Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide The GenBank accession number for the sequence reported in this paper is AF035937.

Abstract

This study reports the organization of the wbp gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent Pseudomonas aeruginosa serotype O6. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of wzx (encoding the O antigen flippase/translocase) and wzz (which modulates O antigen chain length distribution) genes, typical of a wzy-dependent pathway. However, a complete wzy gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, wbpO, wbpP, wbpV and wbpM, and four putative glycosyltransferase genes, wbpR, wbpT, wbpU and wbpL, were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of wbpO, wbpP, wbpV, wbpL and wbpM were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The wbpL mutant was deficient in both A-band and B-band LPS. WbpL O6 is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and Aband synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the wbp O6 genes was by complementation analysis. Two genes from Salmonella typhi, wcdA and wcdB, responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of wbpO and wbpP. wcdA and wcdB restored B-band synthesis in wbpO and wbpP mutants respectively, implying that wbpO and wbpP are involved in UDP-GalNAcA synthesis. Although wbpV has homology to wbpK of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of wbpN (located downstream of wbpM) did not abrogate LPS synthesis in either O5 or O6 ; therefore, it has been renamed orf48.5. These results establish the organization of genes involved in P. aeruginosa B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.

Vi Antigen Biosynthesis inSalmonella typhi: Characterization of UDP-N-acetylglucosamine C-6 Dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic Acid C-4 Epimerase (TviC)

“…Interestingly, the order of the same two reactions is reversed in the biosynthesis of O-antigen in P. aeruginosa O6 in which UDP-GalNAcA (5) is the substrate for the corresponding glycosyltransferases, WbpU and WbpT. 18,30 In this pathway, UDP-GlcNAc (2) is first epimerized by WbpP to give UDP-GalNAc (3), which is then oxidized by WbpO to form UDP-GalNAcA (5) (analogous to route B in Scheme 1). Apparently, a different path has been evolved in Salmonella typhi to make Vi antigen.…”

Identification of the Vibrio vulnificus wbpP Gene and Evaluation of Its Role in Virulence

“…Among these, the gene product of P. aeruginosa wbpP has been biochemically and genetically well characterized (1,36). Also, the enzymatic characteristics of WbpP have recently been confirmed (5).…”

“…UDP-N-acetyl-D-galactosaminuronic acid (UDP-GalNAcA), the product of the further dehydrogenation of UDP-GalNAc, is an important intermediate used for the biosynthesis of different uronic acid sugars of surface polysaccharides that contain GalNAcA or its derivatives, not only in P. aeruginosa, but also in other organisms (36). Also, it has been recently reported that the epimerization is performed by the gene products of wbpP (1).…”

“…Thus, the biosynthetic pathways and molecular genetics of surface polysaccharide production have been widely studied in Pseudomonas aeruginosa (1). The common glycolytic metabolite glucose 1-phosphate is first converted to UDP-N-acetyl-D-glucosamine (UDPGlcNAc), the main activated precursor of surface-associated carbohydrate synthesis (1,5). UDP-N-acetyl-D-galactosamine (UDP-GalNAc) is then formed by the C 4 epimerization of UDP-GlcNAc (5).…”

“…Nonetheless, it is generally believed that a similar biosynthetic pathway operates in gram-negative bacteria. Thus, the biosynthetic pathways and molecular genetics of surface polysaccharide production have been widely studied in Pseudomonas aeruginosa (1). The common glycolytic metabolite glucose 1-phosphate is first converted to UDP-N-acetyl-D-glucosamine (UDPGlcNAc), the main activated precursor of surface-associated carbohydrate synthesis (1,5).…”