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 Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence.

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to alpha-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an alpha-D-galactopyranosyluronic acid (alpha-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.

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.

The wavB gene of Vibrio cholerae and the waaE of Klebsiella pneumoniae codify for a beta-1,4-glucosyltransferase involved in the transfer of a glucose residue to the L-glycero-D-manno-heptose I in the lipopolysaccharide inner core.

Vibrio cholerae WavB protein showed some similarity to WaaE of Klebsiella pneumoniae and Serratia marcescens. From previous data obtained by us and by chemical analyses of a K. pneumoniae non-polar waaE mutant from strain 889 (08:K69), its lipopolysaccharide (LPS) core structure has recently been elucidated. We demonstrated that WaaE is a beta-1,4-glucosyltransferase involved in the transfer of a glucose residue to the L-glycero-D-manno-heptose I in the LPS inner core. Complementation of this K. pneumoniae non-polar waaE mutant with gene wavB obtained, either from V. cholerae or V. mimicus, showed a full complementation either by chemical studies or by a biological test (susceptibility to non-immune serum). The V. cholerae wavB gene is located in a putative core oligosaccharide (OS) gene cluster and the V. cholerae OS core structure showed the same beta-1,4-glucose residue attached to Hep I as is observed for the K. pneumoniae 889 OS core structure. No other glucose residue is found in the ligosaccharide core structure of K. pneumoniae 889. We concluded that WavB protein is able to perform the same function as WaaE.