe nanoparticles were allowed to regrow in the absence of nanoparticles. Finally, the kinetics of bacterial growth and biosurfactant production were modeled. The model's predictions agreed with the experimental results.

Aerobic cyanide degradation by bacterial isolates from cassava factory wastewater.

Science.gov (United States)

Kandasamy, Sujatha; Dananjeyan, Balachandar; Krishnamurthy, Kumar; Benckiser, Gero

2015-01-01

Ten bacterial strains that utilize cyanide (CN) as a nitrogen source were isolated from cassava factory wastewater after enrichment in a liquid media containing sodium cyanide (1 mM) and glucose (0.2% w/v). The strains could tolerate and grow in cyanide concentrations of up to 5 mM. Increased cyanide levels in the media caused an extension of lag phase in the bacterial growth indicating that they need some period of acclimatisation. The rate of cyanide removal by the strains depends on the initial cyanide and glucose concentrations. When initial cyanide and glucose concentrations were increased up to 5 mM, cyanide removal rate increased up to 63 and 61 per cent by Bacillus pumilus and Pseudomonas putida. Metabolic products such as ammonia and formate were detected in culture supernatants, suggesting a direct hydrolytic pathway without an intermediate formamide. The study clearly demonstrates the potential of aerobic treatment with cyanide degrading bacteria for cyanide removal in cassava factory wastewaters.

Gene expression in gut symbiotic organ of stinkbug affected by extracellular bacterial symbiont.

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Futahashi, Ryo; Tanaka, Kohjiro; Tanahashi, Masahiko; Nikoh, Naruo; Kikuchi, Yoshitomo; Lee, Bok Luel; Fukatsu, Takema

2013-01-01

The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations.

A gonococcal homologue of meningococcal γ-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent

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Watanabe Haruo

2005-10-01

Full Text Available Abstract Background It has been speculated that the γ-glutamyl transpeptidase (ggt gene is present only in Neisseria meningitidis and not among related species such as Neisseria gonorrhoeae and Neisseria lactamica, because N. meningitidis is the only bacterium with GGT activity. However, nucleotide sequences highly homologous to the meningococcal ggt gene were found in the genomes of N. gonorrhoeae isolates. Results The gonococcal homologue (ggt gonococcal homologue; ggh was analyzed. The nucleotide sequence of the ggh gene was approximately 95 % identical to that of the meningococcal ggt gene. An open reading frame in the ggh gene was disrupted by an ochre mutation and frameshift mutations induced by a 7-base deletion, but the amino acid sequences deduced from the artificially corrected ggh nucleotide sequences were approximately 97 % identical to that of the meningococcal ggt gene. The analyses of the sequences flanking the ggt and ggh genes revealed that both genes were localized in a common DNA region containing the fbp-ggt (or ggh-glyA-opcA-dedA-abcZ gene cluster. The expression of the ggh RNA could be detected by dot blot, RT-PCR and primer extension analyses. Moreover, the truncated form of ggh-translational product was also found in some of the gonococcal isolates. Conclusion This study has shown that the gonococcal ggh gene is a pseudogene of the meningococcal ggt gene, which can also be designated as Ψggt. The gonococcal ggh (Ψggt gene is the first identified bacterial pseudogene that is transcriptionally active but phenotypically silent.

Directing traffic on DNA-How transcription factors relieve or induce transcriptional interference.

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Hao, Nan; Palmer, Adam C; Dodd, Ian B; Shearwin, Keith E

2017-03-15

Transcriptional interference (TI) is increasingly recognized as a widespread mechanism of gene control, particularly given the pervasive nature of transcription, both sense and antisense, across all kingdoms of life. Here, we discuss how transcription factor binding kinetics strongly influence the ability of a transcription factor to relieve or induce TI.

The transcription factor Mlc promotes Vibrio cholerae biofilm formation through repression of phosphotransferase system components.

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Pickering, Bradley S; Lopilato, Jane E; Smith, Daniel R; Watnick, Paula I

2014-07-01

The phosphoenol phosphotransferase system (PTS) is a multicomponent signal transduction cascade that regulates diverse aspects of bacterial cellular physiology in response to the availability of high-energy sugars in the environment. Many PTS components are repressed at the transcriptional level when the substrates they transport are not available. In Escherichia coli, the transcription factor Mlc (for makes large colonies) represses transcription of the genes encoding enzyme I (EI), histidine protein (HPr), and the glucose-specific enzyme IIBC (EIIBC(Glc)) in defined media that lack PTS substrates. When glucose is present, the unphosphorylated form of EIIBC(Glc) sequesters Mlc to the cell membrane, preventing its interaction with DNA. Very little is known about Vibrio cholerae Mlc. We found that V. cholerae Mlc activates biofilm formation in LB broth but not in defined medium supplemented with either pyruvate or glucose. Therefore, we questioned whether V. cholerae Mlc functions differently than E. coli Mlc. Here we have shown that, like E. coli Mlc, V. cholerae Mlc represses transcription of PTS components in both defined medium and LB broth and that E. coli Mlc is able to rescue the biofilm defect of a V. cholerae Δmlc mutant. Furthermore, we provide evidence that Mlc indirectly activates transcription of the vps genes by repressing expression of EI. Because activation of the vps genes by Mlc occurs under only a subset of the conditions in which repression of PTS components is observed, we conclude that additional inputs present in LB broth are required for activation of vps gene transcription by Mlc. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

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Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

A critique on nuclear factor-kappa B and signal transducer and activator of transcription 3: The key transcription factors in periodontal pathogenesis

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Ranjith Ambili

2017-01-01

Full Text Available Periodontal disease is initiated by microorganisms in dental plaque, and host immunoinflammatory response to the microbial challenge helps in disease progression. Conventional periodontal therapy was mainly targeted on the elimination of microbial component. However, a better understanding of molecular aspects in host response will enable the clinicians to formulate effective host modulation therapy (HMT for the periodontal management. Inflammatory mediators were the main targets for HMT in the past. Transcription factors can regulate the production of multiple mediators simultaneously, and inhibition of these factors will be more beneficial than blocking individual molecule. Two important transcription factors implicated in chronic inflammatory diseases are nuclear factor kappa B (NF-κB and signal transducers and activators of transcription 3. The role of these factors in periodontal disease is a less explored area. This comprehensive review is aimed at unveiling the critical role of NF-κB and signal transducers and activators of transcription 3 in periodontal pathogenesis. An online search was performed using MEDLINE/PubMed database. All publications till 2016 related to NF-κB, signal transducer and activator of transcription 3 (STAT3, and inflammation were included in writing this review. A total of 27,390 references were published based on the search terms used. Out of these, 507 were related to the periodontal research published in English till 2016. Relevant papers were chosen after carefully reading the abstract. This review has attempted to comprehend the existing knowledge regarding the role of transcription factors NF-κB and STAT3 in periodontal disease. Moreover, it also provides a connecting molecular link for the periodontal medicine concept.

Initiation of DNA replication requires actin dynamics and formin activity.

Science.gov (United States)

Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

2017-11-02

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation

Science.gov (United States)

Barchinger, Sarah E.; Pirbadian, Sahand; Baker, Carol S.; Leung, Kar Man; Burroughs, Nigel J.; El-Naggar, Mohamed Y.

2016-01-01

ABSTRACT In limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates that S. oneidensis MR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of the mtrA and mtrC homologs mtrF and mtrD either remains unaffected or decreases under these conditions. The ompW gene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream of mtrC and omcA. The transcriptome and mutant analyses of S. oneidensis MR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires. IMPORTANCE Shewanella oneidensis MR-1 has the capacity to transfer electrons to its external surface

Transcriptional profiling at different sites in lungs of pigs during acute bacterial respiratory infection

DEFF Research Database (Denmark)

Mortensen, Shila; Skovgaard, Kerstin; Hedegaard, Jakob

2011-01-01

The local transcriptional response was studied in different locations of lungs from pigs experimentally infected with the respiratory pathogen Actinobacillus pleuropneumoniae serotype 5B, using porcine cDNA microarrays. This infection gives rise to well-demarcated infection loci in the lung...... of apoptosis and the complement system. Interferon-g was downregulated in both necrotic and bordering areas. Evidence of neutrophil recruitment was seen by the up-regulation of chemotactic factors for neutrophils. In conclusion, we found subsets of genes expressed at different levels in the three selected...... of induced genes as, in unaffected areas a large part of differently expressed genes were involved in systemic reactions to infections, while differently expressed genes in necrotic areas were mainly concerned with homeostasis regulation....

Ionic solution and nanoparticle assisted MALDI-MS as bacterial biosensors for rapid analysis of yogurt.

