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 b iosynthesis in Neisseria meningitidis B are located on a 5 kb DNA frag ment within the chromosomal cps gene cluster. Nucleotide sequence anal ysis revealed four open reading frames (ORFs), which can encode protei ns 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 demons trated by Northern blots. Primer extension analysis revealed that the transcriptional start site is preceded by a nucleotide sequence with h omologies to the sigma(70) consensus promoter sequence of Escherichia coli. Functional analysis of the proteins encoded by the ORFs indicate d that ORF2 encodes the CMP-NeuNAc synthetase, ORF3 encodes the NeuNAc condensing enzyme, and ORF4 encodes the alpha-2,8 polysialyltransfera se. ORF1 encodes an enzyme, which provides a precursor molecule for sy nthesis of monomeric NeuNAc. In E. coli the subcloned ORFs 2-4 were ab le to synthesize a high-molecular-weight alpha-2,8 polysialic acid. In contrast, inactivation of ORF1 in the meningococcal genome resulted i n 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 opero n or within the cps gene cluster.