Miguel Regué

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.

Genetic Characterization of the Klebsiella pneumoniae waa Gene Cluster, Involved in Core Lipopolysaccharide Biosynthesis

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 beta-D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.

A Gene, uge, Is Essential for Klebsiella pneumoniae Virulence

ABSTRACT Klebsiella pneumoniae strains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase (uge) renders a strain with the O−:K− phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The uge gene was present in all the K. pneumoniae strains tested. The K. pneumoniae uge mutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single uge wild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented uge mutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.

A Second Outer-Core Region in Klebsiella pneumoniae Lipopolysaccharide

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 O-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 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.

Uptake of N,N′-Diacetylchitobiose [(GlcNAc)2] via the Phosphotransferase System Is Essential for Chitinase Production by Serratia marcescens 2170

The chiR gene of Serratia marcescens 2170, encoding a LysR-type transcriptional activator, was identified previously as an essential factor for expression of chitinases and a chitin-binding protein, CBP21. To identify other genes that are essential for chitinase production, transposon mutagenesis with mini-Tn5Km1 was carried out, and 25 mutants that were unable to produce chitinases and CBP21 were obtained. Analysis of the mutated gene of one of the mutants, N22, revealed the presence of a pts operon in this bacterium, and a mutation was found in ptsI in the operon. In addition to its inability to produce chitinase, N22 did not grow well on N-acetyl-D-glucosamine (GlcNAc), (GlcNAc)(2), and some other carbon sources, most of which were phosphotransferase system (PTS) sugars. Thus, the inability to produce chitinase was assumed to be caused by the defect in uptake of (GlcNAc)(2) via the PTS, considering that (GlcNAc)(2) is the minimal substrate for chitinase induction and the major product of chitin hydrolysis by chitinases of this bacterium. To confirm this assumption, the chb operon, encoding the (GlcNAc)(2)-specific enzyme II permease, was cloned by reference to its Escherichia coli counterpart, and the Serratia chb operon was shown to comprise chbB, chbC, bglA, chbR, and chbG. Disruption of chbC drastically reduced production of chitinases and CBP21 and impaired growth on colloidal chitin. These results indicate that uptake of (GlcNAc)(2) is mediated by the PTS and that the (GlcNAc)(2)-specific enzyme II permease constitutes its major pathway. Since (GlcNAc)(2) uptake is essential for induction of chitinases and CBP21 production, (GlcNAc)(2) appears to be the key molecule in recognition and utilization of chitin by S. marcescens.

Cloning and sequencing of the Klebsiella pneumoniae O5 wb gene cluster and its role in pathogenesis.

ABSTRACT One representative recombinant clone encoding Klebsiella pneumoniae O5-antigen lipopolysaccharide (LPS) was found upon screening for serum resistance in a cosmid-based genomic library ofK. pneumoniae KT769 (O5:K57) introduced intoEscherichia coli DH5α. A total of eight open reading frames (wbO5 gene cluster) were necessary to produce K. pneumoniae O5-antigen LPS in E. coliK-12. The enzymatic activities proposed for thewbO5 gene cluster are in agreement with the activities proposed for the biosynthesis of K. pneumoniaeO5-antigen LPS. Using the complete DNA sequence of the K. pneumoniae wbO5 gene cluster, we obtained (by single or double recombination) genetically well-characterized mutants devoid only of this O5-antigen LPS. Finally, using these O5−mutants and the corresponding wild-type strains or complemented mutants with the wbO5 gene cluster (O5+strains), we found that the presence of K. pneumoniaeO5-antigen LPS is essential for some pathogenic features like serum resistance, adhesion to uroepithelial cells, and colonization (experimental infections) of the urinary tract in rats.

LysR-type Transcriptional Regulator ChiR Is Essential for Production of All Chitinases and a Chitin-Binding Protein, CBP21, in Serratia marcescens 2170

To identify the genes required for chitinase production by Serratia marcescens 2170, various Tn5 mutants somehow defective in chitinase production were isolated in a previous study. In order to identify the mutated gene in one of the chitinase-deficient mutants, N1, DNA regions flanking the Tn5 insertion were cloned and sequenced. Sequence comparison showed that the mutation occurred in the ORF located between chiB and cbp, which encode chitinase B and chitin-binding protein CBP21, respectively. The ORF encodes a 313-amino acid polypeptide which has significant similarity with various LysR-type transcriptional regulators, and thus the gene was designated chiR. Targeted mutagenesis confirmed that disruption of the chiR gene results in the phenotype of N1. Gel mobility shift assays using partially purified ChiR protein demonstrated that this protein specifically binds to the intergenic region between chiR and cbp. These results strongly suggest that ChiR is a LysR-type transcriptional regulator which is essential for production of all chitinases and CBP21.

Bacteriocin 28b, a chromosomally encoded bacteriocin produced by most Serratia marcescens biotypes

Twenty-six Serratia marcescens strains belonging to fifteen different biotypes were found to produce bacteriocins active against Escherichia coli. On the basis of the pattern of bacteriocin sensitivity of E. coli mutant strains, immunological assays and Southern blot hybridization with a probe for the S. marcescens bss (bacteriocin 28b structural) gene, it was concluded that all these strains apparently produce chromosomally encoded bacteriocins related to bacteriocin 28b. To confirm this conclusion, the genes encoding the bacteriocin produced by one of these strains (S. marcescens JF246) were cloned in plasmid pBR328. E. coli harbouring recombinant plasmid pDG301 produced a bacteriocin active against E. coli and immunologically related to bacteriocin 28b. Immunoblotting experiments showed that bacteriocins 28b and L appear to have the same apparent molecular mass (45 kDa). Furthermore, recombinant plasmid pDG301 DNA hybridized with a bss gene probe.

The UDP N-Acetylgalactosamine 4-Epimerase Gene Is Essential for Mesophilic Aeromonas hydrophila Serotype O34 Virulence

ABSTRACT Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O− phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).