CHARACTERIZATION OF THE GENE CASSETTE REQUIRED FOR BIOSYNTHESIS OF THE (ALPHA-1-]6)-LINKED N-ACETYL-D-MANNOSAMINE-1-PHOSPHATE CAPSULE OF SEROGROUP-A NEISSERIA-MENINGITIDIS

The (alpha 1-->6)-linked N-acetyl-D-mannosamine-1-phosphate meningococ cal capsule of serogroup A Neisseria meningitidis is biochemically dis tinct from the sialic acid-containing capsules produced by other disea se-associated meningococcal serogroups (e.g., B, C, Y, and W-135). We defined the genetic cassette responsible for expression of the serogro up A capsule, The cassette comprised a 4,701-bp nucleotide sequence lo cated between the outer membrane capsule transporter gene, ctrA, and g alE, encoding the UDP-glucose-4-epimerase. Four open reading frames (O RFs) not found in the genomes of the other meningococcal serogroups we re identified, The first serogroup A ORF was separated from ctrA by a 218-bp intergenic region, Reverse transcriptase (RT) PCR and primer ex tension studies of serogroup A mRNA showed that all four ORFs were cot ranscribed in the opposite orientation to ctrA and that transcription of the ORFs was initiated from the intergenic region by a sigma-70-typ e promoter that overlapped the ctrA promoter, The first ORF exhibited 58% amino acid identity with the UDP-N-acetyl-D-glucosamine (UDP-GlcNA c) 2-epimerase of Escherichia call, which is responsible for the conve rsion of UDP-GlcNAc into UDP-N-acetyl-D-mannosamine. Polar or nonpolar mutagenesis of each of the ORFs resulted in an abrogation of serogrou p A capsule production as determined by colony immunoblots and enzyme- linked immunosorbent assay. Replacement of the serogroup A biosyntheti c gene cassette,vith a serogroup B cassette by transformation resulted in capsule switching from a serogroup A capsule to a serogroup B caps ule, These data indicate that assembly of the serogroup A capsule like ly begins with monomeric UDP-GlcNAc and requires proteins encoded by t hree other genes found in the serogroup A N. meningitidis-specific ope ron located between ctrA and galE.