Science.gov (United States)

Lee, Chia-Hsun; Gopal, Judy; Wu, Hui-Fen

2012-01-15

Bacterial analysis from food samples is a highly challenging task because food samples contain intensive interferences from proteins and carbohydrates. Three different conditions of yogurt were analyzed: (1) the fresh yogurt immediately after purchasing, (2) the yogurt after expiry date stored in the refrigerator and (3) the yogurt left outside, without refrigeration. The shelf lives of both these yogurt was compared in terms of the decrease in bacterial signals. AB which initially contained 10(9) cells/mL drastically reduced to 10(7) cells/mL. However, Lin (Feng-Yin) yogurt which initially (fresh) had 10(8) cells/mL, even after two weeks beyond the expiry period showed no marked drop in bacterial count. Conventional MALDI-MS analysis showed limited sensitivity for analysis of yogurt bacteria amidst the complex milk proteins present in yogurt. A cost effective ionic solution, CrO(4)(2-) solution was used to enable the successful detection of bacterial signals (40-fold increased in sensitivity) selectively without the interference of the milk proteins. 0.035 mg of Ag nanoparticles (NPs) were also found to improve the detection of bacteria 2-6 times in yogurt samples. The current approach can be further applied as a rapid, sensitive and effective platform for bacterial analysis from food. Copyright © 2011 Elsevier B.V. All rights reserved.

[Congenital skull base defect causing recurrent bacterial meningitis].

Science.gov (United States)

Berliner, Elihay; Bar Meir, Maskit; Megged, Orli

2012-08-01

Bacterial meningitis is a life threatening disease. Most patients will experience only one episode throughout life. Children who experience bacterial meningitis more than once, require further immunologic or anatomic evaluation. We report a 9 year old child with five episodes of bacterial meningitis due to a congenital defect of the skull base. A two and a half year old boy first presented to our medical center with pneumococcal meningitis. He was treated with antibiotics and fully recovered. Two months later he presented again with a similar clinical picture. Streptococcus pneumoniae grew in cerebrospinal fluid (CSF) culture. CT scan and later MRI of the brain revealed a defect in the anterior middle fossa floor, with protrusion of brain tissue into the sphenoidal sinus. Corrective surgery was recommended but the parents refused. Three months later, a third episode of pneumococcal meningitis occurred. The child again recovered with antibiotics and this time corrective surgery was performed. Five years later, the boy presented once again with clinical signs and symptoms consistent with bacterial meningitis. CSF culture was positive, but the final identification of the bacteria was conducted by broad spectrum 16S ribosomal RNA PCR (16S rRNA PCR) which revealed a sequence of Neisseria lactamica. CT and MRI showed recurrence of the skull base defect with encephalocele in the sphenoid sinus. The parents again refused neurosurgical intervention. A year later the patient presented with bacterial meningitis. CSF culture obtained after initiation of antibiotics was negative, but actinobacillus was identified in the CSF by 16S rRNA PCR. The patient is scheduled for neurosurgical intervention. In patients with recurrent bacterial meningitis caused by organisms colonizing the oropharynx or nasopharynx, an anatomical defect should be carefully sought and surgically repaired.

Bacterial acquisition in juveniles of several broadcast spawning coral species.

Science.gov (United States)

Sharp, Koty H; Ritchie, Kim B; Schupp, Peter J; Ritson-Williams, Raphael; Paul, Valerie J

2010-05-28

Coral animals harbor diverse microorganisms in their tissues, including archaea, bacteria, viruses, and zooxanthellae. The extent to which coral-bacterial associations are specific and the mechanisms for their maintenance across generations in the environment are unknown. The high diversity of bacteria in adult coral colonies has made it challenging to identify species-specific patterns. Localization of bacteria in gametes and larvae of corals presents an opportunity for determining when bacterial-coral associations are initiated and whether they are dynamic throughout early development. This study focuses on the early onset of bacterial associations in the mass spawning corals Montastraea annularis, M. franksi, M. faveolata, Acropora palmata, A. cervicornis, Diploria strigosa, and A. humilis. The presence of bacteria and timing of bacterial colonization was evaluated in gametes, swimming planulae, and newly settled polyps by fluorescence in situ hybridization (FISH) using general eubacterial probes and laser-scanning confocal microscopy. The coral species investigated in this study do not appear to transmit bacteria via their gametes, and bacteria are not detectable in or on the corals until after settlement and metamorphosis. This study suggests that mass-spawning corals do not acquire, or are not colonized by, detectable numbers of bacteria until after larval settlement and development of the juvenile polyp. This timing lays the groundwork for developing and testing new hypotheses regarding general regulatory mechanisms that control bacterial colonization and infection of corals, and how interactions among bacteria and juvenile polyps influence the structure of bacterial assemblages in corals.

Whole genome transcription profiling of Anaplasma phagocytophilum in human and tick host cells by tiling array analysis

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Chavez Adela

2008-07-01

Full Text Available Abstract Background Anaplasma phagocytophilum (Ap is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. Ap alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6 and pathogenesis (human; HL-60 and HMEC-1. Results Detailed, host cell specific transcriptional behavior was revealed. There was extensive differential Ap gene transcription between the tick (ISE6 and the human (HL-60 and HMEC-1 cell lines, with far fewer differentially transcribed genes between the human cell lines, and all disproportionately represented by membrane or surface proteins. There were Ap genes exclusively transcribed in each cell line, apparent human- and tick-specific operons and paralogs, and anti-sense transcripts that suggest novel expression regulation processes. Seven virB2 paralogs (of the bacterial type IV secretion system showed human or tick cell dependent transcription. Previously unrecognized genes and coding sequences were identified, as were the expressed p44/msp2 (major surface proteins paralogs (of 114 total, through elevated signal produced to the unique hypervariable region of each – 2/114 in HL-60, 3/114 in HMEC-1, and none in ISE6. Conclusion Using these methods, whole genome transcription profiles can likely be generated for Ap, as well as other obligate intracellular organisms, in any host cells and for all stages of the cell infection process. Visual representation of comprehensive transcription data alongside an annotated map of the genome renders complex transcription into discernable patterns.

n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

International Nuclear Information System (INIS)

Diakos, Christos; Prieschl, Eva E.; Saeemann, Marcus D.; Boehmig, Georg A.; Csonga, Robert; Sobanov, Yury; Baumruker, Thomas; Zlabinger, Gerhard J.

2006-01-01

Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-α transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling

A short-time scale colloidal system reveals early bacterial adhesion dynamics.

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Christophe Beloin

2008-07-01

Full Text Available The development of bacteria on abiotic surfaces has important public health and sanitary consequences. However, despite several decades of study of bacterial adhesion to inert surfaces, the biophysical mechanisms governing this process remain poorly understood, due, in particular, to the lack of methodologies covering the appropriate time scale. Using micrometric colloidal surface particles and flow cytometry analysis, we developed a rapid multiparametric approach to studying early events in adhesion of the bacterium Escherichia coli. This approach simultaneously describes the kinetics and amplitude of early steps in adhesion, changes in physicochemical surface properties within the first few seconds of adhesion, and the self-association state of attached and free-floating cells. Examination of the role of three well-characterized E. coli surface adhesion factors upon attachment to colloidal surfaces--curli fimbriae, F-conjugative pilus, and Ag43 adhesin--showed clear-cut differences in the very initial phases of surface colonization for cell-bearing surface structures, all known to promote biofilm development. Our multiparametric analysis revealed a correlation in the adhesion phase with cell-to-cell aggregation properties and demonstrated that this phenomenon amplified surface colonization once initial cell-surface attachment was achieved. Monitoring of real-time physico-chemical particle surface properties showed that surface-active molecules of bacterial origin quickly modified surface properties, providing new insight into the intricate relations connecting abiotic surface physicochemical properties and bacterial adhesion. Hence, the biophysical analytical method described here provides a new and relevant approach to quantitatively and kinetically investigating bacterial adhesion and biofilm development.

SMC Progressively Aligns Chromosomal Arms in Caulobacter crescentus but Is Antagonized by Convergent Transcription

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Ngat T. Tran

2017-08-01

Full Text Available The structural maintenance of chromosomes (SMC complex plays an important role in chromosome organization and segregation in most living organisms. In Caulobacter crescentus, SMC is required to align the left and the right arms of the chromosome that run in parallel down the long axis of the cell. However, the mechanism of SMC-mediated alignment of chromosomal arms remains elusive. Here, using genome-wide methods and microscopy of single cells, we show that Caulobacter SMC is recruited to the centromeric parS site and that SMC-mediated arm alignment depends on the chromosome-partitioning protein ParB. We provide evidence that SMC likely tethers the parS-proximal regions of the chromosomal arms together, promoting arm alignment. Furthermore, we show that highly transcribed genes near parS that are oriented against SMC translocation disrupt arm alignment, suggesting that head-on transcription interferes with SMC translocation. Our results demonstrate a tight interdependence of bacterial chromosome organization and global patterns of transcription.

Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function.

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Xiaofei Gao

2009-12-01

Full Text Available Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC, Salmonella, Shigella, Yersinia utilize a type III secretion system (T3SS to inject virulence proteins (effectors into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3, a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kappaB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-kappaB chaperone IkappaBalpha NleH1 repressed the transcription of a RPS3/NF-kappaB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well

PEROXOTITANATE- AND MONOSODIUM METAL-TITANATE COMPOUNDS AS INHIBITORS OF BACTERIAL GROWTH

Energy Technology Data Exchange (ETDEWEB)

Hobbs, D.

