Transcriptional Regulation of opaR, qrr2–4 and aphA by the Master Quorum-Sensing Regulator OpaR in Vibrio parahaemolyticus

Background: Vibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2–4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood. Methodology/Principal Findings: The bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2–4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promote

Comparative transcriptomics in Yersinia pestis: a global view of environmental modulation of gene expression

<p>Abstract</p> <p>Background</p> <p>Environmental modulation of gene expression in <it>Yersinia pestis </it>is critical for its life style and pathogenesis. Using cDNA microarray technology, we have analyzed the global gene expression of this deadly pathogen when grown under different stress conditions <it>in vitro</it>.</p> <p>Results</p> <p>To provide us with a comprehensive view of environmental modulation of global gene expression in <it>Y. pestis</it>, we have analyzed the gene expression profiles of 25 different stress conditions. Almost all known virulence genes of <it>Y. pestis </it>were differentially regulated under multiple environmental perturbations. Clustering enabled us to functionally classify co-expressed genes, including some uncharacterized genes. Collections of operons were predicted from the microarray data, and some of these were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Several regulatory DNA motifs, probably recognized by the regulatory protein Fur, PurR, or Fnr, were predicted from the clustered genes, and a Fur binding site in the corresponding promoter regions was verified by electrophoretic mobility shift assay (EMSA).</p> <p>Conclusion</p> <p>The comparative transcriptomics analysis we present here not only benefits our understanding of the molecular determinants of pathogenesis and cellular regulatory circuits in <it>Y. pestis</it>, it also serves as a basis for integrating increasing volumes of microarray data using existing methods.</p

Genome plasticity of Vibrio parahaemolyticus: microevolution of the 'pandemic group'

<p>Abstract</p> <p>Background</p> <p>Outbreak of <it>V. parahaemolyticus </it>infections occurred since 1996 was linked to a proposed clonal complex, the pandemic group. The whole genome sequence provides an unprecedented opportunity for dissecting genome plasticity and phylogeny of the populations of <it>V. parahaemolyticus</it>. In the present work, a whole-genome cDNA microarray was constructed to compare the genomic contents of a collection of 174 strains of <it>V. parahaemolyticus</it>.</p> <p>Results</p> <p>Genes that present variably in the genome accounted for about 22% of the whole gene pool on the genome. The phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the phylogenetic structure of the 174 strains. Strains were assigned into five complexes (C1 to C5), and those in each complex were related genetically and phylogenetically. C3 and C4 represented highly virulent clinical clones. C2 and C3 constituted two different clonal complexes 'old-O3:K6 clone' and 'pandemic clone', respectively. C3 included all the 39 pandemic strains tested (<it>trh</it>^-, <it>tdh</it>⁺and GS-PCR⁺), while C2 contained 12 pre-1996 'old' O3:K6 strains (<it>trh</it>⁺, <it>tdh</it>^-and GS-PCR^-) tested herein. The pandemic clone (post-1996 'new' O3:K6 and its derivates O4:K68, O1:K25, O1:KUT and O6:K18) might be emerged from the old-O3:K6 clone, which was promoted by acquisition of <it>toxRS</it>/new sequence and genomic islands. A phylogenetic intermediate O3:K6 clade (<it>trh</it>^-, <it>tdh</it>^-and GS-PCR⁺) was identified between the pandemic and old-O3:K6 clones.</p> <p>Conclusion</p> <p>A comprehensive overview of genomic contents in a large collection of global isolates from the microarray-based comparative genomic hybridization data enabled us to construct a phylogenetic structure of <it>V. parahaemolyticus </it>and an evolutionary history of the pandemic group (clone) of this pathogen.</p

Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>The osmotic regulator OmpR in <it>Escherichia coli </it>regulates differentially the expression of major porin proteins OmpF and OmpC. In <it>Yersinia enterocolitica </it>and <it>Y. pseudotuberculosis</it>, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in <it>Y. pestis </it>are not yet fully understood.</p> <p>Results</p> <p><it>Y. pestis </it>OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The <it>ompR </it>mutation likely did not affect the virulence of <it>Y. pestis </it>strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the <it>ompR </it>mutation, indicating the global regulatory role of OmpR in <it>Y. pestis</it>. Real-time RT-PCR or <it>lacZ </it>fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. <it>ompC</it>, <it>F</it>, <it>X</it>, and <it>R </it>were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner.</p> <p>Conclusion</p> <p>OmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. <it>Y. pestis </it>OmpR regulates <it>ompC</it>, <it>F</it>, <it>X</it>, and <it>R </it>directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and × at high osmolarity in <it>Y. pestis</it>, in contrast to the reciprocal regulation of them in <it>E. coli</it>. The main difference is that <it>ompF </it>expression is not repressed at high osmolarity in <it>Y. pestis</it>, which is likely due to the absence of a promoter-distal OmpR-binding site for <it>ompF</it>.</p

