title: Characterization of the gene cassette required for biosynthesis of the (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate capsule of serogroup A Neisseria meningitidis.
abstract: The (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate meningococcal capsule of serogroup A Neisseria meningitidis is biochemically distinct from the sialic acid-containing capsules produced by other disease-associated meningococcal serogroups (e.g., B, C, Y, and W-135). We defined the genetic cassette responsible for expression of the serogroup A capsule. The cassette comprised a 4,701-bp nucleotide sequence located between the outer membrane capsule transporter gene, ctrA, and galE, encoding the UDP-glucose-4-epimerase. Four open reading frames (ORFs) not found in the genomes of the other meningococcal serogroups were identified. The first serogroup A ORF was separated from ctrA by a 218-bp intergenic region. Reverse transcriptase (RT) PCR and primer extension studies of serogroup A mRNA showed that all four ORFs were cotranscribed in the opposite orientation to ctrA and that transcription of the ORFs was initiated from the intergenic region by a sigma-70-type promoter that overlapped the ctrA promoter. The first ORF exhibited 58% amino acid identity with the UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) 2-epimerase of Escherichia coli, which is responsible for the conversion of UDP-GlcNAc into UDP-N-acetyl-D-mannosamine. Polar or nonpolar mutagenesis of each of the ORFs resulted in an abrogation of serogroup A capsule production as determined by colony immunoblots and enzyme-linked immunosorbent assay. Replacement of the serogroup A biosynthetic gene cassette with a serogroup B cassette by transformation resulted in capsule switching from a serogroup A capsule to a serogroup B capsule. These data indicate that assembly of the serogroup A capsule likely begins with monomeric UDP-GlcNAc and requires proteins encoded by three other genes found in the serogroup A N. meningitidis-specific operon located between ctrA and galE.
authors.affiliation: Department of Medicine, Emory University School of Medicine, and Department of Veterans Affairs Medical Center, Atlanta 30303, Georgia, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Swartley
authors.forename: L J
authors.initials: LJ
authors.lastname: Liu
authors.forename: Y K
authors.initials: YK
authors.lastname: Miller
authors.forename: L E
authors.initials: LE
authors.lastname: Martin
authors.forename: S
authors.initials: S
authors.lastname: Edupuganti
authors.forename: D S
authors.initials: DS
authors.lastname: Stephens
mesh_terms: D016667:Bacterial Capsules / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006595:Hexosamines / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009345:Neisseria meningitidis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D016366:Open Reading Frames; D009876:Operon; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014157:Transcription Factors; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014534:UDPglucose 4-Epimerase / Q000235:genetics* / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C097706:CtrA protein, Caulobacter; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D006595:Hexosamines; D012333:RNA, Messenger; D014157:Transcription Factors; D014539:Uridine Diphosphate Sugars; C059191:uridine diphosphate N-acetylmannosamine; D014537:Uridine Diphosphate N-Acetylglucosamine; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; C083982:wecB protein, E coli; D014534:UDPglucose 4-Epimerase; C020551:galactose epimerase; C002022:N-acetylmannosamine