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Pathogenicity and Medical Microbiology |
Departments of Medicine, and Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA1
Department of Veterans Affairs Medical Center, Atlanta, GA 30033, USA2
The Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA3
Author for correspondence: David S. Stephens. Tel: +1 404 728 7688. Fax: +1 404 329 2210. e-mail: dstep01{at}emory.edu
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, Kdo2-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 Kdo2-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.
Keywords: meningococcus, lipopolysaccharides, heptose biosynthesis
Abbreviations: ABC, ATP-binding cassette; ES-MS, electrospray mass spectrometry; Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-2-octulosonic acid; LOS, lipooligosaccharide; NHS, normal human serum
The GenBank accession number for the sequence of the ice-2 operon in Neisseria meningitidis NMB is AF036242.
a Present address: BioResource International, Inc., 840 Main Campus Drive, Suite 3560, Raleigh, NC 27606, USA.
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