title: Genetic and functional analyses of the lgtH gene, a member of the beta-1,4-galactosyltransferase gene family in the genus Neisseria.
abstract: Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Three galactosyltransferase genes, lgtB, lgtE and lgtH, responsible for the biosynthesis of LOS oligosaccharide chains, were analysed in five Neisseria species. The function of lgtH in Neisseria meningitidis 6,275 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 lgtH encodes a beta-1,4-galactosyltransferase, and that the glucose moiety linked to heptose (Hep) in the alpha chain is the acceptor site in the biosynthesis of Neisseria LOS. To understand the sequence diversity and relationships of lgtB, lgtE and lgtH, the entire lgt-1 locus was further sequenced in three N. meningitidis strains and three commensal Neisseria strains, and compared with the previously reported lgt genes from Neisseria 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 lgtB, 12 lgtE and 14 lgtH sequences revealed a distinct evolutionary history of these genes in Neisseria. For example, the splits graph of lgtE displayed a network evolution, indicating frequent DNA recombination, whereas splits graphs of lgtB and lgtH 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.
authors.affiliation: Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, FDA, 8800 Rockville Pike, Bethesda, MD 20892, USA.
authors.forename: Peixuan
authors.initials: P
authors.lastname: Zhu
authors.affiliation: Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, FDA, 8800 Rockville Pike, Bethesda, MD 20892, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Boykins
authors.affiliation: Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, FDA, 8800 Rockville Pike, Bethesda, MD 20892, USA.
authors.forename: Chao-Ming
authors.initials: CM
authors.lastname: Tsai
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D014644:Genetic Variation; D008070:Lipopolysaccharides / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000235:genetics* / Q000378:metabolism; D009343:Neisseria / Q000201:enzymology / Q000235:genetics*; D009345:Neisseria meningitidis / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; D009237:N-Acetyllactosamine Synthase