Pathways Leading from BarA/SirA to Motility andVirulence Gene Expression inSalmonella

ABSTRACT The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.

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SoxS represses flagellar gene expression through RflP in Salmonella enterica Serovar Typhimurium

10.1101/2020.05.29.124750 ◽

2020 ◽

Author(s):

Srinivas S. Thota ◽

Brittany N. Henry ◽

Lon M. Chubiz

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Transcription Factors ◽

Salmonella Enterica ◽

Response Regulator ◽

Flagellar Gene ◽

Treatment And Prevention ◽

Intracellular Signals ◽

Serovar Typhimurium ◽

Close Relatives

ABSTRACTFlagellar gene expression is subject to regulation by many global transcription factors in response to environmental and nutritional signals. One of the primary ways this occurs in Salmonella enterica serovar Typhimurium, and its close relatives, is through controlling levels of FlhD4C2 (the flagellar master regulator) via transcriptional, post-transcriptional, and post-translational mechanisms. Recently, we found the homologous transcription factors MarA, Rob, and SoxS repress flhDC expression by distinct mechanisms. MarA and Rob, regulators involved in inducible multidrug resistance, repressed flhDC transcription by interacting directly with the flhDC promoter. Alternatively, SoxS, the oxidative stress response regulator, altered FlhD4C2 levels independent of flhDC transcription by post-transcriptional or post-translational mechanism. Here, using a forward genetic screen, we discovered that SoxS-dependent repression of flagellar gene expression occurs through RflP, an anti-FlhD4C2 factor that targets FlhD4C2 for proteolytic degradation. Elevated soxS expression resulted in concomitant increases in rflP expression, indicating SoxS may work through RflP at the level of rflP transcription. Mapping of the rflP promoter and a bioinformatic search yielded a putative SoxS binding site proximal to the rflP transcription start site. Comparison of the rflP promoter region in S. Typhimurium and Escherichia coli indicate substantial differences, providing a possible mechanism for differential expression of rflP between these species.IMPORTANCESalmonella enterica is a major cause of foodborne illness. Understanding environmental and intracellular signals used by Salmonella to control expression of virulence-associated traits is critical to advancing treatment and prevention of Salmonella-related disease. Reduced expression of flagella at key points during Salmonella infection aids in evasion of the host innate immune system. Within macrophages Salmonella is non-flagellated and exposed to oxidative stress. SoxS-dependent repression of flagellar genes may provide a potential link between oxidative stress and reductions in flagellar expression.

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Activation of hilA expression at low pH requires the signal sensor CpxA, but not the cognate response regulator CpxR, in Salmonella enterica serovar Typhimurium

Microbiology ◽

10.1099/mic.0.26229-0 ◽

2003 ◽

Vol 149 (10) ◽

pp. 2809-2817 ◽

Cited By ~ 30

Author(s):

Shu-ichi Nakayama ◽

Akira Kushiro ◽

Takashi Asahara ◽

Ryu-ichiro Tanaka ◽

Lan Hu ◽

...

Keyword(s):

Mutant Strain ◽

Cell Invasion ◽

Salmonella Enterica ◽

Response Regulator ◽

Low Ph ◽

Apparent Difference ◽

Virulence Determinants ◽

Serovar Typhimurium ◽

Cognate Response Regulator ◽

Invasion Capacity

A two-component regulatory system, cpxR–cpxA, plays an important role in the pH-dependent regulation of virF, a global activator for virulence determinants including invasion genes, in Shigella sonnei. The authors examined whether the cpxR–cpxA homologues have some function in the expression of Salmonella enterica serovar Typhimurium invasion genes via the regulation of hilA, an activator for these genes. In a Salmonella cpxA mutant, the hilA expression level was reduced to less than 10 % of that in the parent strain at pH 6·0. This mutant strain also showed undetectable synthesis of an invasion gene product, SipC, at pH 6·0 and reduced cell invasion capacity – as low as 20 % of that of the parent. In this mutant, the reduction in hilA expression was much less marked at pH 8·0 than at pH 6·0 – no less than 50 % of that in the parent, and no significant reduction was observed in either SipC synthesis or cell invasion rate, compared to the parent. Unexpectedly, a Salmonella cpxR mutant strain and the parent showed no apparent difference in all three characteristics described above at either pH. These results indicate that in Salmonella, the sensor kinase CpxA activates hilA, and consequently, invasion genes and cell invasion capacity at pH 6·0. At pH 8·0, however, CpxA does not seem to have a large role in activation of these factors. Further, the results show that this CpxA-mediated activation does not require its putative cognate response regulator, CpxR. This suggests that CpxA may interact with regulator(s) other than CpxR to achieve activation at low pH.

