Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis

A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confe r bacteriocin 28b resistance to Escherichia coli K-12, The recombinant cosm id contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD g enes, as was found in E, roll K-12. PCR amplification analysis showed that this cluster is conserved in representative K, pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K, pneumoniae subsp, ozaenae, for which the core chemical str ucture is known, Complementation analysis of known waa mutants from E, coli K-12 and/or Salmonella enterica led to the identification of genes involve d in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae, h: pneumoniae orf10 mutants showed a tw o-log-fold reduction in a mice virulence assay and a strong decrease in cap sule amount. Analysis of a constructed K pneumoniae waaE deletion mutant su ggests that the WaaE protein is involved in the transfer of the branch beta -D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature share d by K, pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.