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Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.
Subject(s)
Vibrio parahaemolyticus/metabolism , Escherichia coli/metabolism , Bacterial Proteins/metabolism , Transcription Factors/genetics , Gene Expression Regulation, BacterialABSTRACT
Objective:To study the transcriptional regulation of pilABCD by the master quorum sensing (QS) regulator OpaR in Vibrio parahaemolyticus. Methods:Total RNAs were extracted from the wild type (WT) and opaR mutant (Δ opaR) strain. Quantitative real-time PCR (qPCR) was employed to calculate the transcriptional variation of pilA (the first gene of pilABCD operon) between WT and Δ opaR. The regulatory DNA region of pilABCD was cloned into the corresponding restriction endonuclease sites of pHRP309 harboring a promoterless lacZ reporter gene. The recombinant pHRP309 plasmid was then transferred into WT and Δ opaR, respectively, to detect the β-galactosidase activity in cellular extracts using a β-Galactosidase Enzyme Assay System (Promega). The primer extension assay was applied to map the transcription start site of pilABCD using the total RNAs extracted from the WT strain as the template. The regulatory DNA region of pilABCD was amplified by PCR, and the over-expressed His-OpaR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Thereafter, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-OpaR to the target DNA in vitro, and the DNase I footprinting assay was further employed to detect the DNA-binding sites of His-OpaR within the target DNA. Results:The results of qPCR and LacZ fusion assays showed that OpaR activated the transcription of pilABCD, leading to a gradual increase in the expression level of pilA with the extension of culture time. The primer extension assay detected only one transcription start site located at 155 bp upstream of pilA. The results of EMSA and DNase Ⅰ footprinting assays showed that His-OpaR protected two DNA regions located from -246 to -197 bp and -181 to -131 bp upstream of pilA. Conclusions:Vibrio parahaemolyticus OpaR activated the transcription of pilABCD in a direct manner.
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Objective To study the transcriptional regulation of vp1667 by H-NS in Vibrio parahaemolyticus.Methods Total RNAs were extracted from Δhns and WT strains.Quantitative RT-PCR was carried out to calculate the transcriptional variation of vp1667 between Δhns and WT.Primer extension assay was also employed to detect the transcription start site and the promoter activity (i.e.the amount of primer extension products) of vp1667 in Δhns and that in WT.The promoter DNA region of vp1667 was amplified, purified, and cloned into the corresponding restriction endonuclease sites of pHRP309 that harbors a gentamicin resistance gene and a promoterless lacZ reporter gene.The recombinant pHRP309 plasmid was transformed into Δhns and WT, respectively, while β-galactosidase activity in cellular extracts was measured using a β-galactosidase enzyme assay system.The over-expressed His-H-NS was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns.The electrophoretic mobility shift assay (EMSA) and DNaseⅠ footprinting were then applied to analyze the DNA-binding activity of His-H-NS to vp1667 promoter region in vitro.Results and Conclusion The primer extension assay detected one transcription start site for vp1667, which was located at 28 bp upstream of vp1667, and its transcribed activity was under the negative control of the H-NS.The EMSA and DNaseⅠ footprinting assay results showed that His-H-NS was unable to bind to the promoter-proximal DNA region of vp1667, suggesting that H-NS indirectly inhibits the transcription of vp1667.
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Objective To investigate the transcriptional autoregulation of PhoP/PhoQ under different growth conditions in Yersinia pestis.Methods The entire promoter region of YPO1635 was amplified and cloned into the pRW50 vector containing a promoterless lacZ reporter gene.The recombinant LacZ reporter plasmid was transformed into the wild-type strain (WT) and the phoP mutant strain (ΔphoP),respectively,to measure the promoter activity (the β-galactosidase activity) of the target gene in WT and ΔphoP by using the β-galactosidase enzyme assay system.Total RNAs were extracted from WT and ΔphoP strains,and primer extension assay was employed to detect the promoter activity by examining the amount of primer extension products of YPO1635 in WT and ΔphoP.Results The LacZ fusion results showed that the transcription of YPO1635 was positively regulated by PhoP under L-TMH and brain-heart infusion(BHI) conditions,but it was not regulated in H-TMH medium.The primer extension assay detected two transcriptional start sites located at 90 and 118 bp upstream of the translation initiation site of phoP,named P1 and P2,respectively.Under low Mg2+ TMH conditions,the promoter activity of P1 rather than P2 was positively regulated by PhoP.Under high Mg2+ TMH conditions,the promoter activities of both P1 and P2 showed no obvious difference in the WT and ΔphoP strains.Under rich BHI conditions,both promoters were under negative control of PhoP.Conclusion Different autoregualtion patterns of PhoP/PhoQ under different growth conditions would help Y.pestis to quickly adapt to the changing living environment.