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Multiple Factors Independently RegulatehilA and Invasion Gene Expression in Salmonella enterica Serovar Typhimurium

Journal of Bacteriology ◽

10.1128/jb.182.7.1872-1882.2000 ◽

2000 ◽

Vol 182 (7) ◽

pp. 1872-1882 ◽

Cited By ~ 167

Author(s):

Robin L. Lucas ◽

C. Phoebe Lostroh ◽

Concetta C. DiRusso ◽

Michael P. Spector ◽

Barry L. Wanner ◽

...

Keyword(s):

Gene Expression ◽

Salmonella Enterica ◽

Inorganic Phosphate ◽

Response Regulator ◽

Regulatory System ◽

Negative Control ◽

Host Cells ◽

Uptake System ◽

Intracellular Signals ◽

Serovar Typhimurium

HilA activates the expression of Salmonella entericaserovar Typhimurium invasion genes. To learn more about regulation ofhilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In addition to expected mutations, we identified Tn5 insertions inpstS, fadD, flhD, flhC, and fliA. Analysis of the pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting thathilA expression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of the fadD mutant indicates that hilA is regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulatehilA expression. flhDC and fliAencode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies with flhCand fliA mutants indicate that FliZ, which is encoded in an operon with fliA, activates expression of hilA, linking regulation of hilA with motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulate hilA expression and invasion. In summary, our screen has identified several distinct pathways that can modulate S. enterica serovar Typhimurium's ability to express hilA and invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.

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Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region

Journal of Bacteriology ◽

10.1128/jb.00430-08 ◽

2008 ◽

Vol 190 (19) ◽

pp. 6467-6474 ◽

Cited By ~ 4

Author(s):

Oliwia Makarewicz ◽

Svetlana Neubauer ◽

Corinna Preusse ◽

Rainer Borriss

Keyword(s):

Bacillus Amyloliquefaciens ◽

Promoter Region ◽

Response Regulator ◽

Dependent Manner ◽

Coding Region ◽

Mobility Shift ◽

Dnase I Footprinting ◽

Regulator Protein ◽

Transition State Regulator ◽

Gel Mobility Shift

ABSTRACT We have previously identified the phyC gene of Bacillus amyloliquefaciens FZB45, encoding extracellular phytase, as a member of the PhoP regulon, which is expressed only during phosphate starvation. Its σA-dependent promoter is positively and negatively regulated by the phosphorylated PhoP response regulator in a phosphate-dependent manner (O. Makarewicz, S. Dubrac, T. Msadek, and R. Borriss, J. Bacteriol. 188:6953-6965, 2006). Here, we provide experimental evidence that the transcription of phyC underlies a second control mechanism exerted by the global transient-phase regulator protein, AbrB, which hinders its expression during exponential growth. Gel mobility shift and DNase I footprinting experiments demonstrated that AbrB binds to two different regions in the phyC promoter region that are separated by about 200 bp. One binding site is near the divergently orientated yodU gene, and the second site is located downstream of the phyC promoter and extends into the coding region of the phyC gene. Cooperative binding to the two distant binding regions is necessary for the AbrB-directed repression of phyC transcription. AbrB does not affect the transcription of the neighboring yodU gene.

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Regulation of d-Xylose Metabolism in Caulobacter crescentus by a LacI-Type Repressor

Journal of Bacteriology ◽

10.1128/jb.01342-07 ◽

2007 ◽

Vol 189 (24) ◽

pp. 8828-8834 ◽

Cited By ~ 28

Author(s):

Craig Stephens ◽

Beat Christen ◽

Kelly Watanabe ◽

Thomas Fuchs ◽

Urs Jenal

Keyword(s):

