Abstract:
ObjectiveThis study aimed to investigate the structure and function of paar (AL536_RS29530), an upstream gene of VflT6SS2 core cluster and its effect on the function of VflT6SS2 in Vibrio fluvialis 85003.
MethodsThe deletion mutant of paar was constructed through homologous recombination mediated by suicide plasmid. The complementation plasmid constructed by cloning the coding sequence of paar by PCR into plasmid pSRKTc was mobilized into the deletion mutant by conjugation. Western Blot analysis was used to detect the relative expression and secretion of the Hcp effector of the derivative strains. Bacterial killing assay was used to measure the change of antibacterial virulence in derivative strains, in comparison with wild type (WT) strain. The relative expression of tssB2 and hcp mRNA was detected by quantitative reverse transcription PCR. Luminescence activity of promoter reporting system was used to test the promoter activities of VflT6SS2 core cluster and hcp-vgrG clusters in the wild type and the deletion mutant.
ResultsThe accurate deletion of paar coding sequence and its complemented strain were successfully constructed. The expression and secretion of Hcp and the antibacterial virulence severely decreased in the deletion mutant, but restored when its trans-complemented plasmid was introduced. Significantly, deletion of paar decreased the mRNA levels of tssB2 and hcp, while promoter activities of VflT6SS2 core cluster and hcp-vgrG clusters showed no significant difference between the wild type and the deletion mutant. It suggested that paar might regulate VflT6SS2 at posttranscriptional level.
Conclusionpaar (AL536_RS29530) is an integral part of VflT6SS2 in V. fluvialis, which contributes to the function of VflT6SS2. Deletion of paar decreased expression and antibacterial virulence of VflT6SS2. Further study on its specific regulation mechanism is needed.
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