Genes Regulated by TorR, the Trimethylamine Oxide Response Regulator of Shewanella oneidensis

ABSTRACT The torECAD operon encoding the trimethylamine oxide (TMAO) respiratory system of Shewanella oneidensis is positively controlled by the TorS/TorR two-component system when TMAO is available. Activation of the tor operon occurs upon binding of the phosphorylated response regulator TorR to a single operator site containing the direct repeat nucleotide sequence TTCATAN4TTCATA. Here we show that the replacement of any nucleotide of one TTCATA hexamer prevented TorR binding in vitro, meaning that TorR specifically interacts with this DNA target. Identical direct repeat sequences were found in the promoter regions of torR and of the new gene torF (SO4694), and they allowed TorR binding to both promoters. Real-time PCR experiments revealed that torR is negatively autoregulated, whereas torF is strongly induced by TorR in response to TMAO. Transcription start site location and footprinting analysis indicate that the operator site at torR overlaps the promoter −10 box, whereas the operator site at torF is centered at −74 bp from the start site, in agreement with the opposite role of TorR in the regulation of the two genes. Since torF and torECAD are positively coregulated by TorR, we propose that the TorF protein plays a role related to TMAO respiration.

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Molecular Characterization of the Mg2+-Responsive PhoP-PhoQ Regulon in Salmonella enterica

Journal of Bacteriology ◽

10.1128/jb.185.21.6287-6294.2003 ◽

2003 ◽

Vol 185 (21) ◽

pp. 6287-6294 ◽

Cited By ~ 84

Author(s):

Sergio Lejona ◽

Andrés Aguirre ◽

María Laura Cabeza ◽

Eleonora García Véscovi ◽

Fernando C. Soncini

Keyword(s):

Transcriptional Regulation ◽

Salmonella Enterica ◽

Response Regulator ◽

Consensus Sequence ◽

Direct Interaction ◽

Direct Repeat ◽

Infection Process ◽

Promoter Regions

ABSTRACT The PhoP/PhoQ two-component system controls the extracellular magnesium deprivation response in Salmonella enterica. In addition, several virulence-associated genes that are mainly required for intramacrophage survival during the infection process are under the control of its transcriptional regulation. Despite shared Mg2+ modulation of the expression of the PhoP-activated genes, no consensus sequence common to all of them could be detected in their promoter regions. We have investigated the transcriptional regulation and the interaction of the response regulator PhoP with the promoter regions of the PhoP-activated loci phoPQ, mgtA, slyB, pmrD, pcgL, phoN, pagC, and mgtCB. A direct repeat of the heptanucleotide sequence (G/T)GTTTA(A/T) was identified as the conserved motif recognized by PhoP to directly control the gene expression of the first five loci, among which the first four are ancestral to enterobacteria. On the other hand, no direct interaction of the response regulator with the promoter of phoN, pagC, or mgtCB was apparent by either in vitro or in vivo assays. These loci are Salmonella specific and were probably acquired by horizontal DNA transfer. Besides, sequence analysis of pag promoters revealed the presence of a conserved PhoP box in 6 out of the 12 genes analyzed. Our results strongly suggest that the expression of a set of Mg2+-controlled genes is driven by PhoP via unknown intermediate regulatory mechanisms that could also involve ancillary factors.

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Effects of ISSo2 Insertions in Structural and Regulatory Genes of the Trimethylamine Oxide Reductase of Shewanella oneidensis

Journal of Bacteriology ◽

10.1128/jb.185.6.2042-2045.2003 ◽

2003 ◽

Vol 185 (6) ◽

pp. 2042-2045 ◽

Cited By ~ 24

Author(s):

Christophe Bordi ◽

Chantal Iobbi-Nivol ◽

Vincent Méjean ◽

Jean-Claude Patte

Keyword(s):

Growth Analysis ◽

Response Regulator ◽

Shewanella Oneidensis ◽

Regulatory Genes ◽

Trimethylamine Oxide ◽

Tmao Reductase

ABSTRACT We have isolated three Shewanella oneidensis mutants specifically impaired in trimethylamine oxide (TMAO) respiration. The mutations arose from insertions of an ISSo2 element into torA, torR, and torS, encoding, respectively, the TMAO reductase TorA, the response regulator TorR, and the sensor TorS. Although TorA is not the sole enzyme reducing TMAO in S. oneidensis, growth analysis showed that it is the main respiratory TMAO reductase. Use of a plasmid-borne torE′-lacZ fusion confirmed that the TorS-TorR phosphorelay mediates TMAO induction of the torECAD operon.