Gene Expression ◽

Energy Source ◽

Regulatory Mechanism ◽

Caulobacter Crescentus ◽

Heterologous Gene Expression ◽

Xylose Metabolism ◽

Mobility Shift ◽

Inducible Promoters ◽

Gel Mobility Shift ◽

Operator Binding

ABSTRACT In the oligotrophic freshwater bacterium Caulobacter crescentus, d-xylose induces expression of over 50 genes, including the xyl operon, which encodes key enzymes for xylose metabolism. The promoter (P xylX ) controlling expression of the xyl operon is widely used as a tool for inducible heterologous gene expression in C. crescentus. We show here that P xylX and at least one other promoter in the xylose regulon (P xylE ) are controlled by the CC3065 (xylR) gene product, a LacI-type repressor. Electrophoretic gel mobility shift assays showed that operator binding by XylR is greatly reduced in the presence of d-xylose. The data support the hypothesis that there is a simple regulatory mechanism in which XylR obstructs xylose-inducible promoters in the absence of the sugar; the repressor is induced to release DNA upon binding d-xylose, thereby freeing the promoter for productive interaction with RNA polymerase. XylR also has an effect on glucose metabolism, as xylR mutants exhibit reduced expression of the Entner-Doudoroff operon and their ability to utilize glucose as a sole carbon and energy source is compromised.

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A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7363 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7363-7376 ◽

Cited By ~ 85

Author(s):

A Cvekl ◽

C M Sax ◽

E H Bresnick ◽

J Piatigorsky

Keyword(s):

Gene Expression ◽

Promoter Activity ◽

Cellular Differentiation ◽

Ocular Lens ◽

Mobility Shift ◽

Specific Expression ◽

Negative Element ◽

Cis Acting ◽

Direct Data ◽

Gel Mobility Shift

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.

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A Dopamine-Responsive Signal Transduction Controls Transcription ofSalmonella entericaSerovar Typhimurium Virulence Genes

mBio ◽

10.1128/mbio.02772-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 2

Author(s):

Dezhi Yang ◽

Ying Kong ◽

Wei Sun ◽

Wei Kong ◽

Yixin Shi

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Family Member ◽

Virulence Genes ◽

Enteric Bacteria ◽

Virulence Determinants ◽

Transcription Activator ◽

Content Type ◽

Serovar Typhimurium ◽

Target Promoters

ABSTRACTWe have shown that the ligand-responsive MarR family member SlyA plays an important role in transcription activation of multiple virulence genes inSalmonella entericaserovar Typhimurium by responding to guanosine tetraphosphate (ppGpp). Here, we demonstrate that another MarR family member, EmrR, is required for virulence ofS.Typhimurium and another enteric bacterium,Yersinia pestis. EmrR is found to activate transcription of an array of virulence determinants, includingSalmonellapathogenicity island 2 (SPI-2) genes and several divergent operons, which have been shown to be activated by SlyA and the PhoP/PhoQ two-component system. We studied the regulatory effect of EmrR on one of these genetic loci, i.e., thepagC-pagDdivergent operon, and characterized a catecholamine neurotransmitter, dopamine, as an EmrR-sensed signal. Dopamine acts on EmrR to reduce its ability to bind to the target promoters, thus functioning as a negative signal to downregulate this EmrR-activated transcription. EmrR can bind to AT-rich sequences, which particularly overlap the SlyA and PhoP binding sites in thepagC-pagDdivergent promoter. EmrR is a priming transcription regulator that binds its target promoters prior to successive transcription activators, by which it displaces universal silencer H-NS from these promoters and facilitates successive regulators to bind these regions. Regulation of theSalmonella-specific gene inEscherichia coliandY. pestisreveals that EmrR-dependent regulation is conserved in enteric bacteria. These observations suggest that EmrR is a transcription activator to control the expression of virulence genes, including the SPI-2 genes. Dopamine can act on the EmrR-mediated signal transduction, thus downregulating expression of these virulence factors.IMPORTANCEIn this study, MarR family regulator EmrR is identified as a novel virulence factor of enteric bacteria, here exemplified bySalmonella entericaserovar Typhimurium andYersinia pestis. EmrR exerts an essential effect as a transcription activator for expression of virulence determinants, includingSalmonellapathogenicity island 2 genes and a set of horizontally acquired genetic loci that formed divergent operons. EmrR senses the neurotransmitter dopamine and is subsequently released from target promoters, resulting in downregulation of the virulence gene expression. Through this action on EmrR, dopamine can weakenSalmonellaresistance against host defense mechanisms. This provides an explanation for the previous observation that dopamine inhibits bacterial infection in animal gastrointestinal tracts. Our findings provide evidence that this neurotransmitter can modulate bacterial gene expression through interaction with virulence regulator EmrR.

