1887

Abstract

Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic . Three galactosyltransferase genes, , and , responsible for the biosynthesis of LOS oligosaccharide chains, were analysed in five species. The function of in 6275 was determined by mutagenesis and chemical characterization of the parent and mutant LOS chains. The chemical characterization included SDS-PAGE, immunoblot, hexose and mass spectrometry analyses. Compared with the parent LOS, the mutant LOS lacked galactose, and its oligosaccharide decreased by three or four sugar units in matrix-assisted laser desorption ionization (MALDI)-MS analysis. The results show that encodes a -1,4-galactosyltransferase, and that the glucose moiety linked to heptose (Hep) in the chain is the acceptor site in the biosynthesis of LOS. To understand the sequence diversity and relationships of , and , the entire locus was further sequenced in three strains and three commensal strains, and compared with the previously reported genes from species. Comparison of the protein sequences of the three enzymes LgtB, LgtE and LgtH showed a conserved N-terminal region, and a highly variable C-terminal region, suggesting functional constraint for substrate and acceptor specificity, respectively. The analyses of allelic variation and evolution of 23 , 12 and 14 sequences revealed a distinct evolutionary history of these genes in . For example, the splits graph of displayed a network evolution, indicating frequent DNA recombination, whereas splits graphs of and displayed star-tree-like evolution, indicating the accumulation of point mutations. The data presented here represent examples of the evolution and variation of prokaryotic glycosyltransferase gene families. These imply the existence of multiple enzyme isoforms for biosynthesis of a great diversity of oligosaccharides in nature.

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2006-01-01
2020-04-07
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