title: gmhX, a novel gene required for the incorporation of L-glycero-D-manno-heptose into lipooligosaccharide in Neisseria meningitidis.
abstract: Lipooligosaccharide (LOS) is a critical virulence factor of Neisseria meningitidis. A Tn916 insertion mutant, designated 469, was found to exhibit a markedly truncated LOS of 2.9 kDa when compared by Tricine/SDS-PAGE to the parental LOS (4.6 kDa). Electrospray mass spectrometry analysis of 469 LOS revealed that it consisted of the deep rough, heptose-deficient structure, Kdo(2)-lipid A. Sequencing of chromosomal DNA flanking the Tn916 insertion in mutant 469 revealed that the transposon had inserted into an ORF predicted to encode a 187 aa protein with sequence homology to the histidinol-phosphate phosphatase domain of Escherichia coli HisB and to a family of genes of unknown function. The gene, designated gmhX, is part of a polycistronic operon (ice-2) containing two other genes, nlaB and orfC. nlaB encodes a lysophosphatidic-acid acyltransferase and orfC is predicted to encode a N-acetyltransferase. Specific polar and non-polar gmhX mutations in the parental strain, NMB, exhibited the truncated LOS structure of mutant 469, and repair of gmhX mutants by homologous recombination with the wild-type gmhX restored the LOS parental phenotype. GmhX mutants demonstrated increased sensitivity to polymyxin B. GmhX mutants and other Kdo(2)-lipid A mutants also demonstrated increased sensitivity to killing by normal human serum but were not as sensitive as inner-core mutants containing heptose. In the genomes of Helicobacter pylori and Synechocystis, gmhX homologues are associated with heptose biosynthesis genes; however, in N. meningitidis, gmhX was found in a location distinct from that of gmhA, rfaD, rfaE, aut and rfaC. GmhX is a novel enzyme required for the incorporation of L-glycero-D-manno-heptose into meningococcal LOS, and is a candidate for the 2-D-glycero-manno-heptose phosphatase of the heptose biosynthesis pathway.
authors.affiliation: Department of Veterans Affairs Medical Center, Atlanta, GA 30033, USA2.|Departments of Medicine, and Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA1.
authors.forename: Giles C
authors.initials: GC
authors.lastname: Shih
authors.affiliation: Department of Veterans Affairs Medical Center, Atlanta, GA 30033, USA2.|Departments of Medicine, and Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA1.
authors.forename: Charlene M
authors.initials: CM
authors.lastname: Kahler
authors.affiliation: The Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA3.
authors.forename: Russell W
authors.initials: RW
authors.lastname: Carlson
authors.affiliation: The Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA3.
authors.forename: M Mahbubur
authors.initials: MM
authors.lastname: Rahman
authors.affiliation: Department of Veterans Affairs Medical Center, Atlanta, GA 30033, USA2.|Departments of Medicine, and Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA1.
authors.forename: David S
authors.initials: DS
authors.lastname: Stephens
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004251:DNA Transposable Elements; D005798:Genes, Bacterial; D006539:Heptoses / Q000235:genetics / Q000378:metabolism*; D008050:Lipid A / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D009345:Neisseria meningitidis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D009876:Operon; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D006539:Heptoses; D008050:Lipid A; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; C088602:3-deoxy-2-octulosonic acid(2)-lipid IV(A); C026312:glycero-manno-heptose; C438389:2-D-glycero-manno-heptose phosphatase; D010744:Phosphoric Monoester Hydrolases