Cross-regulation in Vibrio parahaemolyticus: compensatory activation of polar flagellar genes by the lateral flagellar regulator LafK.
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ABSTRACT:
SUBMITTER: Kim YK
PROVIDER: S-EPMC419961 | biostudies-other | 2004 Jun
REPOSITORIES: biostudies-other
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Project description:Vibrio parahaemolyticus possesses dual flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (i.e., swimming) with a motor that is powered by the sodium motive force. Multiple proton-driven lateral flagella enable translocation over surfaces (i.e., swarming). The polar flagellum is produced continuously, while production of lateral flagella is induced when the organism is grown on surfaces. This work describes the isolation of mutants with insertions in the structural and regulatory laf genes. A Tn5-based lux transcriptional reporter transposon was constructed and used for mutagenesis and subsequent transcriptional analysis of the laf regulon. Twenty-nine independent insertions were distributed within 16 laf genes. DNA sequence analysis identified 38 laf genes in two loci. Among the mutants isolated, 11 contained surface-induced lux fusions. A hierarchy of laf gene expression was established following characterization of the laf::lux transcriptional fusion strains and by mutational and primer extension analyses of the laf regulon. The laf system is like many enteric systems in that it is a proton-driven, peritrichous flagellar system; however, laf regulation was different from the Salmonella-Escherichia coli paradigm. There is no apparent flhDC counterpart that encodes master regulators known to control flagellar biosynthesis and swarming in many enteric bacteria. A potential sigma(54)-dependent regulator, LafK, was demonstrated to control expression of early genes, and a lateral-specific sigma(28) factor controls late flagellar gene expression. Another notable feature was the discovery of a gene encoding a MotY-like product, which previously had been associated only with the architecture of sodium-type polar flagellar motors.
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Project description:Vibrio parahaemolyticus has dual flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum propels the swimmer cell in liquid environments. Numerous unsheathed lateral flagella move the swarmer cell over surfaces. The polar flagellum is produced continuously, whereas the synthesis of lateral flagella is induced under conditions that impede the function of the polar flagellum, e.g., in viscous environments or on surfaces. Thus, the organism possesses two large gene networks that orchestrate polar and lateral flagellar gene expression and assembly. In addition, the polar flagellum functions as a mechanosensor controlling lateral gene expression. In order to gain insight into the genetic circuitry controlling motility and surface sensing, we have sought to define the polar flagellar gene system. The hierarchy of regulation appears to be different from the polar system of Caulobacter crescentus or the peritrichous system of enteric bacteria but is pertinent to many Vibrio and Pseudomonas species. The gene identity and organization of 60 potential flagellar and chemotaxis genes are described. Conserved sequences are defined for two classes of polar flagellar promoters. Phenotypic and genotypic analysis of mutant strains with defects in swimming motility coupled with primer extension analysis of flagellar and chemotaxis transcription provides insight into the polar flagellar organelle, its assembly, and regulation of gene expression.
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Project description:Vibrio campbellii is a gram-negative bacterial pathogen that is both free-living and a pathogen of marine organisms and exhibits swimming motility via a single, polar flagellum. Swimming motility is a critical virulence factor in V. campbellii pathogenesis, and disruption of the flagellar motor significantly decreases host mortality. However, while V. campbelli encodes homologs of flagellar and chemotaxis genes conserved by other members of the Vibrionaceae, the regulatory network governing these genes have not been explored. We systematically deleted all 63 known flagellar and chemotaxis genes in V. campbellii and examined their effects on motility compared to their homologs in other Vibrios. We specifically focused on assessing the roles of the core flagellar regulators of the flagellar regulatory hierarchy established in other Vibrios: rpoN, flrA, flrC, and fliA. Although V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other Vibrios, we observed two critical differences: the σ54-dependent regulator FlrA is dispensable for motility, and Class II gene expression is independent of σ54 regulation. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae.