2011-01-19

Sodium titanates are ion-exchange materials that effectively bind a variety of metal ions over a wide pH range. Sodium titanates alone have no known adverse biological effects but metal-exchanged titanates (or metal titanates) can deliver metal ions to mammalian cells to alter cell processes in vitro. In this work, we test a hypothesis that metal-titanate compounds inhibit bacterial growth; demonstration of this principle is one prerequisite to developing metal-based, titanate-delivered antibacterial agents. Focusing initially on oral diseases, we exposed five species of oral bacteria to titanates for 24 h, with or without loading of Au(III), Pd(II), Pt(II), and Pt(IV), and measuring bacterial growth in planktonic assays through increases in optical density. In each experiment, bacterial growth was compared with control cultures of titanates or bacteria alone. We observed no suppression of bacterial growth by the sodium titanates alone, but significant (p < 0.05, two-sided t-tests) suppression was observed with metal-titanate compounds, particularly Au(III)-titanates, but with other metal titanates as well. Growth inhibition ranged from 15 to 100% depending on the metal ion and bacterial species involved. Furthermore, in specific cases, the titanates inhibited bacterial growth 5- to 375-fold versus metal ions alone, suggesting that titanates enhanced metal-bacteria interactions. This work supports further development of metal titanates as a novel class of antibacterials.

Orchestration of Floral Initiation by APETALA1

NARCIS (Netherlands)

Kaufmann, K.; Muino Acuna, J.M.

2010-01-01

The MADS-domain transcription factor APETALA1 (AP1) is a key regulator of Arabidopsis flower development. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiling and genome-wide binding

Bacterial meningitis in newborn and infant: correlation between organism, CT findings and clinical outcome

International Nuclear Information System (INIS)

Choi, Hye Young; Park, Young Seo; Yoo, Shi Joon; Suh, Dae Chul; Chung, Young Kyo

1993-01-01

Acute bacterial meningitis often results in significant neurologic complications regardless of the antibiotics treatment Computed tomographic (CT) finding of tuberculous meningitis is fairly well known but not the findings of bacterial meningitis. This study was performed to determine the incidence of causative organisms and to correlate between the organisms and computed tomographic (CT) findings with clinical outcome of bacterial meningitis in newborns and infants. We analyzed the brain CT and clinical records of 15 infants who had been diagnosed as bacterial meningitis by CSF culture. We found that the most common organisms were Group B streptococcus in neonates without no neurologic complications in all but one and Hemophilus influenza in infants whose clinical outcomes were poor in all except one. CT findings related with poor prognosis in this study were cerebral edema, basal cisternal obliteration and enhancement, and cerebral infarction on initial CT and ventriculomegaly on follow-up CT. We concluded that CT diagnosed intracranial complications of bacterial meningitis well and could contributed to better treatment of bacterial meningitis

Engineering bacterial translation initiation - Do we have all the tools we need?

Science.gov (United States)

Vigar, Justin R J; Wieden, Hans-Joachim

2017-11-01

Reliable tools that allow precise and predictable control over gene expression are critical for the success of nearly all bioengineering applications. Translation initiation is the most regulated phase during protein biosynthesis, and is therefore a promising target for exerting control over gene expression. At the translational level, the copy number of a protein can be fine-tuned by altering the interaction between the translation initiation region of an mRNA and the ribosome. These interactions can be controlled by modulating the mRNA structure using numerous approaches, including small molecule ligands, RNAs, or RNA-binding proteins. A variety of naturally occurring regulatory elements have been repurposed, facilitating advances in synthetic gene regulation strategies. The pursuit of a comprehensive understanding of mechanisms governing translation initiation provides the framework for future engineering efforts. Here we outline state-of-the-art strategies used to predictably control translation initiation in bacteria. We also discuss current limitations in the field and future goals. Due to its function as the rate-determining step, initiation is the ideal point to exert effective translation regulation. Several engineering tools are currently available to rationally design the initiation characteristics of synthetic mRNAs. However, improvements are required to increase the predictability, effectiveness, and portability of these tools. Predictable and reliable control over translation initiation will allow greater predictability when designing, constructing, and testing genetic circuits. The ability to build more complex circuits predictably will advance synthetic biology and contribute to our fundamental understanding of the underlying principles of these processes. "This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier

Specificity and transcriptional activity of microbiota associated with low and high microbial abundance sponges from the Red Sea

KAUST Repository

Moitinho-Silva, Lucas

2013-08-20

Marine sponges are generally classified as high microbial abundance (HMA) and low microbial abundance (LMA) species. Here, 16S rRNA amplicon sequencing was applied to investigate the diversity, specificity and transcriptional activity of microbes associated with an LMA sponge (Stylissa carteri), an HMA sponge (Xestospongia testudinaria) and sea water collected from the central Saudi Arabia coast of the Red Sea. Altogether, 887 068 denoised sequences were obtained, of which 806 661 sequences remained after quality control. This resulted in 1477 operational taxonomic units (OTUs) that were assigned to 27 microbial phyla. The microbial composition of S. carteri was more similar to that of sea water than to that of X. testudinaria, which is consistent with the observation that the sequence data set of S. carteri contained many more possibly sea water sequences (~24%) than the X. testudinaria data set (~6%). The most abundant OTUs were shared between all three sources (S. carteri, X. testudinaria, sea water), while rare OTUs were unique to any given source. Despite this high degree of overlap, each sponge species contained its own specific microbiota. The X. testudinaria-specific bacterial taxa were similar to those already described for this species. A set of S. carteri-specific bacterial taxa related to Proteobacteria and Nitrospira was identified, which are likely permanently associated with S. carteri. The transcriptional activity of sponge-associated microorganisms correlated well with their abundance. Quantitative PCR revealed the presence of Poribacteria, representing typical sponge symbionts, in both sponge species and in sea water; however, low transcriptional activity in sea water suggested that Poribacteria are not active outside the host context. © 2013 John Wiley & Sons Ltd.

Collective decisions among bacterial viruses

Science.gov (United States)

Joh, Richard; Mileyko, Yuriy; Voit, Eberhard; Weitz, Joshua

2010-03-01

For many temperate bacteriophages, the decision of whether to kill hosts or enter a latent state depends on the multiplicity of infection. In this talk, I present a quantitative model of gene regulatory dynamics to describe how phages make collective decisions within host cells. Unlike most previous studies, the copy number of viral genomes is treated as a variable. In the absence of feedback loops, viral mRNA transcription is expected to be proportional to the viral copy number. However, when there are nonlinear feedback loops in viral gene regulation, our model shows that gene expression patterns are sensitive to changes in viral copy number and there can be a domain of copy number where the system becomes bistable. Hence, the viral copy number is a key control parameter determining host cell fates. This suggests that bacterial viruses can respond adaptively to changes in population dynamics, and can make alternative decisions as a bet-hedging strategy. Finally, I present a stochastic version of viral gene regulation and discuss speed-accuracy trade-offs in the context of cell fate determination by viruses.

Bacterial mycophagy: definition and diagnosis of a unique bacterial-fungal interaction

NARCIS (Netherlands)

Leveau, J.H.J.; Preston, G.M.

2008-01-01

This review analyses the phenomenon of bacterial mycophagy, which we define as a set of phenotypic behaviours that enable bacteria to obtain nutrients from living fungi and thus allow the conversion of fungal into bacterial biomass. We recognize three types of bacterial strategies to derive

Time-resolved dual RNA-seq reveals extensive rewiring of lung epithelial and pneumococcal transcriptomes during early infection

NARCIS (Netherlands)

Aprianto, Rieza; Slager, Jelle; Holsappel, Siger; Veening, Jan-Willem

2016-01-01

BACKGROUND: Streptococcus pneumoniae, the pneumococcus, is the main etiological agent of pneumonia. Pneumococcal infection is initiated by bacterial adherence to lung epithelial cells. The exact transcriptional changes occurring in both host and microbe during infection are unknown. Here, we

Development and validation of quantitative PCR assays to measure cytokine transcript levels in the Florida manatee (Trichechus manatus latirostris)

Science.gov (United States)

Ferrante, Jason; Hunter, Margaret; Wellehan, James F.X.

2018-01-01

Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees (Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α, and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher (Puse as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

Energy spectrum scaling in an agent-based model for bacterial turbulence

Science.gov (United States)

Mikel-Stites, Maxwell; Staples, Anne

2017-11-01

Numerous models have been developed to examine the behavior of dense bacterial swarms and to explore the visually striking phenomena of bacterial turbulence. Most models directly impose fluid dynamics physics, either by modeling the active matter as a fluid or by including interactions between the bacteria and a fluid. In this work, however, the `turbulence' is solely an emergent property of the collective behavior of the bacterial population, rather than a consequence of imposed fluid dynamics physical modeling. The system is simulated using a two dimensional Vicsek-style model, with the addition of individual repulsion to simulate bacterial collisions and physical interactions, and without the common flocking or sensing behaviors. Initial results indicate the presence of k-1 scaling in a portion of the kinetic energy spectrum that can be considered analogous to the inertial subrange in turbulent energy spectra. This result suggests that the interaction of large numbers of individual active bacteria may also be a contributing factor in the emergence of fluid dynamics phenomena, in addition to the physical interactions between bacteria and their fluid environment.

