Molecular analysis of the biosynthesis pathway of the alpha-2,8 polysialic acid capsule by Neisseria meningitidis serogroup B.
The genes encoding all enzymes necessary for capsular polysaccharide biosynthesis in Neisseria meningitidis B are located on a 5 kb DNA fragment within the chromosomal cps gene cluster. Nucleotide sequence analysis revealed four open reading frames (ORFs), which can encode proteins with molecular masses of 41.4 kDa, 24.9 kDa, 38.3 kDa, and 54.4 kDa, respectively. These ORFs constitute a transcriptional unit as demonstrated by Northern blots. Primer extension analysis revealed that the transcriptional start site is preceded by a nucleotide sequence with homologies to the sigma 70 consensus promoter sequence of Escherichia coli. Functional analysis of the proteins encoded by the ORFs indicated that ORF2 encodes the CMP-NeuNAc synthetase, ORF3 encodes the NeuNAc condensing enzyme, and ORF4 encodes the alpha-2,8 polysialyltransferase. ORF1 encodes an enzyme, which provides a precursor molecule for synthesis of monomeric NeuNAc. In E. coli the subcloned ORFs 2-4 were able to synthesize a high-molecular-weight alpha-2,8 polysialic acid. In contrast, inactivation of ORF1 in the meningococcal genome resulted in a complete loss of capsule production. A regulatory enzyme, the CMP-NeuNAc hydrolase, which cleaves CMP-NeuNAc to CMP and NeuNAc, was not found as a part of the capsular polysaccharide biosynthesis gene operon or within the cps gene cluster.
Edwards
U
U
Institute für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Müller
A
A
Hammerschmidt
S
S
Gerardy-Schahn
R
R
Frosch
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D000216:N-Acylneuraminate Cytidylyltransferase; D009345:Neisseria meningitidis / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D019998:Racemases and Epimerases; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012703:Serotyping; D012794:Sialic Acids / Q000096:biosynthesis*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D011135:Polysaccharides, Bacterial; D012794:Sialic Acids; C071290:alpha(2-8)polysialic acid; D000216:N-Acylneuraminate Cytidylyltransferase; C091607:SiaB protein, Neisseria meningitidis; D019998:Racemases and Epimerases; C091606:NeuC protein, Neisseria meningitidis