Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide

Department of Microbiology, University of Guelph, Guelph, Ontario , Canada N1G 2W1 1 Author for correspondence: Joseph S. Lam. Tel: +1 519 824 4120 Ext. 3823. Fax: +1 519 837 1802. e-mail: jlam{at}uoguelph.ca This study reports the organization of the wbp gene cluster and characterization of a numbe...

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Bibliographic Details
Published in:Microbiology (Society for General Microbiology) 1999-12, Vol.145 (12), p.3505-3521
Main Authors: Belanger, Myriam, Burrows, Lori L, Lam, Joseph S
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Bacteriology
Biological and medical sciences
Blotting, Southern
Cloning, Molecular
DNA, Bacterial - genetics
double prime O antigen
Fundamental and applied biological sciences. Psychology
Genes, Bacterial
Genetic Complementation Test
Genetics
glycosyltransferase
Glycosyltransferases - genetics
Glycosyltransferases - metabolism
lipopolysaccharides
Lipopolysaccharides - metabolism
Microbiology
Molecular Sequence Data
Multigene Family
O Antigens - biosynthesis
O Antigens - genetics
Plasmids - genetics
Polymerase Chain Reaction
Pseudomonas aeruginosa
Pseudomonas aeruginosa - classification
Pseudomonas aeruginosa - genetics
Pseudomonas aeruginosa - immunology
Pseudomonas aeruginosa - metabolism
Sequence Alignment
Sequence Analysis, DNA
Transformation, Bacterial
wbpL gene
wbpM gene
wbpO gene
wbpP gene
wbpR gene
wbpT gene
wbpU gene
wbpV gene
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Summary:Department of Microbiology, University of Guelph, Guelph, Ontario , Canada N1G 2W1 1 Author for correspondence: Joseph S. Lam. Tel: +1 519 824 4120 Ext. 3823. Fax: +1 519 837 1802. e-mail: jlam{at}uoguelph.ca 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 A-band 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. Keywords: Pseudomonas aeruginosa , lipopolysaccharide, rfb
ISSN:1350-0872
1465-2080
DOI:10.1099/00221287-145-12-3505