A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli 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 structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch -D-Glc to the O-4 position of L-glycero-D-manno-heptose
The gene cluster (waa) involved in Serratia marcescens
-Glc-(136)-␣-Glc-(134))-␣-D-GlcN-(134)-␣-D-GalA-[(241)-␣-D,D-Hep-(241)-␣-Hep]-(133)-␣-L,D-Hep[(741)-␣-L,D-Hep]-(133)-␣-L,D-Hep-[(441)--D-Glc]-(135)-Kdo.The D configuration of the -Glc, ␣-GclN, and ␣-GalA residues was deduced from genetic data and thus is tentative. Furthermore, other oligosaccharides were identified by ion cyclotron resonance-Fourier-transformed electrospray ionization mass spectrometry, which presumably contained in addition one residue of D-glycero-D-talooct-2-ulosonic acid (Ko) or of a hexuronic acid. Several ions were identified that differed from others by a mass of ؉80 Da, suggesting a nonstoichiometric substitution by a monophosphate residue. However, none of these molecular species could be isolated in substantial amounts and structurally analyzed. On the basis of the structure shown above and the analysis of nonpolar mutants, functions are suggested for the genes involved in core biosynthesis.
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