1887

Abstract

This study reports the organization of the gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent serotype O6. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of (encoding the O antigen flippase/translocase) and (which modulates O antigen chain length distribution) genes, typical of a - dependent pathway. However, a complete gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, , , and , and four putative glycosyltransferase genes, , , and , were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of , , , and were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The mutant was deficient in both A-band and B-band LPS. WbpL 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 genes was by complementation analysis. Two genes from , and , responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of and . and restored B-band synthesis in and mutants respectively, implying that and are involved in UDP-GalNAcA synthesis. Although has homology to 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 (located downstream of ) did not abrogate LPS synthesis in either O5 or O6; therefore, it has been renamed . These results establish the organization of genes involved in B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.

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1999-12-01
2020-07-12
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