Sequence And Functional Analysis Of The Plasmid PEIB202 And WaaL Gene In Edwardsiella Tarda

Edwardsiella tarda, a Gram-negative bacteria belonging to Enterobacteriaceae, is responsible for hemorrhagic septicemia in fish, causing a disastrous effect on aquaculture industry. In our previous study, a virulent E. tarda strain EIB202 was isolated from moribund Scophthalmus maximum, and an inherent 43.7 kb plasmid pEIB202 was found in EIB202. In this study, the sequence of pEIB202 was analyzed. The plasmid pEIB202 possesses six genes probably involved in resistance to antibiotics, and ten genes encoding an incomplete set of components involved in the type IV secretion system (T4SS). The plasmid curing mutant EIB202Δp was screened by counter-selectable marker sacB gene. Compared to the wild type, except for the elimination of resistance to chloramphenicol and tetracycline, EIB202Δp displayed similar phenotypes in growth rate, virulence and extracellular proteins secretion profiles. It was demonstrated that pEIB202 was involved in the multi-resistance of EIB202 but might have indirect effect on the pathogenesis of EIB202.Lipopolysaccharide (LPS), the major constitute of the outer membrane of Gram-negative bacteria, plays an essential role in pathogenesis. Genes probably involved in LPS biosynthesis were analyzed based on the genome sequence. waaL gene, which is responsible for attaching the O-polysaccharide chains to the core-lipid A region, was in-frame deleted in the plasmid curing strain EIB202Ap. According to the SDS-PAGE profile, the AwaaL mutant revealed an O-antigen side chains absence in the LPS production, confirming the ligase activity of WaaL in LPS biosynthesis of E. tarda. The AwaaL mutant also exhibited an increased sensitivity to serum, osmotic pressure, hydrogen peroxide and polymyxin B, indicating essential roles of waaL gene in stress adaptation. Besides, the AwaaL mutant was attenuated in virulence, and showed an impaired ability in internalization of EPC cells, increased LD50 value and a comparatively poor ability of proliferation in vivo, demonstrated that waaL gene contributed to the stress adaptation and virulence in E. tarda, meriting as a target for vaccine development against edwardsiellosis.