Abstract:The type Ⅵ secretion system (T6SS), a protein secretion system generally found in gram-negative bacteria, plays an essential role in virulence, interspecific competition, and environmental adaptability. Hemolysin-coregulated protein (Hcp), an extracellular component of T6SS, is released into the medium and therefore may serve as a marker of a functional T6SS apparatus. RpoS, the σ subunit of RNA polymerase, is involved in the regulation of bacterial growth and stress response. To explore the regulatory effect of rpoS on Vibrio anguillarum T6SS, an MHK3∆rpoS mutant strain was constructed; phenotypic changes were detected using the lacZ reporter gene to construct fusion strains and the changes in hcp1 and hcp2 expression at the transcriptional level were detected using the ONPG (o-nitrophenyl-ß-D-galactopyranoside) method. Western blotting was performed to quantitatively analyze the changes in the mutant Hcp expression at the translational level. The change in the bactericidal activity of the mutant strain was detected via a bacterial antagonistic experiment. The results showed that the growth, mobility, gelatinase activity, and caseinase activity of the MHK3∆rpoS mutant strain showed no significant difference compared with the wild-type strain MHK3 (P>0.05). The biofilm-forming ability significantly increased at the prophase after mutation in rpoS (P<0.05). The transcriptional levels of hcp1 and hcp2 in each growth phase were significantly higher in MHK3∆rpoS than in MHK3 (P<0.01), with highest increases of 1.79-fold and 1.94-fold compared with MHK3, respectively. At the translational level, the secretion of both intracellular and extracellular Hcp in MHK3∆rpoS significantly increased (P<0.05), with highest increases of 1.59-fold and 1.31-fold, respectively. Meanwhile, the bactericidal activity of MHK3∆rpoS against Escherichia coli E5 was about 1% of MHK3. Studies have shown that rpoS has no significant regulatory effect on some phenotypes of MHK3; however, it negatively regulates the biofilm-forming ability at the prophase. This is different from the results of the research on V. anguillarum M3 that posits that rpoS-mediated regulation of the same phenotype in different strains varies. Furthermore, at the levels of transcription and translation, rpoS negatively regulates the expression of Hcp; however, it shows a positive regulatory effect on the bactericidal activity of MHK3. This indicates that the strength of the bactericidal activity of the strain is not directly related to the expression and secretion of Hcp. This study provides innovative ideas and enriches the theoretical basis for further elucidating the regulatory mechanism of T6SS and T6SS-mediated bactericidal activity.