Direct and negative regulation of the sycO-ypkA-ypoJ operon by cyclic AMP receptor protein (CRP) in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>Pathogenic yersiniae, including <it>Y. pestis</it>, share a type III secretion system (T3SS) that is composed of a secretion machinery, a set of translocation proteins, a control system, and six Yop effector proteins including YpkA and YopJ. The cyclic AMP receptor protein (CRP), a global regulator, was recently found to regulate the laterally acquired genes (<it>pla </it>and <it>pst</it>) in <it>Y. pestis</it>. The regulation of T3SS components by CRP is unknown.</p> <p>Results</p> <p>The <it>sycO</it>, <it>ypkA </it>and <it>yopJ </it>genes constitute a single operon in <it>Y. pestis</it>. CRP specifically binds to the promoter-proximate region of <it>sycO</it>, and represses the expression of the <it>sycO-ypkA-yopJ </it>operon. A single CRP-dependent promoter is employed for the <it>sycO-ypkA-yopJ </it>operon, but two CRP binding sites (site 1 and site 2) are detected within the promoter region. A CRP box homologue is found in site 1 other than site 2. The determination of CRP-binding sites, transcription start site and core promoter element (-10 and -35 regions) promotes us to depict the structural organization of CRP-dependent promoter, giving a map of CRP-promoter DNA interaction for <it>sycO-ypkA-yopJ</it>.</p> <p>Conclusion</p> <p>The <it>sycO-ypkA-yopJ </it>operon is under the direct and negative regulation of CRP in <it>Y. pestis</it>. The <it>sycO-ypkA-yopJ </it>promoter-proximate regions are extremely conserved in <it>Y. pestis</it>, <it>Y. pseudotuberculosis </it>and <it>Y. enterocolitica</it>. Therefore, data presented here can be generally applied to the above three pathogenic yersiniae.</p

Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>The zinc uptake regulator Zur is a Zn²⁺-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in <it>Y. pestis</it>.</p> <p>Results</p> <p>We constructed a <it>zur </it>null mutant of <it>Y. pestis </it>biovar <it>microtus </it>strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of <it>Y. pestis </it>upon exposure to zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons <it>znuA, znuCB </it>and <it>ykgM</it>-<it>RpmJ2</it>. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes.</p> <p>Conclusion</p> <p>Zur as a repressor directly controls the transcription of <it>znuA, znuCB </it>and <it>ykgM</it>-<it>RpmJ2 </it>in <it>Y. pestis </it>by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in <it>Y. pestis </it>likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.</p

Regulatory effects of cAMP receptor protein (CRP) on porin genes and its own gene in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>The cAMP receptor protein (CRP) is a global bacterial regulator that controls many target genes. The CRP-cAMP complex regulates the <it>ompR-envZ </it>operon in <it>E. coli </it>directly, involving both positive and negative regulations of multiple target promoters; further, it controls the production of porins indirectly through its direct action on <it>ompR-envZ</it>. Auto-regulation of CRP has also been established in <it>E. coli</it>. However, the regulation of porin genes and its own gene by CRP remains unclear in <it>Y. pestis</it>.</p> <p>Results</p> <p><it>Y. pestis </it>employs a distinct mechanism indicating that CRP has no regulatory effect on the <it>ompR-envZ </it>operon; however, it stimulates <it>ompC </it>and <it>ompF </it>directly, while repressing <it>ompX</it>. No transcriptional regulatory association between CRP and its own gene can be detected in <it>Y. pestis</it>, which is also in contrast to the fact that CRP acts as both repressor and activator for its own gene in <it>E. coli</it>. It is likely that <it>Y. pestis </it>OmpR and CRP respectively sense different signals (medium osmolarity, and cellular cAMP levels) to regulate porin genes independently.</p> <p>Conclusion</p> <p>Although the CRP of <it>Y. pestis </it>shows a very high homology to that of <it>E. coli</it>, and the consensus DNA sequence recognized by CRP is shared by the two bacteria, the <it>Y. pestis </it>CRP can recognize the promoters of <it>ompC</it>, <it>F</it>, and <it>X </it>directly rather than that of its own gene, which is different from the relevant regulatory circuit of <it>E. coli</it>. Data presented here indicate a remarkable remodeling of the CRP-mediated regulation of porin genes and of its own one between these two bacteria.</p

Identification and characterization of PhoP regulon members in Yersinia pestis biovar Microtus

