副溶血弧菌LacZ报告基因融合实验方法的建立与应用

目的:建立副溶血弧菌(Vibrio parahaemolyticus,VP)的LacZ报告基因融合实验方法。方法:PCR扩增靶基因的整个启动子区序列,并将其直接克隆入pHRP309质粒中,构建重组质粒;将重组质粒VPA1513转入VP野生株(WT)和opaR突变株(ΔopaR)中,而后通过比较两者β半乳糖苷酶活性的差异,确定OpaR对VPA1513的调控关系,以检验实验的稳定性。结果:构建出9个VP生物膜或毒力相关基因的LacZ重组质粒;OpaR对VPA1513的转录具有抑制作用。结论:建立了VP的LacZ报告基因融合实验方法,为后续转录调控机制的研究奠定了基础。     

A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi

Two independent quorum-sensing systems control the expression of bioluminescence (lux) in the marine bacterium Vibrio harveyi. Each system is composed of an autoinducer (AI-1 or AI-2) and its cognate sensor (LuxN or LuxQ). The sensors are two-component hybrid kinases, containing both sensor kinase domains and response regulator domains. Sensory information from the two systems is relayed by a phosphotransfer mechanism to a shared integrator protein called LuxO. LuxO is a member of the response regulator class of the two-component family of signal transduction proteins, and LuxO acts negatively to control luminescence. In this report, missense and in frame deletion mutations were constructed in luxO that encoded proteins mimicking either the phosphorylated or the unphosphorylated form, and these mutations were introduced into the V. harveyi chromosome at the luxO locus. Phenotypical analyses of the resulting mutant V. harveyi strains indicate that the phosphorylated form of LuxO is the repressor, and that the unphosphorylated form of the protein is inactive. Analysis of the lux phenotypes of V. harveyi strains containing single and double luxN and luxQ mutations indicate that LuxN and LuxQ have two activities on LuxO. They act as LuxO protein kinases at low cell density in the absence of autoinducers, and they switch to LuxO protein phosphatases at high cell density in the presence of autoinducers. Furthermore, the timing and potency of inputs from the two systems into regulation of quorum sensing are different.     

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

Vibrio parahaemolyticus isolates display variation in colony morphology, alternating between opaque (OP) and translucent (TR) cell types. Phase variation is the consequence of genetic alterations in the locus encoding the quorum sensing output regulator OpaR. Here, we show that both cell types form stable, but distinguishable biofilms that differ with respect to attachment and detachment profiles to polystyrene, pellicle formation and stability at the air/medium interface, and submerged biofilm architecture and dispersion at a solid/liquid interface. The pellicle, which is a cohesive mat of cells, was exploited to identify mutants having altered or defective biofilm formation. Transposon insertion mutants were obtained with defects in genes affecting multiple cell surface characteristics, including extracellular polysaccharide, mannose-sensitive haemagglutinin type 4 pili and polar (but not lateral) flagella. Other insertions disrupted genes coding for potential secreted proteins or transporters of secreted proteins, specifically haemolysin co-regulated protein and an RTX toxin-like membrane fusion transporter, as well as potential modifiers of cell surface molecules (nagAC operon). The pellicle screen also identified mutants with lesions in regulatory genes encoding H-NS, a CsgD-like repressor and an AraC-like protein. This work initiates the characterization of V. parahaemolyticus biofilm formation in the OP and TR cell types and identifies a diverse repertoire of cell surface elements that participate in determining multicellular architecture.     

Quorum regulatory small RNAs repress type VI secretion in Vibrio cholerae

Type VI secretion is critical for Vibrio cholerae to successfully combat phagocytic eukaryotes and to survive in the presence of competing bacterial species. V. cholerae type VI secretion system genes are encoded in one large and two small clusters. In V. cholerae, type VI secretion is controlled by quorum sensing, the cell–cell communication process that enables bacteria to orchestrate group behaviours. The quorum‐sensing response regulator LuxO represses type VI secretion genes at low cell density and the quorum‐sensing regulator HapR activates type VI secretion genes at high cell density. We demonstrate that the quorum regulatory small RNAs (Qrr sRNAs) that function between LuxO and HapR in the quorum‐sensing cascade are required for these regulatory effects. The Qrr sRNAs control type VI secretion via two mechanisms: they repress expression of the large type VI secretion system cluster through base pairing and they repress HapR, the activator of the two small type VI secretion clusters. This regulatory arrangement ensures that the large cluster encoding many components of the secretory machine is expressed prior to the two small clusters that encode the secreted effectors. Qrr sRNA‐dependent regulation of the type VI secretion system is conserved in pandemic and non‐pandemic V. cholerae strains.     

Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter

Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a delta hapR mutant and decreased in a delta luxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between -85 and -58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at -85 and -77. Introducing the -77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin-co-regulated pilus (TCP) and cholera toxin (CT) that occurs in a delta luxO mutant, indicating that HapR function at aphA is critical for density-dependent regulation of virulence genes.     

Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.     

Purification and characterization of a luxO promoter binding protein LuxT from Vibrio harveyi

Bioluminescence in the marine bacterium Vibrio harveyi is cell density dependent and is regulated by small molecules (autoinducers) excreted by the bacteria. The autoinducer signals are relayed to a central regulator, LuxO, which acts in its phosphorylated form as a repressor of the lux operon at the early stages of cell growth. We report in these studies the purification to homogeneity of a luxO DNA binding protein (LuxT) from V. harveyi after five major chromatography steps, including a highly effective DNA affinity chromatography step and reverse-phase HPLC. Regeneration of binding activity was accomplished after HPLC and SDS-PAGE by renaturation of LuxT from guanidine hydrochloride. It was also demonstrated that the functional LuxT was a dimer of 17 kDa that bound tightly (K(d) = 2 nM) to the luxO promoter. The sequences of three tryptic peptides obtained on digestion of the purified protein did not match any sequences in the Protein Data Bank, indicating that LuxT is a new V. harveyi lux regulatory protein.     

Regulation of quorum sensing in Vibrio harveyi by LuxO and sigma-54

The bioluminescent marine bacterium Vibrio harveyi controls light production (lux) by an elaborate quorum-sensing circuit. V. harveyi produces and responds to two different autoinducer signals (AI-1 and AI-2) to modulate the luciferase structural operon (luxCDABEGH) in response to changes in cell-population density. Unlike all other Gram-negative quorum-sensing organisms, V. harveyi regulates quorum sensing using a two-component phosphorylation-dephosphorylation cascade. Each autoinducer is recognized by a cognate hybrid sensor kinase (called LuxN and LuxQ). Both sensors transduce information to a shared phosphorelay protein called LuxU, which in turn conveys the signal to the response regulator protein LuxO. Phospho-LuxO is responsible for repression of luxCDABEGH expression at low cell density. In the present study, we demonstrate that LuxO functions as an activator protein via interaction with the alternative sigma factor, sigma54 (encoded by rpoN). Our results suggest that LuxO, together with sigma54, activates the expression of a negative regulator of luminescence. We also show that phenotypes other than lux are regulated by LuxO and sigma54, demonstrating that in Vibrio harveyi, quorum sensing controls multiple processes.     

Quorum sensing and alternative sigma factor RpoN regulate type VI secretion system I (T6SSVA1) in fish pathogen <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Type VI secretion system (T6SS) is a highly conserved bacterial protein secretion system and is precisely regulated in Gram-negative pathogens. In Vibrio alginolyticus, an important fish pathogen, two complete T6SS gene clusters (T6SSVA1 and T6SSVA2) were identified. In this study, expression of a hemolysin coregulated protein (Hcp1), which is one of the hallmarks of T6SS, was found to be strictly regulated in this bacterium. We showed that the expression of Hcp1 was growth phase-dependent and the production of Hcp1 reached a maximum in the exponential phase. The expression of Hcp1 was positively and negatively regulated by quorum sensing regulators LuxO and LuxR, respectively. In addition, we observed that Hcp1 expression required the alternative sigma factor RpoN and the enhancer-binding protein VasH, which is encoded in T6SSVA1 gene cluster. Moreover, LuxR, RpoN, and VasH could positively regulate the expression of other T6SS genes. Taken together, we demonstrated that the expression of T6SS in V. alginolyticus was under the regulation of quorum sensing and alternative sigma factor.