gmhX, a novel gene required for the incorporation of L-glycero-D-manno-heptose into lipooligosaccharide in Neisseria meningitidis

Lipooligosaccharide (LOS) is a critical virulence factor of Neisseria menin gitidis. A Tn916 insertion mutant, designated 469 was found to exhibit a ma rkedly 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 LO S revealed that it consisted of the deep rough, heptose-deficient structure , Kdo(2)-lipid A. Sequencing of chromosomal DNA flanking the Tn916 insertio n in mutant 469 revealed that the transposon had inserted into an ORF predi cted to encode a 187 aa protein with sequence homology to the histidinol-ph osphate phosphatase domain of Escherichia coli HisB and to a family of gene s of unknown function. The gene, designated gmhX, is part of a polycistroni c operon (ice-2) containing two other genes, nIaB and orfC. nIaB encodes a lysophosphaticlic-acid acyltransferase and orfC is predicted to encode a N- acetyltransferase. Specific polar and non-polar gmhX mutations in the paren tal strain, NMB, exhibited the truncated LOS structure of mutant 469, and r epair of gmhX mutants by homologous recombination with the wild-type gmhX r estored the LOS parental phenotype. GmhX mutants demonstrated increased sen sitivity to polymyxin B. GmhX mutants and other Kdo(2)-lipid A mutants also demonstrated increased sensitivity to killing by normal human serum but we re not as sensitive as inner-core mutants containing heptose. In the genome s of Helicobacter pylori and Synechocystis, gmhX homologues are associated with heptose biosynthesis genes, however, in N. meningitidis, gmhX was foun d 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-hepto se into meningococcal LOS, and is a candidate for the 2-D-glycero-manno-hep tose phosphatase of the heptose biosynthesis pathway.