Identification of a Wzy Polymerase Required for Group IV Capsular Polysaccharide and Lipopolysaccharide Biosynthesis in Vibrio vulnificus

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Abstract
The estuarine bacterium Vibrio vulnificus is a human and animal pathogen. The expression of capsular polysaccharide (CPS) is essential for virulence. We used a new mini-Tn 10 delivery vector, pNKTXI-SceI, to generate a mutant library and identify genes essential for CPS biosynthesis. Twenty-one acapsular mutants were isolated, and the disrupted gene in one mutant, coding for a polysaccharide polymerase ( wzy ), is described here. A wecA gene initiating glycosyltransferase was among the genes identified in the region flanking the wzy gene. This, together with the known structure of the CPS, supports a group IV capsule designation for the locus; however, its overall organization mirrored that of group I capsules. This new arrangement may be linked to our finding that the CPS region appears to have been recently acquired by horizontal transfer. Alcian Blue staining and immunoblotting with antisera against the wild-type strain indicated that the wzy ::Tn 10 mutant failed to produce CPS and was attenuated relative to the wild type in a septicemic mouse model. Interestingly, immunoblotting revealed that the mutant was also defective in lipopolysaccharide (LPS) production. However, the core-plus-one O-antigen pattern typical of wzy mutations was apparent. CPS production, LPS production, and virulence were restored following complementation with the wild-type wzy gene. Hence, Wzy participates in both CPS and LPS biosynthesis and is required for virulence in strain 27562. To our knowledge, this is the first functional demonstration of a Wzy polysaccharide polymerase in V. vulnificus and is the first to show a link between LPS and CPS biosynthesis.

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