Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis - PubMed (original) (raw)

Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis

M Regué et al. J Bacteriol. 2001 Jun.

Abstract

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.

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Figures

FIG. 1

FIG. 1

Comparative structure of the chemotype Rc core LPS of E. coli and S. enterica (A) (20) versus K. pneumoniae strain RFK-11 (B) (43, 45). In K. pneumoniae RFK-11 (O8−:K−), only two Kdo_p_ residues were detected (42). In K. pneumoniae R20 (O1−:K20−), a Glc_p_N residue substituted with a tetraglycan of α-1,2-linked

d

-_glycero_-

d

-_manno_-heptopyranose was found instead of Glc_p_ll (44). Dashed arrows denote modifications that are either nonstoichiometric or are confined to a particular core LPS type. P, phosphate; PPEtn, ethanolamine pyrophosphate.

FIG. 2

FIG. 2

Physical map of plasmids used in this study. Plasmids conferring high- and low-level bacteriocin 28b resistance on E. coli NM554 are denoted by asterisks and underlined letters, respectively. The right-side _Bgl_II site corresponds to the junction between the insert and vector cosmid in recombinant cosmid pNUR8.

FIG. 3

FIG. 3

E. coli NM554 harboring plasmids pNUR8 (lane 1), pNUR5 (lane 3), vector pLA2917 (lane 2), pBG3 (lane 4), pBG1 (lane 5), pNUC4 (lane 6), and pBG2 (lane 7). Similar results were obtained using the E. coli DH5α background.

FIG. 4

FIG. 4

SDS-Tricine-PAGE analysis of LPS from S. enterica serovar Typhimurium SA1377 (_waaC_630) (lane 1), SA1377 (pB1) (lane 2), SA1377 (pBG1) (lane 3), SA1377 (pGEMT-WaaC) (lane 4), SL3789 (waaF511) (lane 5), SL3789 (pB1) (lane 6), and SL3789 (pGEMT-WaaF) (lane 7).

FIG. 5

FIG. 5

SDS-PAGE analysis of LPS from K. pneumoniae 52145 (wild type) (lane 1), NC20 (waaL) (lane 2), and NC20 (pGEMT-WaaL).

FIG. 6

FIG. 6

SDS-Tricine-PAGE analysis of LPS from K. pneumoniae 52145 (wild type) (lane 1), NC16 (waaE) (lane 2), NC16 (pGEMT-WaaE) (lane 3), and NC16 (pGLU) (lane 4).

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