CalR activates the transcription of type Ⅵ secretion system 1 genes in Vibrio parahaemolyticus

Abstract:

[Objective] To study the transcriptional regulation of calR gene by the master quorum sensing (QS) regulators AphA and OpaR in Vibrio parahaemolyticus, and to investigate the regulatory actions of CalR on the transcription of type VI secretion system 1 (T6SS1) genes. [Methods] Total RNAs were extracted from the wild-type (WT) strain and the regulatory gene mutants. Quantitative real-time PCR was carried out to calculate the transcriptional variation of each target gene between WT and the mutants. Primer extension assay was employed to detect the transcription start site and the expression variation of each target gene between WT and the mutants. The promoter region of each target gene was cloned into the restriction endonuclease sites of pHRP309, which harbors a gentamicin resistance gene and a promoterless lacZ reporter gene. Thereafter, each recombinant pHRP309 plasmid was transferred into WT and the mutants. The transformants were cultured and then lysed, and a β-Galactosidase Enzyme Assay System (Promega) was used for quantifying the β-galactosidase activity in cell lysates. The over-expressed His-recombinant regulatory proteins were purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay (EMSA) and DNase I footprinting were adopted to analyze the binding activity of each His-recombinant protein to its target promoter DNA region in vitro. [Results] The expression level of calR gene was up-regulated gradiently with the increase in OD₆₀₀ value from 0.05 to 1.20. AphA operating at a low cell density had no regulatory activity on calR gene transcription, whereas OpaR operating at a high cell density indirectly activated the transcription of calR gene. Moreover, CalR bound to the promoter regions of the T6SS1 operons vp1388-1390, vp1393-1406, vp1400-1406, and vp1409-1407 to activate their transcription under the non-inducing growth condition. [Conclusion] The transcription of calR gene was indirectly activated by OpaR. CalR was required for the basal transcription of T6SS1 genes under the non-inducing growth condition.