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Objective To study the impact of QseBC on the motility of Salmonella enterica serovar Typhi ( S.Typhi ) . Methods The motility of wild-type ( WT) and null mutants (ΔqseB and ΔqseC) at mid-log phase was investigated by swimming assay.Quantitative RT-PCR was carried out to calculate the transcriptional variation of flhD and qseB among WT,ΔqseB andΔqseC.QseB overexpressing strain was constructed to compare its motility and flhD expression with the wild-type control.Results The result of motility assay showed that the motility of ΔqseB was similar to that of the WT strain , while the motility of ΔqseC was much lower than that of WT .qRT-PCR revealed that compared with WT , the expression of flhD was significantly decreased in ΔqseC while the expression of qseB was increased considerably .The motility of QseB overex-pressing strain was lower .Conclusion The expression of flhD may be regulated by QseBC which has an effect on the motil-ity of S.typhi, and the overexpression of QseB may inhibit the motility .
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Objective To investigate the regulation of swimming motility by H-NS in Vibrio parahaemolyticus(VP). Methods VP was inoculated into the semi-solid swimming agar plate containing 1% Oxoid tryptone, 2% NaCl, 0.5%Difco Noble Agar, and 0.1% arabinose followed by incubation at 37℃ for 4.5 h before the diameters of bacterial lawns were measured.Total RNAs were extracted from the wild-type (WT) strains and the hns null mutant (Δhns), and the quantitative real-time( RT)-PCR( qRT-PCR) was carried out to calculate the transcriptional variation of flaA between WT andΔhns strains.The entire promoter DNA region of flaA was amplified and cloned into the lacZ fusion vector pHRP309 containing a promoterless lacZ gene. The recombinant lacZ reporter plasmid was transformed into WT and Δhns, respectively, to measure the β-galactosidase activities in cellular extracts using the β-galactosidase enzyme assay system. Results and Conclusion The phenotype results showed that swimming motility of VP was enhanced by H-NS.The qRT-PCR and LacZ fusion results indicated that the transcription of flaA was positively regulated by H-NS.Collectively, H-NS promotes the swimming motility of VP, at least partly, by activating the transcription of flaA.
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<p><b>OBJECTIVE</b>To establish a method for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.</p><p><b>METHODS</b>The entire coding region of opaR or aphA was amplified by PCR and cloned into pBAD33. The recombinant plasmid was transformed into δopaR and δaphA (the opaR or aphA null mutant strain, respectively) separately to construct the complemented mutant strain C-δaphA and C-δopaR, respectively. RT-PCR was used to verify the transcription of opaR and aphA in the corresponding complemented mutant strains. Primer extension experiments were performed to determine the relative mRNA levels of mfpA (a gene previously characterized to be negatively regulated by AphA and positively by OpaR) in the wild-type strain, δopaR, δaphA, C-δaphA, and C-δopaR.</p><p><b>RESULTS</b>opaR and aphA were transcribed in the corresponding complemented mutant strains, and their mRNA levels were comparable to those detected in the wild-type strains.</p><p><b>CONCLUSION</b>A method has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.</p>
Subject(s)
Bacterial Proteins , Genetics , Gene Expression , Genetic Complementation Test , Methods , Plasmids , Genetics , Promoter Regions, Genetic , Vibrio parahaemolyticus , GeneticsABSTRACT
Objective To investigate the effect of salinity and temperature on motility of Vibrio parahaemolyticus. Methods V.parahaemolyticus was inoculated on swarming or swimming agar plates containing different amounts of salinity (0.5%, 1.0%, 2.0%, and 4.0% NaCl, respectively), followed by incubation at 26 or 37℃, before the diameters of bacterial lawns were measured .Results and Conclusion The swarming motility was not affected by salinity , while the swimming motility was positively correlated with salinity .Maximum swimming occurred in 2.0% NaCl, and displayed a slight decline in salinity of 4.0%.Both swimming and swarming were affected by temperature , and the motility was signifi-cantly enhanced in 37℃vs 26℃.These results indicate that both salinity and temperature can modulate the motility of V. parahaemolyticus.
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0.05 ;Ti milled/VITA vs Ni Cr/VITA P