Translation elicits a growth rate-dependent, genome-wide, differential protein production in Bacillus subtilis.

Science.gov (United States)

Borkowski, Olivier; Goelzer, Anne; Schaffer, Marc; Calabre, Magali; Mäder, Ulrike; Aymerich, Stéphane; Jules, Matthieu; Fromion, Vincent

2016-05-17

Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

WRKY transcription factors

Science.gov (United States)

Bakshi, Madhunita; Oelmüller, Ralf

2014-01-01

WRKY transcription factors are one of the largest families of transcriptional regulators found exclusively in plants. They have diverse biological functions in plant disease resistance, abiotic stress responses, nutrient deprivation, senescence, seed and trichome development, embryogenesis, as well as additional developmental and hormone-controlled processes. WRKYs can act as transcriptional activators or repressors, in various homo- and heterodimer combinations. Here we review recent progress on the function of WRKY transcription factors in Arabidopsis and other plant species such as rice, potato, and parsley, with a special focus on abiotic, developmental, and hormone-regulated processes. PMID:24492469

Reconstruction of the core and extended regulons of global transcription factors.

Directory of Open Access Journals (Sweden)

Yann S Dufour

2010-07-01

Full Text Available The processes underlying the evolution of regulatory networks are unclear. To address this question, we used a comparative genomics approach that takes advantage of the large number of sequenced bacterial genomes to predict conserved and variable members of transcriptional regulatory networks across phylogenetically related organisms. Specifically, we developed a computational method to predict the conserved regulons of transcription factors across alpha-proteobacteria. We focused on the CRP/FNR super-family of transcription factors because it contains several well-characterized members, such as FNR, FixK, and DNR. While FNR, FixK, and DNR are each proposed to regulate different aspects of anaerobic metabolism, they are predicted to recognize very similar DNA target sequences, and they occur in various combinations among individual alpha-proteobacterial species. In this study, the composition of the respective FNR, FixK, or DNR conserved regulons across 87 alpha-proteobacterial species was predicted by comparing the phylogenetic profiles of the regulators with the profiles of putative target genes. The utility of our predictions was evaluated by experimentally characterizing the FnrL regulon (a FNR-type regulator in the alpha-proteobacterium Rhodobacter sphaeroides. Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks. Our findings also predict that, at least for the FNR-type regulators, there is a core set of target genes conserved across many species. In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors. The comparative genomics approach we developed is readily applicable to other

Effects of Fe nanoparticles on bacterial growth and biosurfactant production

Energy Technology Data Exchange (ETDEWEB)

Liu Jia; Vipulanandan, Cumaraswamy, E-mail: cvipulanandan@uh.edu [University of Houston, Department of Civil and Environmental Engineering (United States); Cooper, Tim F. [University of Houston, Department of Biology and Biochemistry (United States); Vipulanandan, Geethanjali [University of Houston, Department of Biomedical Engineering (United States)

2013-01-15

Environmental conditions can have a major impact on bacterial growth and production of secondary products. In this study, the effect of different concentrations of Fe nanoparticles on the growth of Serratia sp. and on its production of a specific biosurfactant was investigated. The Fe nanoparticles were produced using the foam method, and the needle-shaped nanoparticles were about 30 nm in diameter. It was found that Fe nanoparticles can have either a positive or a negative impact on the bacterial growth and biosurfactant production, depending on their concentration. At 1 mg/L of Fe nanoparticle concentration the bacterial growth increased by 57 % and biosurfactant production increased by 63 %. When the Fe nanoparticle concentration was increased to 1 g/L, the bacterial growth decreased by 77 % and biosurfactant activity was undetectable. The biosurfactant itself was not directly affected by Fe nanoparticles over the range of concentrations studied, indicating that the observed changes in biosurfactant activity resulted indirectly from the effect of nanoparticles on the bacteria. These negative effects with nanoparticle exposures were temporary, demonstrated by the restoration of biosurfactant activity when the bacteria initially exposed to Fe nanoparticles were allowed to regrow in the absence of nanoparticles. Finally, the kinetics of bacterial growth and biosurfactant production were modeled. The model's predictions agreed with the experimental results.

Effects of Fe nanoparticles on bacterial growth and biosurfactant production

International Nuclear Information System (INIS)

Liu Jia; Vipulanandan, Cumaraswamy; Cooper, Tim F.; Vipulanandan, Geethanjali

2013-01-01

Environmental conditions can have a major impact on bacterial growth and production of secondary products. In this study, the effect of different concentrations of Fe nanoparticles on the growth of Serratia sp. and on its production of a specific biosurfactant was investigated. The Fe nanoparticles were produced using the foam method, and the needle-shaped nanoparticles were about 30 nm in diameter. It was found that Fe nanoparticles can have either a positive or a negative impact on the bacterial growth and biosurfactant production, depending on their concentration. At 1 mg/L of Fe nanoparticle concentration the bacterial growth increased by 57 % and biosurfactant production increased by 63 %. When the Fe nanoparticle concentration was increased to 1 g/L, the bacterial growth decreased by 77 % and biosurfactant activity was undetectable. The biosurfactant itself was not directly affected by Fe nanoparticles over the range of concentrations studied, indicating that the observed changes in biosurfactant activity resulted indirectly from the effect of nanoparticles on the bacteria. These negative effects with nanoparticle exposures were temporary, demonstrated by the restoration of biosurfactant activity when the bacteria initially exposed to Fe nanoparticles were allowed to regrow in the absence of nanoparticles. Finally, the kinetics of bacterial growth and biosurfactant production were modeled. The model’s predictions agreed with the experimental results.

Transcriptional portrait of Actinobacillus pleuropneumoniae during acute disease--potential strategies for survival and persistence in the host.

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Kirstine Klitgaard

Full Text Available BACKGROUND: Gene expression profiles of bacteria in their natural hosts can provide novel insight into the host-pathogen interactions and molecular determinants of bacterial infections. In the present study, the transcriptional profile of the porcine lung pathogen Actinobacillus pleuropneumoniae was monitored during the acute phase of infection in its natural host. METHODOLOGY/PRINCIPAL FINDINGS: Bacterial expression profiles of A. pleuropneumoniae isolated from lung lesions of 25 infected pigs were compared in samples taken 6, 12, 24 and 48 hours post experimental challenge. Within 6 hours, focal, fibrino hemorrhagic lesions could be observed in the pig lungs, indicating that A. pleuropneumoniae had managed to establish itself successfully in the host. We identified 237 differentially regulated genes likely to encode functions required by the bacteria for colonization and survival in the host. This group was dominated by genes involved in various aspects of energy metabolism, especially anaerobic respiration and carbohydrate metabolism. Remodeling of the bacterial envelope and modifications of posttranslational processing of proteins also appeared to be of importance during early infection. The results suggested that A. pleuropneumoniae is using various strategies to increase its fitness, such as applying Na+ pumps as an alternative way of gaining energy. Furthermore, the transcriptional data provided potential clues as to how A. pleuropneumoniae is able to circumvent host immune factors and survive within the hostile environment of host macrophages. This persistence within macrophages may be related to urease activity, mobilization of various stress responses and active evasion of the host defenses by cell surface sialylation. CONCLUSIONS/SIGNIFICANCE: The data presented here highlight the importance of metabolic adjustments to host conditions as virulence factors of infecting microorganisms and help to provide insight into the mechanisms

Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

International Nuclear Information System (INIS)

Calzone, F.J.; Britten, R.J.; Davidson, E.J.

1987-01-01

An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements

Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

Science.gov (United States)

Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

2014-07-01

Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.

The TLR2 Antagonist Staphylococcal Superantigen-Like Protein 3 Acts as a Virulence Factor to Promote Bacterial Pathogenicity in vivo

NARCIS (Netherlands)

Koymans, Kirsten J.; Goldmann, Oliver; Karlsson, Christofer A.Q.; Sital, Wiedjai; Thänert, Robert; Bisschop, Adinda; Vrieling, Manouk; Malmström, Johan; van Kessel, Kok P M; De Haas, Carla J.C.; Van Strijp, Jos A.G.; Medina, Eva

2017-01-01

Toll-like receptor (TLR) signaling is important in the initiation of immune responses and subsequent instigation of adaptive immunity. TLR2 recognizes bacterial lipoproteins and plays a central role in the host defense against bacterial infections, including those caused by Staphylococcus aureus.