<p>Abstract</p> <p>Background</p> <p>The transcription regulator PhoP has been shown to be important for <it>Y. pestis </it>survival in macrophages and under various <it>in vitro </it>stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in <it>Y. pestis</it>. A series of biochemical experiments were done herein to study members of the PhoP regulon of <it>Y. pestis </it>biovar <it>Microtus</it>.</p> <p>Results</p> <p>By using gel mobility shift assay and quantitative RT-PCR, a total of 30 putative transcription units were characterized as direct PhoP targets. The primer extension assay was further used to determine the transcription start sites of 18 PhoP-dependent promoters and to localize the -10 and -35 elements. The DNase I footprinting was used to identify the PhoP-binding sites within 17 PhoP-dependent promoters, enabling the identification of PhoP box and matrix that both represented the conserved signals for PhoP recognition in <it>Y. pestis</it>. Data presented here providing a good basis for modeling PhoP-promoter DNA interactions that is crucial to the PhoP-mediated transcriptional regulation.</p> <p>Conclusion</p> <p>The proven direct PhoP targets include nine genes encoding regulators and 21 genes or operons with functions of detoxification, protection against DNA damages, resistance to antimicrobial peptides, and adaptation to magnesium limitation. We can presume that PhoP is a global regulator that controls a complex regulatory cascade by a mechanism of not only directly controlling the expression of specific genes, but also indirectly regulating various cellular pathways by acting on a set of dedicated regulators. These results help us gain insights into the PhoP-dependent mechanisms by which <it>Y. pestis </it>survives the antibacterial strategies employed by host macrophages.</p

Genotyping and Phylogenetic Analysis of Yersinia pestis by MLVA: Insights into the Worldwide Expansion of Central Asia Plague Foci

BACKGROUND: The species Yersinia pestis is commonly divided into three classical biovars, Antiqua, Medievalis, and Orientalis, belonging to subspecies pestis pathogenic for human and the (atypical) non-human pathogenic biovar Microtus (alias Pestoides) including several non-pestis subspecies. Recent progress in molecular typing methods enables large-scale investigations in the population structure of this species. It is now possible to test hypotheses about its evolution which were proposed decades ago. For instance the three classical biovars of different geographical distributions were suggested to originate from Central Asia. Most investigations so far have focused on the typical pestis subspecies representatives found outside of China, whereas the understanding of the emergence of this human pathogen requires the investigation of strains belonging to subspecies pestis from China and to the Microtus biovar. METHODOLOGY/PRINCIPAL FINDINGS: Multi-locus VNTR analysis (MLVA) with 25 loci was performed on a collection of Y. pestis isolates originating from the majority of the known foci worldwide and including typical rhamnose-negative subspecies pestis as well as rhamnose-positive subspecies pestis and biovar Microtus. More than 500 isolates from China, the Former Soviet Union (FSU), Mongolia and a number of other foci around the world were characterized and resolved into 350 different genotypes. The data revealed very close relationships existing between some isolates from widely separated foci as well as very high diversity which can conversely be observed between nearby foci. CONCLUSIONS/SIGNIFICANCE: The results obtained are in full agreement with the view that the Y. pestis subsp. pestis pathogenic for humans emerged in the Central Asia region between China, Kazakhstan, Russia and Mongolia, only three clones of which spread out of Central Asia. The relationships among the strains in China, Central Asia and the rest of the world based on the MLVA25 assay provide an unprecedented view on the expansion and microevolution of Y. pestis

A Glimpse of Streptococcal Toxic Shock Syndrome from Comparative Genomics of S. suis 2 Chinese Isolates

BACKGROUND: Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen, causing more than 200 cases of severe human infection worldwide, with the hallmarks of meningitis, septicemia, arthritis, etc. Very recently, SS2 has been recognized as an etiological agent for streptococcal toxic shock syndrome (STSS), which was originally associated with Streptococcus pyogenes (GAS) in Streptococci. However, the molecular mechanisms underlying STSS are poorly understood. METHODS AND FINDINGS: To elucidate the genetic determinants of STSS caused by SS2, whole genome sequencing of 3 different Chinese SS2 strains was undertaken. Comparative genomics accompanied by several lines of experiments, including experimental animal infection, PCR assay, and expression analysis, were utilized to further dissect a candidate pathogenicity island (PAI). Here we show, for the first time, a novel molecular insight into Chinese isolates of highly invasive SS2, which caused two large-scale human STSS outbreaks in China. A candidate PAI of ∼89 kb in length, which is designated 89K and specific for Chinese SS2 virulent isolates, was investigated at the genomic level. It shares the universal properties of PAIs such as distinct GC content, consistent with its pivotal role in STSS and high virulence. CONCLUSIONS: To our knowledge, this is the first PAI candidate from S. suis worldwide. Our finding thus sheds light on STSS triggered by SS2 at the genomic level, facilitates further understanding of its pathogenesis and points to directions of development on some effective strategies to combat highly pathogenic SS2 infections