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The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to L-glycero-D-manno-heptopyranose II (LD-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of D-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to LD-HeppII. [ABSTRACT FROM AUTHOR]

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 β-Glcp(1–6)-α-Glcp(1, while in core type 1 the GlcpN residue is substituted at the O-6 position by either the disaccharide α-Hep(1–4)-α-Kdo(2 or a Kdo residue (Kdo is 3-deoxy-o-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. [ABSTRACT FROM AUTHOR]

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 α-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an α-D-galactopyranosyluronic acid (α-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. [ABSTRACT FROM AUTHOR]

A recombinant clone encoding enzymes for Klebsiella pneumoniae O12-antigen iipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of K, pneumoniae KT776 (O12:K80) introduced into Escherichia coli DH5α. A total of eight open reading frames (ORFs) (wb[sub O12] gene cluster) were necessary to produce K. pneumoniae O12-antigen LPS in E. coli K-12. A complete analysis of the K. pneumoniae wb[sub O12] cluster revealed an interesting coincidence with the wb[sub O4] cluster of Serratia marcescens from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of K. pneumoniae strain KT776 (O12) and to study complementation between the two enterobacterial wb clusters using mutants of S. marcescens N28b (O4) obtained previously. Both wb gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The wzm-wzt genes and the wbbA or wbbB genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (wzm-wzt-wbbA or wzm-wzt-wbbB) into mutants unable to produce O antigen allowed production of the specific O antigen. The K. pneumoniae O12 WbbL protein performs the same function as WbbL from S. marcescens O4 in either the S. marcescens O4 or E. coli K-12 genetic background. [ABSTRACT FROM AUTHOR]

To determine the function of the waaE gene in the biosynthesis of the innercore 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 α-L-glycero-D-manno-heptopyranose I (L,D-Heppl) at the 0-4 position by a β-D-glucopyranose (β-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 β-D-Glcp-(1 → 4)α-L,D-Heppl (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 [β-D-Glcp-(1 → 4)-α-L,DHeppl] in the inner-core LPS of Enterobacteriaceae members with unknown LPS-core structures. [ABSTRACT FROM AUTHOR]

The article studies the ionic interaction of Klebsiella pneumoniae K2 capsule and core lipopolysaccharide (LPS). Previous studies have described the complete structures of LPS core types 1 and 2 from K. pneumoniae. They are characterized by a lack of phosphoryl residues, but they contain galacturonic acid residues, which contribute to the necessary negative charges.

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-HeppIIIα(1→7)-L,D-HeppIIα(1→3)-L,DHeppIα(1→5)-KdopI(4←2)αKdopII] (L,D-Hepp, L-glycero-D-manno-heptopyranose; 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: β-Glc-(1→6)-α-Glc-(1→))-α-D-GlcN-(1→4)-α-D-GalA-[(2←1)-α-D,D-Hep-(2←1)-αHep]-(1→3)-α-L,D-Hep[(7←1)-α-L,D-Hep]-(1→3)-α-L,D-Hep-[(4←1)-β-D-Glc]-(1→5)-Kdo. The D configuration of the β-Glc, α-GclN, and α-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. [ABSTRACT FROM AUTHOR]