The spatial biology of transcription and translation in rapidly growing Escherichia coli

Directory of Open Access Journals (Sweden)

Somenath eBakshi

2015-07-01

Full Text Available Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP in live, rapidly growing E. coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0-3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20-30 min nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription

Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus

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Kovaleva Galina

2011-06-01

Full Text Available Abstract Background Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in bacteria is one of the critical tasks of modern genomics. The Shewanella genus is comprised of metabolically versatile gamma-proteobacteria, whose lifestyles and natural environments are substantially different from Escherichia coli and other model bacterial species. The comparative genomics approaches and computational identification of regulatory sites are useful for the in silico reconstruction of transcriptional regulatory networks in bacteria. Results To explore conservation and variations in the Shewanella transcriptional networks we analyzed the repertoire of transcription factors and performed genomics-based reconstruction and comparative analysis of regulons in 16 Shewanella genomes. The inferred regulatory network includes 82 transcription factors and their DNA binding sites, 8 riboswitches and 6 translational attenuators. Forty five regulons were newly inferred from the genome context analysis, whereas others were propagated from previously characterized regulons in the Enterobacteria and Pseudomonas spp.. Multiple variations in regulatory strategies between the Shewanella spp. and E. coli include regulon contraction and expansion (as in the case of PdhR, HexR, FadR, numerous cases of recruiting non-orthologous regulators to control equivalent pathways (e.g. PsrA for fatty acid degradation and, conversely, orthologous regulators to control distinct pathways (e.g. TyrR, ArgR, Crp. Conclusions We tentatively defined the first reference collection of ~100 transcriptional regulons in 16 Shewanella genomes. The resulting regulatory network contains ~600 regulated genes per genome that are mostly involved in metabolism of carbohydrates, amino acids, fatty acids, vitamins, metals, and stress responses. Several reconstructed regulons including NagR for N-acetylglucosamine catabolism were experimentally validated in S

APOBEC3G inhibits elongation of HIV-1 reverse transcripts.

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Kate N Bishop

2008-12-01

Full Text Available APOBEC3G (A3G is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation.

The Clinical Differentiation of Bacterial and Fungal Keratitis: A Photographic Survey

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Dalmon, Cyril; Porco, Travis C.; Lietman, Thomas M.; Prajna, N. Venkatesh; Prajna, Lalitha; Das, Mano Ranjan; Kumar, J. Arun; Mascarenhas, Jeena; Margolis, Todd P.; Whitcher, John P.; Jeng, Bennie H.; Keenan, Jeremy D.; Chan, Matilda F.; McLeod, Stephen D.; Acharya, Nisha R.

2012-01-01

Purpose. The purpose of this study was to determine whether clinical signs of infectious keratitis can be used to identify the causative organism. Methods. Eighty photographs of eyes with culture-proven bacterial keratitis or smear-proven fungal keratitis were randomly selected from 2 clinical trials. Fifteen cornea specialists from the F. I. Proctor Foundation and the Aravind Eye Care System assessed the photographs for prespecified clinical signs of keratitis, and they identified the most likely causative organism. Results. Clinicians were able to correctly distinguish bacterial from fungal etiology 66% of the time (P < 0.001). The Gram stain, genus, and species were accurately predicted 46%, 25%, and 10% of the time, respectively. The presence of an irregular/feathery border was associated with fungal keratitis, whereas a wreath infiltrate or an epithelial plaque was associated with bacterial keratitis. Conclusions. Cornea specialists correctly differentiated bacterial from fungal keratitis more often than chance, but in fewer than 70% of cases. More specific categorization led to less successful clinical distinction. Although certain clinical signs of infectious keratitis may be associated with a bacterial or fungal etiology, this study highlights the importance of obtaining appropriate microbiological testing during the initial clinical encounter. (ClinicalTrials.gov number, NCT00324168.) PMID:22395880

Incidence of bacterial diseases associated with irrigation methods on onions (Allium cepa).

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Chorolque, A; Pozzo Ardizzi, C; Pellejero, G; Aschkar, G; García Navarro, F J; Jiménez Ballesta, R

2018-04-24

In the last decade, diseases of bacterial origin in onions have increased and this has led to significant losses in production. These diseases are currently observed in both the Old and New Worlds. The aim of the experimental work reported here was to evaluate whether the irrigation method influences the incidence of diseases of bacterial origin. In cases where the inoculum was natural, the initial incidence of Soft Bacterial Rot was not manifested in any treatment in the first year, whereas at the end of the conservation period all treatments had increased incidences of infection. Sprinkler irrigation (8%) was statistically differentiated from the other treatments, for which the final incidence was similar (4.5%). For all irrigation treatments, the final incidence of Bacterial Soft Rot decreased or remained stable towards the end of the cycle, with the exception of sprinkler irrigation in 2015, which increased. It can be inferred from the results that the irrigation method does have an influence on the incidence of diseases of bacterial origin in the post-harvest stage for onions. This article is protected by copyright. All rights reserved.

A computer investigation of chemically mediated detachment in bacterial biofilms.

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Hunt, Stephen M; Hamilton, Martin A; Sears, John T; Harkin, Gary; Reno, Jason

2003-05-01

A three-dimensional computer model was used to evaluate the effect of chemically mediated detachment on biofilm development in a negligible-shear environment. The model, BacLAB, combines conventional diffusion-reaction equations for chemicals with a cellular automata algorithm to simulate bacterial growth, movement and detachment. BacLAB simulates the life cycle of a bacterial biofilm from its initial colonization of a surface to the development of a mature biofilm with cell areal densities comparable to those in the laboratory. A base model founded on well established transport equations that are easily adaptable to investigate conjectures at the biological level has been created. In this study, the conjecture of a detachment mechanism involving a bacterially produced chemical detachment factor in which high local concentrations of this detachment factor cause the bacteria to detach from the biofilm was examined. The results show that the often observed 'mushroom'-shaped structure can occur if detachment events create voids so that the remaining attached cells look like mushrooms.

A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells.

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Tetsuo Yasugi

Full Text Available Neurogenesis is initiated by a set of basic Helix-Loop-Helix (bHLH transcription factors that specify neural progenitors and allow them to generate neurons in multiple rounds of asymmetric cell division. The Drosophila Daughterless (Da protein and its mammalian counterparts (E12/E47 act as heterodimerization factors for proneural genes and are therefore critically required for neurogenesis. Here, we demonstrate that Da can also be an inhibitor of the neural progenitor fate whose absence leads to stem cell overproliferation and tumor formation. We explain this paradox by demonstrating that Da induces the differentiation factor Prospero (Pros whose asymmetric segregation is essential for differentiation in one of the two daughter cells. Da co-operates with the bHLH transcription factor Asense, whereas the other proneural genes are dispensible. After mitosis, Pros terminates Asense expression in one of the two daughter cells. In da mutants, pros is not expressed, leading to the formation of lethal transplantable brain tumors. Our results define a transcriptional feedback loop that regulates the balance between self-renewal and differentiation in Drosophila optic lobe neuroblasts. They indicate that initiation of a neural differentiation program in stem cells is essential to prevent tumorigenesis.

Interplay of Gene Expression Noise and Ultrasensitive Dynamics Affects Bacterial Operon Organization

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Ray, J. Christian J; Igoshin, Oleg A.

2012-01-01

Bacterial chromosomes are organized into polycistronic cotranscribed operons, but the evolutionary pressures maintaining them are unclear. We hypothesized that operons alter gene expression noise characteristics, resulting in selection for or against maintaining operons depending on network architecture. Mathematical models for 6 functional classes of network modules showed that three classes exhibited decreased noise and 3 exhibited increased noise with same-operon cotranscription of interacting proteins. Noise reduction was often associated with a decreased chance of reaching an ultrasensitive threshold. Stochastic simulations of the lac operon demonstrated that the predicted effects of transcriptional coupling hold for a complex network module. We employed bioinformatic analysis to find overrepresentation of noise-minimizing operon organization compared with randomized controls. Among constitutively expressed physically interacting protein pairs, higher coupling frequencies appeared at lower expression levels, where noise effects are expected to be dominant. Our results thereby suggest an important role for gene expression noise, in many cases interacting with an ultrasensitive switch, in maintaining or selecting for operons in bacterial chromosomes. PMID:22956903

A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

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Lorimer, H E; Brewer, B J; Fangman, W L

1995-09-01

Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

Bacterial acquisition in juveniles of several broadcast spawning coral species.

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Koty H Sharp

Full Text Available Coral animals harbor diverse microorganisms in their tissues, including archaea, bacteria, viruses, and zooxanthellae. The extent to which coral-bacterial associations are specific and the mechanisms for their maintenance across generations in the environment are unknown. The high diversity of bacteria in adult coral colonies has made it challenging to identify species-specific patterns. Localization of bacteria in gametes and larvae of corals presents an opportunity for determining when bacterial-coral associations are initiated and whether they are dynamic throughout early development. This study focuses on the early onset of bacterial associations in the mass spawning corals Montastraea annularis, M. franksi, M. faveolata, Acropora palmata, A. cervicornis, Diploria strigosa, and A. humilis. The presence of bacteria and timing of bacterial colonization was evaluated in gametes, swimming planulae, and newly settled polyps by fluorescence in situ hybridization (FISH using general eubacterial probes and laser-scanning confocal microscopy. The coral species investigated in this study do not appear to transmit bacteria via their gametes, and bacteria are not detectable in or on the corals until after settlement and metamorphosis. This study suggests that mass-spawning corals do not acquire, or are not colonized by, detectable numbers of bacteria until after larval settlement and development of the juvenile polyp. This timing lays the groundwork for developing and testing new hypotheses regarding general regulatory mechanisms that control bacterial colonization and infection of corals, and how interactions among bacteria and juvenile polyps influence the structure of bacterial assemblages in corals.

