Phenotype analysis of mutant strains on hemolytic ability mediated by Tn5 in pathogenic Vibrio parahaemolyticus

Abstract:

[Objective] We established the mutation library of pathogenic Vibrio parahaemolyticus (Vp), analyzed the phenotype characteristic of hemolytic ability changed mutations, to provide the research basis for deeper exploration and understanding of the regulation system and mechanism of tdh. [Methods] In this study, the Mini-Tn5-Km2 was inserted randomly into the genome of pathogenic Vp (ATCC 33846) by biparental mating. The conjugants (KmRAmpS) were investigated using thiosulfate citrate bile salts sucrose agar medium with kanamycin (Km) and ampicillin (Amp) respectively. After detection of the Km gene by polymerase chain reaction, library of mutations was established, which the Tn5 was inserted in different gene locus randomly. The hemolytic activity was evaluated with human blood agar. Different characters of these conjugants were evaluated, including growth rate, biofilm formation and motility ability. [Results] Library of mutations was successfully established including 490 mutants. After evaluating hemolytic activity, the phenotype of 5 strains was changed, including 2 up-regulated strains and 3 down-regulated strains. There was significant difference between the 5 conjugants and parent strain on growth rate, biofilm formation and motility ability. Among 3 down-regulated strains, the biofilm formation ability of 2 conjugants was increased (P<0.05). The residual 3 strains demonstrated the significant difference (P<0.05) on growth rate, biofilm formation ability and motility, comparing with their parent strain. [Conclusion] Tn5 transposon can be used to establish the library of Vp mutations; Hemolytic ability of Vp is related to the phenotype; The five conjugants will be contributed on investigation of regulation system and mechanism of tdh in further study.