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Up to now only one major type of core oligosaccharide has been found in the lipopolysaccharide of all Klebsiella pneumoniae strains analyzed. Applying a different screening approach, we identified a novel Klebsiella pneumoniae core (type 2). Both Klebsiella core types share the same inner core and the outer-core-proximal disaccharide, GlcN-(1,4)-GalA, but they differ in the GlcN substituents. In core type 2, the GlcpN residue is substituted at the O-4 position by the disaccharide [beta]-Glcp(1-6)-[alpha]-Glcp(1, while in core type 1 the GlcpN residue is substituted at the 0-6 position by either the disaccharide [alpha]-Hep(1-4)-[alpha]-Kdo(2 or a Kdo residue (Kdo is 3-deoxy-d-manno-octulosonic acid). This difference correlates with the presence of a three-gene region in the corresponding core biosynthetic clusters. Engineering of both core types by interchanging this specific region allowed studying the effect on virulence. The replacement of Klebsiella core type 1 in a highly type 2 virulent strain (52145) induces lower virulence than core type 2 in a murine infection model.

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-HeppIII[alpha](1 [right arrow] 7)-L,D-HeppII[alpha](1[arrow right] 3)-L,D-Hepp[alpha](1[right arrow] 5)-KdopI(4 [left arrow] 2)[alpha]KdopII] (L,D-Hepp, L-glycero-D-manno-eptopyranose; Kdo, 3-deoxy-D-manno-oct2-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 [right arrow] 6)-[alpha]-Glc-(1 [right arrow] 4))-[alpha]-D-GlcN-(1 [right arrow] 4)-[alpha]-D-GalA-[(2 [left arrow] 1) -[alpha]-D,D-Hep- (2[left arrow] 1)-[alpha]Hep]-(1 [right arrow] 3)-[alpha]-L,D-Hep[(7 [left arrow] 1)-[alpha]-L,D-Hep]-(1 [right arrow] 3)-[alpha]-L,D-Hep-[(4 [left arrow] 1)-[beta]-D-Glc]-(1 [right arrow] 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-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.

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 virulent 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.

Results show that a recombinat cosmid carries the waa gene cluster mediating Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis as revealed by genetic complementation analyses of waa mutants.

Results show that eight open reading frames of wb(sub)012 gene cluster code for Klebsiella pneumoniae O12-antigen lipopolysaccharide in Escherichia coli K-12. The wb gene cluster belongs to ABC 2 transporter-dependent pathways mediating O-antigen heteropolysaccharides synthesis.