RsaM: a transcriptional regulator of Burkholderia spp. with novel fold

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Michalska, Karolina [Midwest Center for Structural Genomics, Argonne National Laboratory, IL USA; Structural Biology Center, Biosciences Division, Argonne National Laboratory, IL USA; Chhor, Gekleng [Midwest Center for Structural Genomics, Argonne National Laboratory, IL USA; Clancy, Shonda [Midwest Center for Structural Genomics, Argonne National Laboratory, IL USA; Jedrzejczak, Robert [Midwest Center for Structural Genomics, Argonne National Laboratory, IL USA; Babnigg, Gyorgy [Midwest Center for Structural Genomics, Argonne National Laboratory, IL USA; Winans, Stephen C. [Department of Microbiology, Cornell University, Ithaca NY USA; Joachimiak, Andrzej [Midwest Center for Structural Genomics, Argonne National Laboratory, IL USA; Structural Biology Center, Biosciences Division, Argonne National Laboratory, IL USA; Department of Biochemistry and Molecular Biology, University of Chicago, IL USA

2014-07-04

Burkholderia cepacia complex (Bcc) is a set of closely related bacterial species that are notorious pathogens of cystic fibrosis patients, responsible for life-threatening lung infections. Expression of several virulence factors of Bcc is controlled by a mechanism known as quorum sensing (QS). QS is a means of bacterial communication used to coordinate gene expression in a cell-density-dependent manner. The system involves the production of diffusible signaling molecules (N-acyl-L-homoserine lactones, AHLs), that bind to cognate transcriptional regulators and influence their ability to regulate gene expression. One such system that is highly conserved in Bcc consists of CepI and CepR. CepI is AHL synthase, while CepR is an AHL-dependent transcription factor. In most members of the Bcc group, the cepI and cepR genes are divergently transcribed and separated by additional genes. One of them, bcam1869, encodes the BcRsaM protein, which was recently postulated to modulate the abundance or activity of CepI or CepR. Here we show the crystal structure of BcRsaM from B. cenocepacia J2315. It is a single-domain protein with unique topology and presents a novel fold. The protein is a dimer in the crystal and in solution. This regulator has no known DNA binding motifs and direct binding of BcRsaM to the cepI promoter could not be detected in in vitro assays. Therefore, we propose that the modulatory action of RsaM might result from interactions with other components of the QS machinery rather than from direct association with the DNA promoter.

Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo.

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Yan Coulombe

2010-05-01

Full Text Available The genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation.We have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation.Our observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.

Potential Environmental Factors Affecting Oil-Degrading Bacterial Populations in Deep and Surface Waters of the Northern Gulf of Mexico.

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Liu, Jiqing; Bacosa, Hernando P; Liu, Zhanfei

2016-01-01

Understanding bacterial community dynamics as a result of an oil spill is important for predicting the fate of oil released to the environment and developing bioremediation strategies in the Gulf of Mexico. In this study, we aimed to elucidate the roles of temperature, water chemistry (nutrients), and initial bacterial community in selecting oil degraders through a series of incubation experiments. Surface (2 m) and bottom (1537 m) waters, collected near the Deepwater Horizon site, were amended with 200 ppm light Louisiana sweet crude oil and bacterial inoculums from surface or bottom water, and incubated at 4 or 24°C for 50 days. Bacterial community and residual oil were analyzed by pyrosequencing and gas chromatography-mass spectrometry (GC-MS), respectively. The results showed that temperature played a key role in selecting oil-degrading bacteria. Incubation at 4°C favored the development of Cycloclasticus, Pseudoalteromonas , Sulfitobacter , and Reinekea , while 24°C incubations enhanced Oleibacter, Thalassobius, Phaeobacter, and Roseobacter. Water chemistry and the initial community also had potential roles in the development of hydrocarbon-degrading bacterial communities. Pseudoalteromonas , Oleibacter , and Winogradskyella developed well in the nutrient-enriched bottom water, while Reinekea and Thalassobius were favored by low-nutrient surface water. We revealed that the combination of 4°C, crude oil and bottom inoculum was a key factor for the growth of Cycloclasticus , while the combination of surface inoculum and bottom water chemistry was important for the growth of Pseudoalteromonas . Moreover, regardless of the source of inoculum, bottom water at 24°C was a favorable condition for Oleibacter. Redundancy analysis further showed that temperature and initial community explained 57 and 19% of the variation observed, while oil and water chemistry contributed 14 and 10%, respectively. Overall, this study revealed the relative roles of temperature, water

Empirical treatment of bacterial keratitis: an international survey of corneal specialists.

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Austin, Ariana; Schallhorn, Julie; Geske, Mike; Mannis, Mark; Lietman, Tom; Rose-Nussbaumer, Jennifer

2017-08-01

New antibiotic agents and changing susceptibility patterns may have changed the empirical treatment of bacterial keratitis. Our objective in this study was to survey cornea specialists' practice patterns in the initial treatment of bacterial ulcers. This study consisted of a short online survey emailed to members of the Cornea Society listserv for an international sample of cornea specialists. Data collection began July 2014 and ended October 2014. A total of 1009 surveys were emailed, and we received 140 (14%) responses. The majority of US clinicians surveyed (n=83, 80%) chose fortified antibiotics empirically, with 55% (n=57) selecting fortified vancomycin and 16% (n=17) using fluoroquinolone alone. International respondents were twice as likely to use fluoroquinolone monotherapy (31%, n=11, p=0.07) and less likely to use fortified vancomycin (33%, n=12, p=0.03). Forty-five per cent (n=46) of US respondents reported that their initial antibiotic choice covered methicillin-resistant Staphylococcus aureus , compared with 22% (n=8) of international respondents (p<0.01). Overall, respondents who were concerned about availability of antibiotics and toxicity were 20.86 (p<0.001) and 7.48 (p<0.001) times more likely to choose fluoroquinolone monotherapy, respectively. If respondents' primary considerations were broad spectrum coverage or antibiotic resistance they had 7.10 (p<0.001) and 12.51 (p<0.001) times the odds of using fortified vancomycin, respectively. Practice patterns for the initial treatment of bacterial keratitis vary with clinicians in the USA being more likely to use fortified antibiotics versus fluoroquinolone monotherapy and more concerned with resistant organisms than their international peers.

Phytoplankton-bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge.

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Bertrand, Erin M; McCrow, John P; Moustafa, Ahmed; Zheng, Hong; McQuaid, Jeffrey B; Delmont, Tom O; Post, Anton F; Sipler, Rachel E; Spackeen, Jenna L; Xu, Kai; Bronk, Deborah A; Hutchins, David A; Allen, Andrew E

2015-08-11

Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton-bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton-bacterial interactions in both positive and negative feedback loops.

A Rice CPYC-Type Glutaredoxin OsGRX20 in Protection against Bacterial Blight, Methyl Viologen and Salt Stresses

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Xi Ning

2018-02-01

Full Text Available Glutaredoxins (GRXs belong to the antioxidants involved in the cellular stress responses. In spite of the identification 48 GRX genes in rice genomes, the biological functions of most of them remain unknown. Especially, the biological roles of members of GRX family in disease resistance are still lacking. Our proteomic analysis found that OsGRX20 increased by 2.7-fold after infection by bacterial blight. In this study, we isolated and characterized the full-length nucleotide sequences of the rice OsGRX20 gene, which encodes a GRX family protein with CPFC active site of CPYC-type class. OsGRX20 protein was localized in nucleus and cytosol, and its transcripts were expressed predominantly in leaves. Several stress- and hormone-related motifs putatively acting as regulatory elements were found in the OsGRX20 promoter. Real-time quantitative PCR analysis indicated that OsGRX20 was expressed at a significantly higher level in leaves of a resistant or tolerant rice genotype, Yongjing 50A, than in a sensitive genotype, Xiushui 11, exposed to bacterial blight, methyl viologen, heat, and cold. Its expression could be induced by salt, PEG-6000, 2,4-D, salicylic acid, jasmonic acid, and abscisic acid treatments in Yongjing 50A. Overexpression of OsGRX20 in rice Xiushui 11 significantly enhanced its resistance to bacterial blight attack, and tolerance to methyl viologen and salt stresses. In contrast, interference of OsGRX20 in Yongjing 50A led to increased susceptibility to bacterial blight, methyl viologen and salt stresses. OsGRX20 restrained accumulation of superoxide radicals in aerial tissue during methyl viologen treatment. Consistently, alterations in OsGRX20 expression affect the ascorbate/dehydroascorbate ratio and the abundance of transcripts encoding four reactive oxygen species scavenging enzymes after methyl viologen-induced stress. Our results demonstrate that OsGRX20 functioned as a positive regulator in rice tolerance to multiple stresses

Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

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Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

2015-05-26

DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

COMPARATIVE STUDY IN THE MANAGEMENT OF BACTERIAL VAGINOSIS METRONIDAZOLE VS. LACTOBACILLUS

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Narmadha Selvaraj

2017-01-01

Full Text Available BACKGROUND The incidence and morbidity of bacterial vaginosis is high. But most of the patients are asymptomatic and recurrences are common even after effective treatment. Studies comparing the therapeutic efficacy of the drugs indicated for Bacterial vaginosis are also very minimal. Aim of this study was to analyse the epidemiological pattern of bacterial vaginosis and to compare the efficacy of Metronidazole and Lactobacillus in the management of bacterial vaginosis. MATERIALS AND METHODS 100 newly diagnosed Bacterial vaginosis patients were enrolled and randomly allotted into two study groups of Metronidazole for one week and Lactobacillus for two months respectively. The patients are followed up based on Amsel’s criteria and Nugent’s scoring system at the time of initiation of treatment, one week, one month, two months and six months after treatment. Ultrasonogram was done at the end of two months. Statistical Analysis- chi square test with yate’s correction and Fisher’s exact test (2 tailed to calculate the P value. Settings and Design- Prospective, open labeled, comparative clinical trial. RESULTS Lactobacillus group was better than Metronidazole based on Amsel’s criteria at six months follow up period and based on Nugent’s scoring system at two months and six months follow up period. CONCLUSION Epidemiological interpretation in our study shows Bacterial vaginosis is common in the 24-40 years of age; marital status, earlier age at marriage, number of deliveries is directly proportionate to bacterial vaginosis incidence. Irregular menstrual cycle, number of sexual partners, and mode of delivery has no association with the incidence of Bacterial vaginosis. Moderate to profuse genital discharge is the commonest symptom observed. Whiff test (80%, Clue cells (90% and alkaline vaginal pH > 5 has high degree of sensitivity.

Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

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Gorgoglione, Bartolomeo; Carpio, Yamila; Secombes, Christopher J; Taylor, Nick G H; Lugo, Juana María; Estrada, Mario Pablo

2015-12-01

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens. Copyright © 2015

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Phase variation of Opa proteins of Neisseria meningitidis and the effects of bacterial transformation ... Male function for ensuring pollination and reproductive success in Berberis ... Environmental fluctuations do not select for increased variation or ... orthologue of the human ribosomal RNA transcription initiation factor-IA.

Architecture of the RNA polymerase II-Mediator core initiation complex.

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Plaschka, C; Larivière, L; Wenzeck, L; Seizl, M; Hemann, M; Tegunov, D; Petrotchenko, E V; Borchers, C H; Baumeister, W; Herzog, F; Villa, E; Cramer, P

2015-02-19

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

Rhizosphere bacterial carbon turnover is higher in nucleic acids than membrane lipids: implications for understanding soil carbon cycling

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Ashish A. Malik

2015-04-01

Full Text Available Using a pulse-chase 13CO2 plant labeling experiment we compared the flow of plant carbon into macromolecular fractions of root-associated soil microorganisms. Time dependent 13C dilution patterns in microbial cellular fractions were used to calculate their turnover time. The turnover times of microbial biomolecules were found to vary: microbial RNA (19 h and DNA (30 h turned over fastest followed by chloroform fumigation extraction-derived soluble cell lysis products (14 d, while phospholipid fatty acids (PLFAs had the slowest turnover (42 d. PLFA/NLFA 13C analyses suggest that both mutualistic arbuscular mycorrhizal and saprophytic fungi are dominant in initial plant carbon uptake. In contrast, high initial 13C enrichment in RNA hints at bacterial importance in initial C uptake due to the dominance of bacterial derived RNA in total extracts of soil RNA. To explain this discrepancy, we observed low renewal rate of bacterial lipids, which may therefore bias lipid fatty acid based interpretations of the role of bacteria in soil microbial food webs. Based on our findings, we question current assumptions regarding plant-microbe carbon flux and suggest that the rhizosphere bacterial contribution to plant assimilate uptake could be higher. This highlights the need for more detailed quantitative investigations with nucleic acid biomarkers to further validate these findings.

The T-ALL related gene BCL11B regulates the initial stages of human T-cell differentiation.

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Ha, V L; Luong, A; Li, F; Casero, D; Malvar, J; Kim, Y M; Bhatia, R; Crooks, G M; Parekh, C

2017-11-01

The initial stages of T-cell differentiation are characterized by a progressive commitment to the T-cell lineage, a process that involves the loss of alternative (myelo-erythroid, NK, B) lineage potentials. Aberrant differentiation during these stages can result in T-cell acute lymphoblastic leukemia (T-ALL). However, the mechanisms regulating the initial stages of human T-cell differentiation are obscure. Through loss of function studies, we showed BCL11B, a transcription factor recurrently mutated T-ALL, is essential for T-lineage commitment, particularly the repression of NK and myeloid potentials, and the induction of T-lineage genes, during the initial stages of human T-cell differentiation. In gain of function studies, BCL11B inhibited growth of and induced a T-lineage transcriptional program in T-ALL cells. We found previously unknown differentiation stage-specific DNA binding of BCL11B at multiple T-lineage genes; target genes showed BCL11B-dependent expression, suggesting a transcriptional activator role for BCL11B at these genes. Transcriptional analyses revealed differences in the regulatory actions of BCL11B between human and murine thymopoiesis. Our studies show BCL11B is a key regulator of the initial stages of human T-cell differentiation and delineate the BCL11B transcriptional program, enabling the dissection of the underpinnings of normal T-cell differentiation and providing a resource for understanding dysregulations in T-ALL.

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism

OpenAIRE

Reilman, E.; Mars, R. A. T.; van Dijl, J. M.; Denham, Emma

2014-01-01

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology,...

Gram-Negative Bacterial Sensors for Eukaryotic Signal Molecules

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Olivier Lesouhaitier

2009-09-01

Full Text Available Ample evidence exists showing that eukaryotic signal molecules synthesized and released by the host can activate the virulence of opportunistic pathogens. The sensitivity of prokaryotes to host signal molecules requires the presence of bacterial sensors. These prokaryotic sensors, or receptors, have a double function: stereospecific recognition in a complex environment and transduction of the message in order to initiate bacterial physiological modifications. As messengers are generally unable to freely cross the bacterial membrane, they require either the presence of sensors anchored in the membrane or transporters allowing direct recognition inside the bacterial cytoplasm. Since the discovery of quorum sensing, it was established that the production of virulence factors by bacteria is tightly growth-phase regulated. It is now obvious that expression of bacterial virulence is also controlled by detection of the eukaryotic messengers released in the micro-environment as endocrine or neuro-endocrine modulators. In the presence of host physiological stress many eukaryotic factors are released and detected by Gram-negative bacteria which in return rapidly adapt their physiology. For instance, Pseudomonas aeruginosa can bind elements of the host immune system such as interferon-γ and dynorphin and then through quorum sensing circuitry enhance its virulence. Escherichia coli sensitivity to the neurohormones of the catecholamines family appears relayed by a recently identified bacterial adrenergic receptor. In the present review, we will describe the mechanisms by which various eukaryotic signal molecules produced by host may activate Gram-negative bacteria virulence. Particular attention will be paid to Pseudomonas, a genus whose representative species, P. aeruginosa, is a common opportunistic pathogen. The discussion will be particularly focused on the pivotal role played by these new types of pathogen sensors from the sensing to the transduction

Glove and gown effects on intraoperative bacterial contamination.

