Isolation,Identification And Investigation On The Mechanism Of Transposon Mutants Which Hyper Produce Extracellular Polysaccharide In Pantoea Ananatis

Pantoea ananatis(P. ananatis) is one of the gram-negative bacteria and encountered worldwide. P. ananatis was reported as a common colonist of wheat heads at ripening and caused disease symptoms in a wide range of economically important agricultural crops. Especially, "center rot" disease of onion caused by P. ananatis induced that the infected leaves were usually collapsed and hang down beside the neck. However, the major virulent factors of P. ananatis had not been elucidated yet.It was reported that virulence of the plant pathogens were correlated with the ability to produce extracellular polysaccharide (EPS). EPS was considered a virulence determinant in many bacterial plant pathogens because of its central role in wilt induction, host colonization, biofilm formation. It was thus likely to be important in physical interactions with the host. In Pantoea stewartii, which was a vascular pathogen of corn maize, mutations at the EPS biosynthetic gene cluster generally lead to loss of pathogenicity. In Erwinia amylovora, which was the cause of fire blight disease, mutants that lacked the synthesis of EPS were reduced in virulence. From these reports, it was assumed that EPS biosynthesis correlated with the pathogenicity of P. ananatis. The purpose of this study was to isolate and characterize the transposon mutants which caused hyper production of EPS, to extract the plasmid DNA of the mutants and identify the mutated genes, and to investigate the relationship between the mutated genes and excessive production of EPS.In order to identify specific genes which caused hyper production of EPS, P. ananatis strain SK-1 was mutagenized with the transposon Tn5-OT182. Transposon carrier plasmid pOT182 was transformed into SK-1 by electropolation. Transposon mutants of SK-1 were selected by plating on LB agar plates containing tetracycline. Transposon mutants were replica-plated onto TSB agar plates containing tetracycline, a total of 3,000 colonies were screened. As the results,21 mutants, that showed hyper production of EPS, were successfully isolated. In order to identify the mutated gene, chromosomal DNA of mutants was extracted, digested with following restriction enzymes: EcoRⅠ, SacⅠ, XhoⅠor HindⅢThe chromosomal fragments were self-ligated and transformed into Escherichia coli DH5α. Transformants were selected by plating on LB agar plates containing tetracycline. Plasmids were extracted by improved alkali lysis method from the transformants. DNA sequencing was performed by using BigDye Terminator ver. 3.1 and ABI Prism 3100 Genetic Analyzer (Applied Biosystems). The specific primers were used to initiate DNA sequence reactions with plasmids obtained by self-cloning. DNA sequences were compared with the sequences in the DDBJ/EMBL/GenBank databases, whose predicted functions were deduced.As the results, the transposon integration of the mutant 33,91,40,11,46,81,29 and 98 was observed to be located in a gene with strong homology to known proteins. The mutation sites of the EPS hyper-producing mutant 33,91, and 40 were genes that encoded an adhesion-like protein, outer membrane protein and lipoprotein, respectively. Mutation of cell surface structure might cause excessive EPS in SK-1. The mutation site of the EPS hyper-producing mutant 11 was a gene that catalyzed the transfer of a sugar moiety from an activated sugar donor to saccharide or non-saccharide acceptors. Mutation of glycosyltransferase or iron transport lipoprotein in mutant 46 and 81 might affect metabolisms of EPS biosynthesis. Mutation of putative sensor protein or ribonuclease R in mutant 29 and 98 were related to biosynthesizing of nucleic acid, one composition of EPS.