[Regulation mechanism of the quorum sensing regulator AphA on the type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis ].

Objective: To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis . Methods: Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), Δ aphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter- lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB 2, hcp ( tssD 2) and vgrG ( tssI 2), and the quorum sensing regulator HapR in WT and Δ aphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD 2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results: The mRNA expression levels of tssB 2, hcp ( tssD 2), vgrG ( tssI 2), and hapR as well as the protein expression and secretion levels of Hcp in Δ aphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD 2a, tssI 2a, and hapR were higher in Δ aphA strain than in the WT strain, while the promoter activity of tssD 2b showed the opposite trend. The promoter sequence analysis of tssD 2a and tssD 2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD 2b. The promoter activity of tssD 2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR . Conclusions: AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR . AphA showed opposite regulation patterns for tssD 2a and tssD 2b, and AphA could positively regulate the expression of tssD 2b by directly binding to the tssD 2b promoter region (-335 bp to -229 bp).