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The VirR Response Regulator from Clostridium perfringens Binds Independently to Two Imperfect Direct Repeats Located Upstream of the pfoA Promoter

Journal of Bacteriology ◽

10.1128/jb.182.1.57-66.2000 ◽

2000 ◽

Vol 182 (1) ◽

pp. 57-66 ◽

Cited By ~ 42

Author(s):

Jackie K. Cheung ◽

Julian I. Rood

Keyword(s):

Binding Site ◽

Clostridium Perfringens ◽

Response Regulator ◽

Toxin Production ◽

Site Directed Mutagenesis ◽

Direct Repeats ◽

Mobility Shift ◽

Perfringolysin O ◽

Dnase I Footprinting ◽

Gel Mobility Shift

ABSTRACT Regulation of toxin production in the gram-positive anaerobeClostridium perfringens occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the virS gene, while its cognate response regulator is encoded by the virRgene. We have constructed a VirR expression plasmid inEscherichia coli and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the pfoA gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated plc, colA, and pfoRgenes, which encode alpha-toxin, collagenase, and a putativepfoA regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the pfoA promoter. When this region was deleted, VirR was no longer able to bind to the pfoApromoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the pfoA promoter and activating transcription.

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The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx

Development ◽

10.1242/dev.126.1.97 ◽

1999 ◽

Vol 126 (1) ◽

pp. 97-107 ◽

Cited By ~ 10

Author(s):

J.D. Thatcher ◽

C. Haun ◽

P.G. Okkema

Keyword(s):

Gene Expression ◽

Caenorhabditis Elegans ◽

Recognition Sequence ◽

Mobility Shift ◽

Enhancer Activity ◽

Gene Target ◽

Gfp Reporter ◽

Pharyngeal Muscles ◽

Gel Mobility Shift ◽

Dauer Larvae

Gene expression in the pharyngeal muscles of Caenorhabditis elegans is controlled in part by organ-specific signals, which in the myo-2 gene target a short DNA sequence termed the C subelement. To identify genes contributing to these signals, we performed a yeast one-hybrid screen for cDNAs encoding factors that bind the C subelement. One clone recovered was from daf-3, which encodes a Smad most closely related to vertebrate Smad4. We demonstrated that DAF-3 binds C subelement DNA directly and specifically using gel mobility shift and DNase1 protection assays. Mutation of any base in the sequence GTCTG interfered with binding in the gel mobility shift assay, demonstrating that this pentanucleotide is a core recognition sequence for DAF-3 binding. daf-3 is known to promote formation of dauer larvae and this activity is negatively regulated by TGFbeta-like signaling. To determine how daf-3 affects C subelement enhancer activity in vivo, we examined expression a gfp reporter controlled by a concatenated C subelement oligonucleotide in daf-3 mutants and other mutants affecting the TGFbeta-like signaling pathway controlling dauer formation. Our results demonstrate that wild-type daf-3 can repress C subelement enhancer activity during larval development and, like its dauer-promoting activity, daf-3's repressor activity is negatively regulated by TGFbeta-like signaling. We have examined expression of this gfp reporter in dauer larvae and have observed no daf-3-dependent repression of C activity. These results suggest daf-3 directly regulates pharyngeal gene expression during non-dauer development.

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Regulation of osmC Gene Expression by the Two-Component System rcsB-rcsC inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.183.20.5870-5876.2001 ◽

2001 ◽

Vol 183 (20) ◽

pp. 5870-5876 ◽

Cited By ~ 73

Author(s):

Marcela Davalos-Garcia ◽

Annie Conter ◽

Isabelle Toesca ◽

Claude Gutierrez ◽

Kaymeuang Cam

Keyword(s):

Response Regulator ◽

Regulatory Element ◽

In Vitro Transcription ◽

Proximal Promoter ◽

Two Component System ◽

Mobility Shift ◽

Component System ◽

Two Component ◽

Gel Mobility Shift

ABSTRACT The Escherichia coli osmC gene encodes an envelope protein of unknown function whose expression depends on osmotic pressure and growth phase. The gene is transcribed from two overlapping promoters, osmCp 1 andosmCp 2. Several factors regulating these promoters have been reported. The leucine-responsive protein Lrp represses osmCp 1 and activatesosmCp 2, the nucleoid-associated protein H-NS represses both promoters, and the stationary-phase sigma factor ςs specifically recognizesosmCp 2. This work reports the identification of an additional regulatory element, the two-component systemrcsB-rcsC, affecting positively the distal promoter osmCp 1. The response regulator of the system, RcsB, does not affect expression of the proximal promoter osmCp 2. Deletion analysis located the site necessary for RcsB activation just upstream ofosmCp 1. In vitro transcription experiments and gel mobility shift assays demonstrated that RcsB stimulates RNA polymerase binding at osmCp 1.

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