Genome-wide analysis of the RpoN regulon in

Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA.
BMC Genomics (Impact Factor: 3.99). 08/2009; 10(1):331. DOI: 10.1186/1471-2164-10-331
Source: PubMed


The role of the RNA polymerase sigma factor RpoN in regulation of gene expression in Geobacter sulfurreducens was investigated to better understand transcriptional regulatory networks as part of an effort to develop regulatory modules for genome-scale in silico models, which can predict the physiological responses of Geobacter species during groundwater bioremediation or electricity production.
An rpoN deletion mutant could not be obtained under all conditions tested. In order to investigate the regulon of the G. sulfurreducens RpoN, an RpoN over-expression strain was made in which an extra copy of the rpoN gene was under the control of a taclac promoter. Combining both the microarray transcriptome analysis and the computational prediction revealed that the G. sulfurreducens RpoN controls genes involved in a wide range of cellular functions. Most importantly, RpoN controls the expression of the dcuB gene encoding the fumarate/succinate exchanger, which is essential for cell growth with fumarate as the terminal electron acceptor in G. sulfurreducens. RpoN also controls genes, which encode enzymes for both pathways of ammonia assimilation that is predicted to be essential under all growth conditions in G. sulfurreducens. Other genes that were identified as part of the RpoN regulon using either the computational prediction or the microarray transcriptome analysis included genes involved in flagella biosynthesis, pili biosynthesis and genes involved in central metabolism enzymes and cytochromes involved in extracellular electron transfer to Fe(III), which are known to be important for growth in subsurface environment or electricity production in microbial fuel cells. The consensus sequence for the predicted RpoN-regulated promoter elements is TTGGCACGGTTTTTGCT.
The G. sulfurreducens RpoN is an essential sigma factor and a global regulator involved in a complex transcriptional network controlling a variety of cellular processes.

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Available from: Ching Leang, Oct 01, 2015
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    • "Using relatively low cutoffs for the fold-change (1.5- to 2-fold) in transcript levels between the σ54-overexpression strain and wild type or ΔrpoN strains, a considerable portion of the σ54-dependent transcriptome was defined in Escherichia coli[13], Vibrio cholerae[14] and Geobacter sulfurreducens[15]. However, not all previously-identified σ54-dependent operons were detected for E. coli and G. sulfurreducens, and evidence from the V. cholera and G. sulfurreducens studies suggests that overexpression of σ54 may repress expression from some σ54-dependent promoters and alter expression of σ54-independent promoters [13-15]. We hypothesize that a promiscuous and constitutive variant of the bEBP DctD from S. meliloti can activate transcription from all σ54-dependent promoters in S. Typhimurium LT2 at wild-type levels of σ54 under a single growth condition, thereby facilitating global characterization of the σ54 regulon without overexpression of σ54. "
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    ABSTRACT: Background Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ54 regulon in a bacterium is that the σ54 RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ54-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP—the AAA+ ATPase domain of DctD from Sinorhizobium meliloti—to activate transcription from all σ54-dependent promoters for the characterization of the σ54 regulon of Salmonella Typhimurium LT2. Results The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ54-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ54-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ54 binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ54 binding sites suggest that many of these sites are capable of functioning as σ54-dependent promoters. Conclusion Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse σ54-dependent promoters of the S. Typhimurium LT2 σ54 regulon under a single growth condition, this approach is likely to be valuable for examining σ54 regulons in other bacterial species. The S. Typhimurium σ54 regulon included a high number of intragenic σ54 binding sites/promoters, suggesting that σ54 may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.
    BMC Genomics 09/2013; 14(1):602. DOI:10.1186/1471-2164-14-602 · 3.99 Impact Factor
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    • "If FlgZ and its homologs function as RpoN chaperones, such activity may allow RpoN to compete effectively with other sigma factors for binding RNA polymerase. This could be important for RpoN function in members of the Deltaproteobacteria which typically have exceptional numbers of RpoN-dependent activators (Myxococcus xanthus has ~50 different activators) and where RpoN is a significant global regulator in these bacteria [35,36]. "
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    ABSTRACT: Background Helicobacter pylori HP0958 protein (FlgZ) prevents the rapid turnover of RpoN (σ54), a transcription factor required for expression of several flagellar genes in H. pylori. FlgZ possesses a zinc-ribbon domain (DUF164) that contains two conserved CXXC motifs which coordinate a zinc ion and is thought to interact with nucleic acids or proteins. Two conserved cysteine residues in FlgZ (Cys-202 and Cys-223) were replaced with serine to assess their significance in FlgZ function. After confirming the importance of the CXXC motifs in the DUF164 domain of FlgZ, the distribution of DUF164 proteins and RpoN homologs in other bacteria was examined to determine if a correlation existed for the concurrence of the two proteins. Results Levels of RpoN were greatly reduced in H. pylori strains that expressed the FlgZC202S or FlgZC223S variants. The FlgZC202S variant, but not the FlgZC223S variant, accumulated at levels similar to the wild-type protein. DUF164 proteins are not universally distributed and appear to be absent in several major bacterial taxa, including Cyanobacteria as well as Alpha-, Beta- and Gammaproteobacteria. With the exception of the Actinobacteria, members of which generally lack RpoN, genes encoding DUF164 proteins and RpoN are frequently found in the same genome. Interestingly, many of the DUF164 proteins in Actinobacteria and Bacteroidetes lack most or even all of the conserved cysteine residues. Conclusions These findings suggest the importance of the zinc-ribbon domain of FlgZ in protecting RpoN from turnover. Since many bacteria that possess a DUF164 protein also contain RpoN, DUF164 proteins may have roles in RpoN protection or function in other bacteria.
    08/2011; 1(8):1-10. DOI:10.1186/2042-5783-1-8
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    • "However, it was soon after established that Sigma-54 mediated control of transcription is not only connected to nitrogen assimilation but to a wider range of cellular processes and physiology in the enterobacteria [25,33]. Since then, it was shown that its role also encompasses the regulation of for example: flagellar biosynthesis in E. coli [34]; carboxylate uptake, central metabolism and flagellar biosynthesis in Geobacter sulfurreducens [35]; phosphotransferase system (PTS)-mediated carbohydrate uptake in the Gram-positive species Lactobacillus plantarum [23] and Listeria monocytogenes [36]; and PTS-mediated regulation in Gram-positive as well as Gram-negative organisms [37,38]; osmotolerance in Listeria [39]; the utilization of compounds like gamma-aminobutyrate in Bacillus [40], and the less familiar biphenyl in Ralstonia metallidurans [41] and toluene, xylene (see [42]) and choline [43] in Pseudomonas; Type III secretion system mediated pathogenicity in Pseudomonas syringae [44] and Type VI secretion system mediated toxin secretion in e.g. Aeromonas and Marinomonas [45]; the adaptation to cold shock in B. subtilis [24]; the control of Sigma-S [46], lipoprotein biosynthesis and virulence [47] in Borrelia burgdorferi; acid resistance of pathogenic E. coli O157 [48]; biofilm formation by Burkholderia [49]; and motility, biofilm formation, luminescence, and colonization in Vibrio fischeri [50,51]. "
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    ABSTRACT: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm.
    BMC Genomics 08/2011; 12(1):385. DOI:10.1186/1471-2164-12-385 · 3.99 Impact Factor
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