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Identification and Characterization of a Regulatory Sequence Recognized by Mycobacterium tuberculosis Persistence Regulator MprA

Journal of Bacteriology ◽

10.1128/jb.187.1.202-212.2005 ◽

2005 ◽

Vol 187 (1) ◽

pp. 202-212 ◽

Cited By ~ 55

Author(s):

Hongjun He ◽

Thomas C. Zahrt

Keyword(s):

Mycobacterium Tuberculosis ◽

Response Regulator ◽

Regulatory System ◽

Direct Repeat ◽

Regulatory Sequence ◽

Spacer Length ◽

Signaling Systems ◽

Downstream Effector

ABSTRACT Establishment and maintenance of persistent, latent infection by Mycobacterium tuberculosis are dependent on expression of the mprA-mprB regulatory system. Previously, MprA and MprB were shown to participate in phosphotransfer reactions characteristic of two-component signaling systems. To begin identifying downstream effector genes regulated by mprA-mprB during persistent stages of infection, a search for the regulatory sequence(s) recognized by response regulator MprA was carried out. Here, evidence is presented demonstrating that MprA recognizes a 19-bp sequence comprising two loosely conserved 8-bp direct repeat subunits separated by 3 nucleotides. This motif, termed the MprA box, is found upstream of the mprA coding sequence and that of downstream gene pepD (Rv0983). Protein phosphorylation was not required for binding to this DNA sequence by MprA in vitro; however, phosphorylation enhanced DNA binding by MprA and was required for the regulation of mprA and pepD by MprA in vivo. Binding of MprA to the MprA box was dependent on conserved nucleotides contained within repeat subunits and on the spacer length separating these repeats. In addition, recognition of this sequence proceeded via tandem binding of two monomers of MprA. Identification of the genetic determinants regulated by MprA will ultimately enhance our understanding of the mechanisms utilized by M. tuberculosis to undergo latency.

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Structure of the DNA-binding domain of the response regulator SaeR fromStaphylococcus aureus

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715010287 ◽

2015 ◽

Vol 71 (8) ◽

pp. 1768-1776 ◽

Cited By ~ 8

Author(s):

Xiaojiao Fan ◽

Xu Zhang ◽

Yuwei Zhu ◽

Liwen Niu ◽

Maikun Teng ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Response Regulator ◽

Regulatory System ◽

Binding Domain ◽

Dna Binding Domain ◽

Promoter Regions ◽

Winged Helix

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors inStaphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeRDBD) was solved at 2.5 Å resolution. The structure reveals that SaeRDBDexists as a monomer and has the canonical winged helix–turn–helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix–turn–helix module were verified to be important for binding to the P1 promoterin vitroand for the physiological function of SaeRin vivo.

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Phosphorylated AbsA2 Negatively Regulates Antibiotic Production in Streptomyces coelicolor through Interactions with Pathway-Specific Regulatory Gene Promoters

Journal of Bacteriology ◽

10.1128/jb.00305-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5284-5292 ◽

Cited By ~ 67

Author(s):

Nancy L. McKenzie ◽

Justin R. Nodwell

Keyword(s):

Response Regulator ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Regulatory Gene ◽

Gene Clusters ◽

Negative Regulator ◽

Gene Promoters ◽

Promoter Regions ◽

Calcium Dependent

ABSTRACT The AbsA two-component signal transduction system, comprised of the sensor kinase AbsA1 and the response regulator AbsA2, acts as a negative regulator of antibiotic production in Streptomyces coelicolor, for which the phosphorylated form of AbsA2 (AbsA2∼P) is the agent of repression. In this study, we used chromatin immunoprecipitation to show that AbsA2 binds the promoter regions of actII-ORF4, cdaR, and redZ, which encode pathway-specific activators for actinorhodin, calcium-dependent antibiotic, and undecylprodigiosin, respectively. We confirm that these interactions also occur in vitro and that the binding of AbsA2 to each gene is enhanced by phosphorylation. Induced expression of actII-ORF4 and redZ in the hyperrepressive absA1 mutant (C542) brought about pathway-specific restoration of actinorhodin and undecylprodigiosin production, respectively. Our results suggest that AbsA2∼P interacts with as many as four sites in the region that includes the actII-ORF4 promoter. These data suggest that AbsA2∼P inhibits antibiotic production by directly interfering with the expression of pathway-specific regulators of antibiotic biosynthetic gene clusters.

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Mutations to Essential Orphan Response Regulator HP1043 of Helicobacter pylori Result in Growth-Stage Regulatory Defects

Infection and Immunity ◽

10.1128/iai.01193-12 ◽

2013 ◽

Vol 81 (5) ◽

pp. 1439-1449 ◽

Cited By ~ 8

Author(s):

Igor N. Olekhnovich ◽

Serhiy Vitko ◽

Olga Chertihin ◽

Raquel Hontecillas ◽

Monica Viladomiu ◽

...

Keyword(s):

