Functional study on gene AL536_RS29525 of Vibrio fluvialis

  Objective  To investigate the sequence characteristics of gene AL536_RS29525 located upstream of VflT6SS2 major cluster and downstream of paar gene of Vibrio fluvialis, its influence on VflT6SS2 secretion function and bactericidal activity as well as possible regulatory mechanism.

  Methods  Muscle and GeneDoc softwares were used to compare and analyze AL536_RS29525 and homologs. Deletion mutant of AL536_RS29525 was constructed using suicide plasmid based on the homologous recombination technology. Trans-complementation plasmid was constructed by cloning the coding sequence of AL536_RS29525 into plasmid pSRKTc, which was mobilized into the deletion mutant by conjugation. The expression and secretion of Hcp protein were analyzed by Western Blot. Standard bactericidal assay with E. coli as prey was used to measure the bactericidal ability of derivative strains. Luminescence activity of Lux based promoter fusion was used to test the promoter activities of VflT6SS2 major cluster and hcp orphan cluster in ΔAL536_RS29525 mutant strains and wild strains. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the hcp(tssD2) and vipA(tssB2) mRNA level.

  Results  The genomic structure and sequence of AL536_RS29525 homologs were conserved in Vibrio species. The deletion of AL536_RS29525 greatly decreased the expression and secretion of Hcp, and also significantly reduced the bactericidal ability. These defects can be restored by introducing the trans-complementation plasmid PSR-29525 into ΔAL536_RS29525 mutant. The mRNA expression levels of hcp(tssD2) and vipA(tssB2) of ∆AL536_RS29525 were lower than those of wild strains. However, the promoter activities of VflT6SS2 major cluster and hcp orphan were not significantly different between the mutant strains and wild strains.

  Conclusion  AL536_RS29525 is an important component required for the expression and secretion of VflT6SS2 with a critical role in bactericidal activity of V. fluvialis.