Functional Analysis Of Core Genes In T6SS Cluster Of Ralstonia Solanacearum Strain Po82

Bacteria T6SS (Type VI Secretion System) is a novel secretion pathway which was reportedrecently. It is bound up with bacterial pathogenity and virulence on human beings and animals and playsan important role in interaction with the external environment. It widely exists in many ofgram-negative bacteria. Based on our previous phylogenetic research and pathogenicity test, Ralstoniasolanacearum strain Po82, a phylotype II/sequevar4strain (historically known as race2), proved to be apathogenicity variation strain. It affect not only solanaceous plants such as tomato, eggplant andpotato, but banana plants as well. Therefore, studying the core genes of type VI secretion system in R.solanacearum strain Po82will help uncover the mechanism of bacterial pathogenesis.In this study, Firstly, bioinformatics analysis demonstrated that Ralstonia solanacearum Po82strain harbor the T6SS gene cluster, and then eight core genes of T6SS were cloned. Secondly, In orderto research the function of the gene cluster of Type VI secretion system, the core genes deletion mutantswere constructed by using homologous recombination. Phenotype of the mutants, the complementarystrains and the wild Po82strain were compared.1. The gene cloning and bioinformatics analysis of Ralstonia solanacearum Po82Type VIsecretion system gene clusterBioinformatic analysis based on the complete genome sequence of Ralstonia solanacearum Po82strain revealed that the~26.8kb T6SS gene cluster was harbored in megaplasid of Po82strain. TheT6SS gene cluster of Po82strain has an average GC content of66.4%and encodes48ORFs includingall of the known core gene products. Eight core genes of the Type VI secretion system were cloned andcompared with those of GMI1000strain, the consistency is beyond90%. The function of gene and thestructure of the its coding for protein was forecased through bioinformatics. We found that the VgrGand Hcp protein tertiary structure of Ralstonia solanacearum Po82is highly similar to theircounterparter of Escherichia coli CFT073and Pseudomonas aeruginosa, respectively.2. Construction of the core gene deletion mutants, their corresponding complementarystrains and overexpressing mutantsIn order to elucidate the function of the gene cluster of the Type VI secretion system, five coregenes mutations were constructed by using homologous recombinant, and then their correspondingcomplementary strains were constructed.3. Pathogenicity test on the T6SS core gene mutants and their correspondingcomplementary strainPathogenicity tests showed that the disease symptoms caused by mutants were delayed whencompared to the wild-type strains, and the wild-type strains were weaker than the mutants inpathogenicity. The results suggested that the Type VI secretion system of Po82strain played animportant role in pathogenicity. 4. Study on phenotypic characteristics of the T6SS core gene mutantsRelated phenotypic characteristics of hcp, vgrG1and impA gene mutants were tested. The resultsshowed that there were no significant differences among the mutants, their correspondingcomplementary strains and the wild-type strains in the ability of growth, the mobility and the biofilmformation.