RmpA Regulation of Capsular Polysaccharide Biosynthesis inKlebsiella pneumoniaeCG43

Abstract

ABSTRACTSequence analysis of the large virulence plasmid pLVPK inKlebsiella pneumoniaeCG43 revealed the presence of another mucoid factor encoding genermpAbesidesrmpA2. Promoter activity measurement indicated that the deletion ofrmpAreduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carryingrmpArestored CPS production in thermpAorrmpA2mutant but not in thercsBmutant. Transformation of thermpAdeletion mutant with anrcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration ofin vivointeraction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5′ noncoding region ofrmpA. The promoter activity analysis indicated that the deletion offurincreased thermpApromoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, thefurdeletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis inK. pneumoniaeCG43 via an RcsB-dependent manner. The expression ofrmpAis regulated by the availability of iron and is negatively controlled by Fur.