A role for the PhoBR regulatory system homologue in the Vibrio cholerae phosphate-limitation response and intestinal colonization The GenBank accession number for the sequence reported in this paper is AF043352.

Krüger, Wanda M. A. von; Humphreys, Sue; Ketley, Julian M.

doi:10.1099/00221287-145-9-2463

Cited by 54 publications

(64 citation statements)

References 64 publications

(43 reference statements)

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“…The 14 regulators identified in this screen (SI Table 3) include the following known regulators: VieB (VC1651), a response regulator that is expressed in vivo in the mouse and is part of a signal transduction system that contributes to regulation of cholera toxin production (16,17); VarS (VC2453), a sensor kinase involved in virulence and quorum sensing (18,19); and PhoR (VC0720), a sensor kinase that controls expression of the PhoBR regulon in response to phosphate and is important for colonization of the rabbit small intestine (20). Deletion of either VC1349 or VC0303, two response regulators, had no effect on virulence in the mouse model ( Table 2).…”

Section: In Vitro (Lb)mentioning

confidence: 99%

An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers

Lombardo

Michalski

Martinez-Wilson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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In vivo expression technology (IVET) has been widely used to study gene expression of human bacterial pathogens in animal models, but has heretofore not been used in humans to our knowledge. As part of ongoing efforts to understand Vibrio cholerae pathogenesis and develop improved V. cholerae vaccines, we have performed an IVET screen in humans for genes that are preferentially expressed by V. cholerae during infection. A library of 8,734 nontoxigenic V. cholerae strains carrying transcriptional fusions of genomic DNA to a resolvase gene was ingested by five healthy adult volunteers. Transcription of the fusion leads to resolvasedependent excision of a sacB-containing cassette and thus the selectable phenotype of sucrose resistance (Suc R ). A total of Ϸ20,000 Suc R isolates, those carrying putative in vivo-induced fusions, were recovered from volunteer stool samples. Analysis of the fusion junctions from >7,000 Suc R isolates from multiple samples from multiple volunteers identified 217 candidate genes for preferential expression during human infection. Of genes or operons induced in three or more volunteers, the majority of those tested (65%) were induced in an infant mouse model. VC0201 (fhuC), which encodes the ATPase of a ferrichrome ABC transporter, is one of the identified in vivo-induced genes and is required for virulence in the mouse model. gene expression ͉ genetics ͉ vaccinology ͉ virulence O ur understanding of the complex interactions between bacterial pathogens and humans relies heavily on the use of animal and tissue culture model systems that serve as surrogates of human infection. One useful genetic tool for discovering genes and pathways involved in virulence is in vivo expression technology (IVET), which was designed to identify genes of pathogens that are preferentially expressed during infection and has been extensively used in model systems (reviewed by refs. 1 and 2). IVET is a promoter-trapping strategy in which cells carrying a library of transcriptional fusions of genomic DNA to a reporter gene are used to infect a model host and those carrying fusions that are expressed in vivo can be identified by either a genetic screen or selection. This technique allows the identification of genes that may be expressed only under in vivo conditions [in vivo-induced genes (ivi genes)]. Such genes may be very difficult to identify during growth under laboratory conditions, but are likely to be important in the host for survival and virulence. One form of IVET, used in this work, is recombination-based IVET (3, 4) in which the reporter gene encodes a resolvase that effects a permanent genetic change, allowing a direct selection for cells that expressed the resolvase even transiently during infection.Here, we describe the use of recombination-based IVET to identify genes of Vibrio cholerae, the causative agent of the diarrheal disease cholera, that are expressed during human infection. This approach has the potential to identify important virulence genes that may not be expressed in vitro or during in...

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Section: In Vitro (Lb)mentioning

confidence: 99%

An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers

Lombardo

Michalski

Martinez-Wilson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Recent studies have shown that the PhoBR system has many additional roles in bacteria (12,13,18). In particular, roles for PhoBR have been demonstrated in V. cholerae motility, biofilm formation, intestinal colonization, and virulence (9,10,36,37,43,47).…”

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confidence: 99%

“…In a previous work, we have shown that in V. cholerae phoR and phoB genes constitute an operon whose regulatory region contains a putative Pho box sequence located at Ϫ35, as in E. coli (47). Moreover, mutation in phoBR eliminated the expression of Pho regulon genes in strains of V. cholerae O1 under P i limitation.…”

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confidence: 99%

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Fine-Tuning Control ofphoBRExpression in Vibrio cholerae by Binding of PhoB to Multiple Pho Boxes

Diniz

Goulart²,

Barbosa³

et al. 2011

J Bacteriol

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The control of Vibrio cholerae phoBR expression by PhoB involves its binding to Pho boxes at ؊35 (box 1), ؊60 (box 2), and ؊80 (box 3) from the putative phoB translation start site. These loci were located in the sense (box 1) and antisense (boxes 2 and 3) strands of the phoBR regulatory region, and PhoB binds to these individual boxes with distinct affinities. Fusions of sequences containing different combinations of these boxes upstream of the lacZ reporter in a plasmid demonstrated that only those carrying boxes 1, 2, and 3, or 1 alone, activated transcription under inorganic phosphate (P i ) limitation. When a fragment, including only boxes 1 and 2, was fused to lacZ, expression was no longer induced by low P i , suggesting a repressive role for PhoBϳbox2 (PhoB bound to box 2) over the transcriptional activity induced by PhoBϳbox1. The similarity between lacZ expression levels from promoter fragments containing the three boxes or box 1 alone showed that PhoBϳbox3 eliminated the repressive effect imposed by PhoBϳbox2 on phoBR transcription. Complementation assays with a phoBR-containing plasmid demonstrated that the 234-bp promoter fragment carrying the three boxes is absolutely required for operon expression in Vibrio cholerae ⌬phoBR cells. This was observed under P i abundance, when phoBR was expressed at a basal level and, also in low P i conditions, when Pho regulon genes were fully expressed. Thus, under P i limitation, PhoB exerts dual regulatory functions by binding sequentially distinct Pho boxes to enable the fine-tuning and precise control of phoBR expression in V. cholerae cells.

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“…In Mycobacterium tuberculosis, pstS1 and pstS2 have been identified as virulence factors (6,7). Expression of the pst-SCAB operons is generally induced under P i -limited conditions and is mediated by the two-component regulatory systems PhoBR in Gram-negative microorganisms (8)(9)(10), PhoPR in most Gram-positive organisms (11,12), and SenX3-RegX3 in Mycobacterium smegmatis (13). In E. coli and M. smegmatis, repression of the phosphate starvation response requires the Pst transporter, and mutations in pstS result in constitutive target gene expression (1,14).…”

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confidence: 99%

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

et al. 2014

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dBacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains.

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A role for the PhoBR regulatory system homologue in the Vibrio cholerae phosphate-limitation response and intestinal colonization The GenBank accession number for the sequence reported in this paper is AF043352.

Cited by 54 publications

References 64 publications

An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers

An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers

Fine-Tuning Control ofphoBRExpression in Vibrio cholerae by Binding of PhoB to Multiple Pho Boxes

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

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