Abstract:[Objective]To investigate the transcriptional regulation of mshH gene by the master quorum sensing (QS) regulators AphA and OpaR in Vibrio parahaemolyticus. [Methods] Total RNAs were extracted from the wild-type (WT) strain and the regulatory gene mutants (ΔaphA and ΔopaR). Quantitative real-time PCR (qPCR) was employed to compare the transcriptional variation of mshH gene between WT and ΔaphA (or ΔopaR) and to detect the growth phase-dependent transcription of mshH gene. The promoter region of mshH was cloned into the upstream region of the promoterless LacZ reporter gene in pHRP309. The recombinant plasmid was respectively transferred into WT, ΔaphA, and ΔopaR, and the β-galactosidase activities in the extracts of the recombinant strains were determined via a β-galactosidase enzyme assay system (Promega). Thus, the expression levels ofmshH gene between different strains or different growth phases can be compared based on the results of LacZ fusion. The promoter-proximal DNA region ofmshH was amplified by PCR, and the over-expressed His-AphA and His-OpaR were purified simultaneously under native conditions. The electrophoretic mobility shift assay (EMSA) was adopted to detect the DNA-binding activity of His-AphA or His-OpaR, and the DNase I footprinting assay was further employed to detect the DNA-binding sites of the His-recombinant proteins within the target DNA. [Results]mshH gene expression manifested in a growth phase-dependent manner, and the high expression level occurred at high cell density (HCD). At low cell density (LCD), AphA inhibited the transcription of mshH gene, while His-AphA had no binding activity to the promoter-proximal DNA fragment of mshH. At HCD, OpaR activated the transcription of mshH gene. His-OpaR protected two DNA regions located from −160 bp to −80 bp and −58 bp to −19 bp upstream of mshH gene. [Conclusion]AphA indirectly inhibited the transcription of mshH gene at LCD, whereas OpaR activated the transcription ofmshH gene in a direct manner at HCD.