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Ward, William G; Cooper, Joshua M; Lippert, Dylan; Kablawi, Rawan O; Neiberg, Rebecca H; Sherertz, Robert J

2014-03-01

Experiments were performed to determine the risk of bacterial contamination associated with changing outer gloves and using disposable spunlace paper versus reusable cloth gowns. Despite decades of research, there remains a lack of consensus regarding certain aspects of optimal aseptic technique including outer glove exchange while double-gloving and surgical gown type selection. In an initial glove study, 102 surgical team members were randomized to exchange or retain outer gloves 1 hour into clean orthopedic procedures; cultures were obtained 15 minutes later from the palm of the surgeon's dominant gloved hand and from the surgical gown sleeve. Surgical gown type selection was recorded. A laboratory strike-through study investigating bacterial transmission through cloth and paper gowns was performed with coagulase-negative staphylococci. In a follow-up glove study, 251 surgical team members, all wearing paper gowns, were randomized as in the first glove study. Glove study 1 revealed 4-fold higher levels of baseline bacterial contamination (31% vs 7%) on the sleeve of surgical team members wearing cloth gowns than those using paper gowns [odds ratio (95% confidence interval): 4.64 (1.72-12.53); P = 0.0016]. The bacterial strike-through study revealed that 26 of 27 cloth gowns allowed bacterial transmission through the material compared with 0 of 27 paper gowns (P < 0.001). In glove study 2, surgeons retaining outer gloves 1 hour into the case had a subsequent positive glove contamination rate of 23% compared with 13% among surgeons exchanging their original outer glove [odds ratio (95% confidence interval): 1.97 (1.02-3.80); P = 0.0419]. Paper gowns demonstrated less bacterial transmission in the laboratory and lower rates of contamination in the operating room. Disposable paper gowns are recommended for all surgical cases, especially those involving implants, because of the heightened risk of infection. Outer glove exchange just before handling implant materials

The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis

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Gimenez-Ibanez, Selena; Boter, Marta; Fernández-Barbero, Gemma; Chini, Andrea; Rathjen, John P.; Solano, Roberto

2014-01-01

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome. PMID:24558350

BACTERIAL LEACHING OF ELECTRONIC SCRAP: INFLUENCE OF PROCESS PARAMETERS

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Luciana Harue Yamane

2013-03-01

Full Text Available The application of bacterial leaching in the ore treatment is already known and also can be applied such as treatment of electronic waste to copper recovery. This paper investigates the influence of process parameters (pulp density, inoculums volume, rotation speed and initial concentration of ferrous iron on bacterial leaching of copper from printed circuit board of computers using the bacterium Acidithiobacillus ferrooxidans–LR. Printed circuit boards from computers were comminuted using a hammer mill. The powder obtained was magnetically separated and the non-magnetic material used in this study. A shake flask study was carried out on the non-magnetic material using a shaker. The results show that Acidithiobacillus ferrooxidans–LR can leach 99% of copper from printed circuit boards (non–magnetic material under the determined conditions through of the studies.

Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP

International Nuclear Information System (INIS)

Lewis, E.J.; Harrington, C.A.; Chikaraishi, D.M.

1987-01-01

Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH 4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to + 27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained with 773 bases

Transcriptional regulatory elements in the noncoding region of human papillomavirus type 6

International Nuclear Information System (INIS)

Wu, Tzyy-Choou.

1989-01-01

The structure and function of the transcriptional regulatory region of human papillomavirus type 6 (HPV-6) has been investigated. To investigate tissue specific gene expression, a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in plastic-embedded tissue sections of genital and respiratory tract papillomata by using in situ hybridization and immunoperoxidase assays has been developed. This method, using ultrathin sections and strand-specific 3 H labeled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution, and the modified immunocytochemistry provides better sensitivity. The results suggest that genital tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. To study the tissue tropism of HPV-6 at the level of regulation of viral gene expression, the polymerase chain reaction was used to isolate the noncoding region (NCR) of HPV-6 in independent isolates. Nucleotide sequence analysis of molecularly cloned DNA identified base substitutions, deletions/insertions and tandem duplications. Transcriptional regulatory elements in the NCR were assayed in recombinant plasmids containing the bacterial gene for chloramphenicol acetyl transferase

Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

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Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

2015-11-05

Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

Bacterial lung abscess

International Nuclear Information System (INIS)

Groskin, S.A.; Panicek, D.M.; Ewing, D.K.; Rivera, F.; Math, K.; Teixeira, J.; Heitzman, E.R.

1987-01-01

A retrospective review of patients with bacterial lung abscess was carried out. Demographic, clinical, and radiographical features of this patient group are compared with similar data from patients with empyema and/or cavitated lung carcinoma; differential diagnostic points are stressed. The entity of radiographically occult lung abscess is discussed. Complications associated with bacterial lung abscess are discussed. Current therapeutic options and treatment philosophy for patients with bacterial lung abscess are noted

CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation.

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Bruna, Roberto E; Molino, María Victoria; Lazzaro, Martina; Mariscotti, Javier F; García Véscovi, Eleonora

2018-04-15

PrtA is the major secreted metalloprotease of Serratia marcescens Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in Serratia In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of prtA , there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along Serratia strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in S. marcescen s, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a cpxR mutant background, prtA is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of S. marcescens to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of S. marcescens IMPORTANCE We demonstrate that S. marcescens metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in S. marcescens , the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is

Bacterial growth on macrophyte leachate and fate of bacterial production

International Nuclear Information System (INIS)

Findlay, S.; Carlough, L.; Crocker, M.T.; Gill, H.K.; Meyer, J.L.; Smith, P.J.

1986-01-01

The role bacteria play in transferring organic carbon to other trophic levels in aquatic ecosystems depends on the efficiency with which they convert dissolved organic [ 14 C]-labelled carbon into bacterial biomass and on the ability of consumers to graze bacteria. The authors have measured the conversion efficiency for bacteria growing on macrophyte-derived dissolved organic carbon and estimated the amount of bacterial production removed by grazing. Bacteria converted this DOC into new tissue with an efficiency of 53%, substantially higher than the apparent conversion efficiency of macrophyte-derived particulate organic carbon or other types of DOC. Two estimates of grazing indicate that the decline in bacterial numbers after the bloom was probably due to grazing by flagellates. These results show the significance of the bacterial link between DOC and other trophic levels

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

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Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

2017-06-13

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription

Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection

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Walker M Andrew

2010-07-01

Full Text Available Abstract Background Plant cytochrome P450 monooxygenases (CYP mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines. Results Cloning of genomic DNA and cDNA revealed that the CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS in the upstream region and three candidate polyadenylation (PolyA sites in the downstream region of CYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression. Conclusions This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression

Potential role of Arabidopsis PHP as an accessory subunit of the PAF1 transcriptional cofactor.

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Park, Sunchung; Ek-Ramos, Maria Julissa; Oh, Sookyung; van Nocker, Steven

2011-08-01

Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function.

Interplay of Noisy Gene Expression and Dynamics Explains Patterns of Bacterial Operon Organization

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Igoshin, Oleg

2011-03-01

Bacterial chromosomes are organized into operons -- sets of genes co-transcribed into polycistronic messenger RNA. Hypotheses explaining the emergence and maintenance of operons include proportional co-regulation, horizontal transfer of intact ``selfish'' operons, emergence via gene duplication, and co-production of physically interacting proteins to speed their association. We hypothesized an alternative: operons can reduce or increase intrinsic gene expression noise in a manner dependent on the post-translational interactions, thereby resulting in selection for or against operons in depending on the network architecture. We devised five classes of two-gene network modules and show that the effects of operons on intrinsic noise depend on class membership. Two classes exhibit decreased noise with co-transcription, two others reveal increased noise, and the remaining one does not show a significant difference. To test our modeling predictions we employed bioinformatic analysis to determine the relationship gene expression noise and operon organization. The results confirm the overrepresentation of noise-minimizing operon architectures and provide evidence against other hypotheses. Our results thereby suggest a central role for gene expression noise in selecting for or maintaining operons in bacterial chromosomes. This demonstrates how post-translational network dynamics may provide selective pressure for organizing bacterial chromosomes, and has practical consequences for designing synthetic gene networks. This work is supported by National Institutes of Health grant 1R01GM096189-01.

Prediction of bacterial meningitis based on cerebrospinal fluid pleocytosis in children

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Sofia Águeda

Full Text Available Children with cerebrospinal fluid pleocytosis are frequently treated with parenteral antibiotics, but only a few have bacterial meningitis. Although some clinical prediction rules, such as bacterial meningitis score, are of well-known value, the cerebrospinal fluid white blood cells count can be the initial available information. Our aim was to establish a cutoff point of cerebrospinal fluid white blood cell count that could distinguish bacterial from viral and aseptic meningitis. A retrospective study of children aged 29 days to 17 years who were admitted between January 1st and December 31th, 2009, with cerebrospinal fluid pleocytosis (white blood cell > 7 µL-1 was conducted. The cases of traumatic lumbar puncture and of antibiotic treatment before lumbar puncture were excluded. There were 295 patients with cerebrospinal fluid pleocytosis, 60.3% females, medium age 5.0 ± 4.3 years distributed as: 12.2% 1-3 months; 10.5% 3-12 months; 29.8% 12 months to 5 years; 47.5% >5 years. Thirty one children (10.5% were diagnosed with bacterial meningitis, 156 (52.9% viral meningitis and 108 (36.6% aseptic meningitis. Bacterial meningitis was caused by Neisseria meningi tidis (48.4%, Streptococcus pneumoniae (32.3%, other Streptococcus species (9.7%, and other agents (9.7%. cerebrospinal fluid white blood cell count was significantly higher in patients with bacterial meningitis (mean, 4839 cells/µL compared to patients with aseptic meningitis (mean, 159 cells/µL, p < 0.001, with those with aseptic meningitis (mean, 577 cells/µL, p < 0.001 and with all non-bacterial meningitis cases together (p < 0.001. A cutoff value of 321 white blood cell/µL showed the best combination of sensitivity (80.6% and specificity (81.4% for the diagnosis of bacterial meningitis (area under receiver operating characteristic curve 0.837. Therefore, the value of cerebrospinal fluid white blood cell count was found to be a useful and rapid diagnostic test to distinguish