2021-07-26 | GSE167483 | GEO
Project description:Vibrio parahaemolyticus possesses two types of flagella, polar and lateral, powered by distinct energy sources, which are derived from the sodium and proton motive forces, respectively. Although proton-powered flagella in Escherichia coli and Salmonella enterica serovar Typhimurium have been extensively studied, the mechanism of torque generation is still not understood. Molecular knowledge of the structure of the sodium-driven motor is only now being developed. In this work, we identify the switch components, FliG, FliM, and FliN, of the sodium-type motor. This brings the total number of genes identified as pertinent to polar motor function to seven. Both FliM and FliN possess charged domains not found in proton-type homologs; however, they can interact with the proton-type motor of E. coli to a limited extent. Residues known to be critical for torque generation in the proton-type motor are conserved in the sodium-type motor, suggesting a common mechanism for energy transfer at the rotor-stator interface regardless of the driving force powering rotation. Mutants representing a complete panel of insertionally inactivated switch and motor genes were constructed. All of these mutants were defective in sodium-driven swimming motility. Alkaline phosphatase could be fused to the C termini of MotB and MotY without abolishing motility, whereas deletion of the unusual, highly charged C-terminal domain of FliM disrupted motor function. All of the mutants retained proton-driven, lateral motility over surfaces. Thus, although central chemotaxis genes are shared by the polar and lateral systems, genes encoding the switch components, as well as the motor genes, are distinct for each motility system.
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Project description:Proteases play a key role in numerous bacterial physiological events. Microbial proteases are used in the pharmaceutical industry and in biomedical applications. The genus Vibrio comprises protease-producing bacteria. Proteases transform polypeptides into shorter chains for easier utilization. They also function as a virulence factor in pathogens. The mechanism by which protease genes are regulated in Vibrio parahaemolyticus, an emerging world-wide human pathogen, however, still remains unclear. Quorum sensing is the communication system of bacteria. OpaR is the master quorum-sensing regulator in V. parahaemolyticus. In the present study, quantitative reverse transcriptase-polymerase chain reaction and protease gene promoter-fusion reporter assays revealed that OpaR represses seven protease genes-three metalloprotease genes and four serine protease genes-which are involved in environmental survival and bacterial virulence. Furthermore, the electrophoresis mobility shift assay demonstrated that OpaR is bound directly to the promoter region of each of the seven protease genes. DNase I footprinting identified the sequence of these OpaR-binding sites. ChIP-seq analyses revealed 435 and 835 OpaR-binding sites in the late-log and stationary phases, respectively. These OpaR-binding sequences indicated a conserved OpaR-binding motif: TATTGATAAAATTATCAATA. These results advance our understanding of the protease regulation system in V. parahaemolyticus. This study is the first to reveal the OpaR motif within V. parahaemolyticus in vivo, using ChIP-seq, and to provide a database for OpaR direct regulon.
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Project description:Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.
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Project description:Bacteriophage has been recognized as a novel approach to treat bacterial infectious diseases. However, phage resistance may reduce the efficacy of phage therapy. Here, we described a mechanism of bacterial resistance to phage infections. In Gram-negative enteric pathogen Vibrio parahaemolyticus, we found that polar flagella can reduce the phage infectivity. Deletion of polar flagella, but not the lateral flagella, can dramatically promote the adsorption of phage to the bacteria and enhances the phage infectivity to V. parahaemolyticus, indicating that polar flagella play an inhibitory role in the phage infection. Notably, it is the rotation, not the physical presence, of polar flagella that inhibits the phage infection of V. parahaemolyticus. Strikingly, phage dramatically reduces the virulence of V. parahaemolyticus only when polar flagella were absent both in vitro and in vivo. These results indicated that polar flagella rotation is a previously unidentified mechanism that confers bacteriophage resistance.
