title: Two glycosyltransferase genes, lgtF and rfaK, constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of Neisseria meningitidis.
abstract: We have characterized an operon required for inner-core biosynthesis of the lipooligosaccharide (LOS) of Neisseria meningitidis. Using Tn916 mutagenesis, we recently identified the alpha-1,2-N-acetylglucosamine (GlcNAc) transferase gene (rfaK), which when inactivated prevents the addition of GlcNAc and alpha chain to the meningococcal LOS inner core (C. M. Kahler, R. W. Carlson, M. M. Rahman, L. E. Martin, and D. S. Stephens, J. Bacteriol. 178:1265-1273, 1996). During the study of rfaK, a second open reading frame (lgtF) of 720 bp was found upstream of rfaK. An amino acid sequence homology search of the GenBank and EMBL databases revealed that the amino terminus of LgtF has significant homology with a family of beta-glycosyltransferases involved in the biosynthesis of polysaccharides and O antigen of lipopolysaccharides. The chromosomal copy of lgtF was mutagenized with a nonpolar antibiotic resistance cassette to minimize potential polar effects on rfaK. Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and composition analysis of the LOS from the nonpolar lgtF mutant showed that this strain produced a truncated LOS structure which contained a LOS inner core of GlcNAc1Hep2KDO2lipid A but without the addition of lacto-N-neotetraose to HepI or glucose to HepII. These results and the amino acid homology with beta-glycosyltransferases suggest that lgtF encodes the UDP-glucose:LOS-beta-1,4-glucosyltransferase which attaches the first glucose residue to HepI of LOS. Reverse transcriptase PCR and primer extension analysis indicate that both lgtF and rfaK are cotranscribed as a polycistronic message from a promoter upstream of lgtF. This arrangement suggests that completion of the LOS inner core and the initiation of the alpha chain addition are tightly coregulated in N. meningitidis.
authors.affiliation: Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Kahler
authors.forename: R W
authors.initials: RW
authors.lastname: Carlson
authors.forename: M M
authors.initials: MM
authors.lastname: Rahman
authors.forename: L E
authors.initials: LE
authors.lastname: Martin
authors.forename: D S
authors.initials: DS
authors.lastname: Stephens
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002240:Carbohydrate Sequence; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009345:Neisseria meningitidis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D009876:Operon; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C103165:lgtF protein, Neisseria meningitidis; C501545:rfaK protein, Neisseria meningitidis