Abstract:[Background] Many domestic studies have reported in detail the application of gene knockout with suicide vectors in single gene knockout. However, the changes in bacterial resistance after gene knockout and the impact on subsequent gene knockout remain unclear. [Objective] In order to explore the changes in the resistance of Vibrio vulnificus (V. vulnificus) to chloramphenicol and its effect on subsequent gene knockout in the process of gene knockout with pDS132, the vvhA and rtxA1 gene knockout strains were constructed. [Methods] The single gene knockout strains YJ016-ΔvvhA, YJ016-ΔrtxA1 and double gene knockout strains YJ016-ΔvvhAΔrtxA1 of V. vulnificus YJ016 were constructed by the homologous recombination with suicide vector pDS132. The concentrations of chloramphenicol and the proportions of strains successfully recombined on the screening plate were recorded. The minimum inhibitory concentration (MIC) of each strain to chloramphenicol and other antibiotics, and the mutation frequency of chloramphenicol resistance were determined and then analyzed.[Results] The MIC and the mutation frequency of chloramphenicol of the single gene knockout strains were higher than that of the wild type; the diameter of the gentamicin inhibition zone of the double gene knockout strains was smaller than that of the wild type. When the rtxA1 gene was knocked out on YJ016-ΔvvhA and the chloramphenicol concentration of was 2, 4 μg/mL, the proportions of strains with single-crossover recombination were 40% (8/20) and 5% (1/20), respectively; when the vvhA gene was knocked out on YJ016-ΔrtxA1, the proportion was 0% (0/20). [Conclusion] In the process of gene knockout with the suicide vector pDS132, the resistance of V. vulnificus to chloramphenicol increased, which may affect the subsequent screening in single-crossover recombination. The result is helpful for the application of the homologous recombination technique with suicide vector to multiple gene knockout.