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Project description:The gene hierarchy directing biogenesis of peritrichous flagella on the surface of Escherichia coli and other enterobacteria is controlled by the heterotetrameric master transcriptional regulator FlhD(2)C(2). To assess the extent to which FlhD(2)C(2) directly activates promoters of a wider regulon, a computational screen of the E. coli genome was used to search for gene-proximal DNA sequences similar to the 42-44 bp inverted repeat FlhD(2)C(2) binding consensus. This identified the binding sequences upstream of all eight flagella class II operons, and also putative novel FlhD(2)C(2) binding sites in the promoter regions of 39 non-flagellar genes. Nine representative non-flagellar promoter regions were all bound in vitro by active reconstituted FlhD(2)C(2) over the K(D) range 38-356 nM, and of the nine corresponding chromosomal promoter-lacZ fusions, those of the four genes b1904, b2446, wzz(fepE) and gltI showed up to 50-fold dependence on FlhD(2)C(2) in vivo. In comparison, four representative flagella class II promoters bound FlhD(2)C(2) in the K(D) range 12-43 nM and were upregulated in vivo 30- to 990-fold. The FlhD(2)C(2)-binding sites of the four regulated non-flagellar genes overlap by 1 or 2 bp the predicted -35 motif of the FlhD(2)C(2)-activated sigma(70) promoters, as is the case with FlhD(2)C(2)-dependent class II flagellar promoters. The data indicate a wider FlhD(2)C(2) regulon, in which non-flagellar genes are bound and activated directly, albeit less strongly, by the same mechanism as that regulating the flagella gene hierarchy.
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Project description:Here we probe the global response to calcium in the marine bacterium and pathogen Vibrio parahaemolyticus by using transcriptome, reporter and phenotypic analyses. Swarming gene expression and motility were enhanced by calcium. Calcium also stimulated expression and function of one of the organism’s two type three secretion systems (T3SS1 but not the T3SS2). Although low calcium is an inducing signal for the T3SS of many organisms, calcium stimulation of T3SS has not been reported before. EGTA was also a stimulus for T3SS1 expression and activity; however this appeared to be the consequence of iron rather than calcium chelation. LafK, a key regulator of swarming genes, was found necessary for calcium induction of T3SS1 gene transcription and cytotoxicity. Regulation of swarming and T3SS1 were additionally linked by a negative feedback loop propagated by ExsA. Overexpression of exsA was used to probe the extent of the T3SS1 regulon and verify its coincident induction by calcium and EGTA. The calcium transcriptome analysis revealed a calcium-repressed LysR-type transcriptional regulator; CalR was shown to repress swarming and T3SS1 gene expression. Thus in V. parahaemolyticus, calcium influences gene expression and behavior and seems a signal pertinent for surface colonization and virulence.
2010-11-05 | GSE22190 | GEO
Project description:Here we probe the global response to calcium in the marine bacterium and pathogen Vibrio parahaemolyticus by using transcriptome, reporter and phenotypic analyses. Swarming gene expression and motility were enhanced by calcium. Calcium also stimulated expression and function of one of the organismM-bM-^@M-^Ys two type three secretion systems (T3SS1 but not the T3SS2). Although low calcium is an inducing signal for the T3SS of many organisms, calcium stimulation of T3SS has not been reported before. EGTA was also a stimulus for T3SS1 expression and activity; however this appeared to be the consequence of iron rather than calcium chelation. LafK, a key regulator of swarming genes, was found necessary for calcium induction of T3SS1 gene transcription and cytotoxicity. Regulation of swarming and T3SS1 were additionally linked by a negative feedback loop propagated by ExsA. Overexpression of exsA was used to probe the extent of the T3SS1 regulon and verify its coincident induction by calcium and EGTA. The calcium transcriptome analysis revealed a calcium-repressed LysR-type transcriptional regulator; CalR was shown to repress swarming and T3SS1 gene expression. Thus in V. parahaemolyticus, calcium influences gene expression and behavior and seems a signal pertinent for surface colonization and virulence. The gene expression profiles of Vibrio parahaemolyticus cells grown on rich medium or medium supplemented with CaCl2 or EGTA were compared using Affymetrix custom microarrays.
2010-11-05 | E-GEOD-22190 | biostudies-arrayexpress