Genetic and structural characterization of the core region of the lipopolysaccharide from Serratia marcescens N28b (serovar O4).

The gene cluster (waa) involved in Serratia marcescens N28b core lipopolysaccharide (LPS) biosynthesis was identified, cloned, and sequenced. Complementation analysis of known waa mutants from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae led to the identification of five genes coding for products involved in the biosynthesis of a shared inner core structure: [L,D-HeppIIIalpha(1-->7)-L,D-HeppIIalpha(1-->3)-L,D-HeppIalpha(1-->5)-KdopI(4<--2)alphaKdopII] (L,D-Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid). Complementation and/or chemical analysis of several nonpolar mutants within the S. marcescens waa gene cluster suggested that in addition, three waa genes were shared by S. marcescens and K. pneumoniae, indicating that the core region of the LPS of S. marcescens and K. pneumoniae possesses additional common features. Chemical and structural analysis of the major oligosaccharide from the core region of LPS of an O-antigen-deficient mutant of S. marcescens N28b as well as complementation analysis led to the following proposed structure: beta-Glc-(1-->6)-alpha-Glc-(1-->4))-alpha-D-GlcN-(1-->4)-alpha-D-GalA-[(2<--1)-alpha-D,D-Hep-(2<--1)-alpha-Hep]-(1-->3)-alpha-L,D-Hep[(7<--1)-alpha-L,D-Hep]-(1-->3)-alpha-L,D-Hep-[(4<--1)-beta-D-Glc]-(1-->5)-Kdo. The D configuration of the beta-Glc, alpha-GclN, and alpha-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-talo-oct-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.

The inner-core lipopolysaccharide biosynthetic waaE gene: function and genetic distribution among some Enterobacteriaceae.

To determine the function of the waaE gene in the biosynthesis of the inner-core LPS of Klebsiella pneumoniae, a waaE non-polar mutant has been constructed. Data obtained from the comparative chemical analysis of LPS samples obtained from the wild-type, the mutant strain and the complemented mutant demonstrated that the waaE gene is involved in substitution of alpha-L-glycero-D-manno-heptopyranose I (L,D-HeppI) at the O-4 position by a beta-D-glucopyranose (beta-D-Glcp) residue. In addition, DNA amplification and nucleotide sequence determination studies revealed that waaE homologues located between the waaA and coaD genes are present in clinical isolates of Enterobacteriaceae containing the structure beta-D-Glcp-(1-->4)-alpha-L,D-HeppI (K. pneumoniae, Proteus mirabilis and Yersinia enterocolitica), as well as in strains of Serratia marcescens and Enterobacter aerogenes of unknown LPS-core structures. Complementation studies using non-polar waaE mutants prove that all the waaE homologues perform the same function. Furthermore, K. pneumoniae, Ser. marcescens and P. mirabilis non-polar waaE mutants showed reduced adhesion and pathogenicity. In addition, the Ser. marcescens and P. murabilis waaE mutants showed reduced swarming motility and ability to form biofilms in vitro. All these characteristics were rescued by reintroduction of the waaE gene independently of its origin. An easy DNA amplification method to detect this gene was established, which also helps in finding the potential presence of this structural feature [beta-D-Glcp-(1-->4)-alpha-L,D-HeppI] in the inner-core LPS of Enterobacteriaceae members with unknown LPS-core structures.