Gene Expression ◽

Helicobacter Pylori ◽

Dna Binding ◽

Conformational Changes ◽

Growth Stage ◽

Response Regulator ◽

Proteomic Profiling ◽

Promoter Regions ◽

Content Type

ABSTRACTHelicobacter pyloriestablishes lifelong infections of the gastric mucosa, a niche considered hostile to most microbes. While responses to gastric acidity and local inflammation are understood, little is known as to how they are integrated into homeostatic control of cell division and growth-stage gene expression. Here we investigate the essential orphan response regulator HP1043, a member of the OmpR/PhoB subfamily of transcriptional regulators that is unique to theEpsilonproteobacteriaand that lacks phosphorylation domains. To test the hypothesis that conformational changes in the homodimer might lead to defects in gene expression, we sought mutations that might alter DNA-binding efficiency. Two introduced mutations (C215S, C221S) C terminal to the DNA-binding domain of HP1043 (HP1043CC11) resulted in a 2-fold higher affinity for its own promoter by footprinting. Modeling studies with the crystal structure of HP1043 suggested that C215S might affect the helix-turn-helix domain. Genomic replacement of thehp1043allele with thehp1043CC11mutant allele resulted in a 2-fold decrease in protein levels, despite a dramatic increase in mRNA. The mutations did not affectin vitrogrowth rates or colonization efficiency in a mouse model. Proteomic profiling (CC11 mutant strain versus wild type) identified many expression differences, and quantitative PCR further revealed that 11 out of 12 examined genes had lost growth-stage regulation and that 6 of the genes contained HP1043 binding consensus sequences within the promoter regions (fur,cagA,cag23,flhA,flip, andnapA). Our studies show that mutations that affect DNA-binding affinity can be used to identify new members of the HP1043 regulon.

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ResDE-Dependent Regulation of Enterotoxin Gene Expression in Bacillus cereus: Evidence for Multiple Modes of Binding for ResD and Interaction with Fnr

Journal of Bacteriology ◽

10.1128/jb.00321-09 ◽

2009 ◽

Vol 191 (13) ◽

pp. 4419-4426 ◽

Cited By ~ 23

Author(s):

Julia Esbelin ◽

Jean Armengaud ◽

Assia Zigha ◽

Catherine Duport

Keyword(s):

Bacillus Cereus ◽

Response Regulator ◽

Regulatory System ◽

Molecular Evidence ◽

Promoter Regions ◽

Food Borne ◽

Regulator Genes ◽

Hemolysin Bl

ABSTRACT In the food-borne pathogen Bacillus cereus F4430/73, the production of major virulence factors hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of hbl and nhe transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution, regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (p) of the enterotoxin regulator genes resDE, fnr, and plcR and the enterotoxin structural genes nhe and hbl, but with different affinities. Binding of ResD to pplcR, pnhe, and phbl was not dependent on the ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and presDE and pfnr. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an antiactivator of Fnr, while phosphorylated ResD acts as a coactivator of Fnr. Finally, our findings represent the first molecular evidence of the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates B. cereus virulence.

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Biochemical Characterization of RssA-RssB, a Two-Component Signal Transduction System Regulating Swarming Behavior in Serratia marcescens

Journal of Bacteriology ◽

10.1128/jb.187.16.5683-5690.2005 ◽

2005 ◽

Vol 187 (16) ◽

pp. 5683-5690 ◽

Cited By ~ 13

Author(s):

Jun-Rong Wei ◽

Yu-Huan Tsai ◽

Po-Chi Soo ◽

Yu-Tze Horng ◽

Shang-Chen Hsieh ◽

...

Keyword(s):

Signal Transduction ◽

Serratia Marcescens ◽

Biochemical Characterization ◽

Response Regulator ◽

Negative Regulator ◽

Site Directed Mutagenesis ◽

Promoter Regions ◽

Two Component ◽

Two Component Signal Transduction

ABSTRACT Our previous study had identified a pair of potential two-component signal transduction proteins, RssA-RssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37°C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30°C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfFSm ), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.

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Effects of Iron and Temperature on Expression of the Pseudomonas aeruginosa tolQRA Genes: Role of the Ferric Uptake Regulator

Journal of Bacteriology ◽

10.1128/jb.180.11.2836-2841.1998 ◽

1998 ◽

Vol 180 (11) ◽

pp. 2836-2841 ◽

Cited By ~ 13

Author(s):

Eric R. Lafontaine ◽

Pamela A. Sokol

Keyword(s):

Pseudomonas Aeruginosa ◽

Transcriptional Start Site ◽

Regulatory Gene ◽

Iron Regulation ◽

Northern Hybridization ◽

Ferric Uptake Regulator ◽

Start Site ◽

Promoter Regions

ABSTRACT The tolQRA genes have been recently identified inPseudomonas aeruginosa PAO. In this study, we examined the effect of iron and temperature on tolQRA expression. A promoterless lacZ gene was introduced downstream of plasmid-encoded tolQ and tolA, and expression was monitored by measuring β-galactosidase activity of cultures. Addition of 25 μM FeCl3 to the culture medium reduced tolQRA expression by 50 to 60% in PAO but by only 25% in the fur mutant PAO A4. Northern hybridization analysis revealed that iron regulation occurs at the level of transcription and involves the P. aeruginosaferric uptake regulator (Fur). Primer extension analysis was used to identify the proposed transcriptional start site oftolA. Although a putative Fur box was identified 20 bp upstream of the proposed start site, purified Fur did not bind to thetolA or tolQR promoter regions in an in vitro gel retardation assay. Therefore, iron regulation of thetol genes appears to involve an intermediate regulatory gene. Expression of tolQR andtolA was optimal at 37°C and was reduced by 40 to 50% when cultures were grown at either 42 or 25°C. Growth in high-iron medium at 25°C further reduced tolQR andtolA expression.

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