Molecular cloning and analysis of genes for sialic acid synthesis in Neisseria meningitidis group B and purification of the meningococcal CMP-NeuNAc synthetase enzyme.
The gene encoding for the CMP-NeuNAc synthetase enzyme of Neisseria meningitidis group B was cloned by complementation of a mutant of Escherichia coli defective for this enzyme. The gene (neuA) was isolated on a 4.1-kb fragment of meningococcal chromosomal DNA. Determination of the nucleotide sequence of this fragment revealed the presence of three genes, termed neuA, neuB, and neuC, organized in a single operon. The presence of a truncated ctrA gene at one end of the cloned DNA and a truncated gene encoding for the meningococcal sialyltransferase at the other confirmed that the cloned DNA corresponded to region A and part of region C of the meningococcal capsule gene cluster. The predicted amino acid sequence of the meningococcal NeuA protein was 57% homologous to that of NeuA, the CMP-NeuNAc synthetase encoded by E. coli K1. The predicted molecular mass of meningococcal NeuA protein was 24.8 kDa, which was 6 kDa larger than that formerly predicted (U. Edwards and M. Frosch, FEMS Microbiol. Lett. 96:161-166, 1992). Purification of the recombinant meningococcal NeuA protein together with determination of the N-terminal amino acid sequence confirmed that this 24.8-kDa protein was indeed the meningococcal CMP-NeuNAc synthetase. The predicted amino acid sequences of the two other encoded proteins were homologous to those of the NeuC and NeuB proteins of E. coli K1, two proteins involved in the synthesis of NeuNAc. These results indicate that common steps exist in the biosynthesis of NeuNAc in these two microorganisms.
Ganguli
S
S
Department of Microbiology, University of Leicester, United Kingdom.
Zapata
G
G
Wallis
T
T
Reid
C
C
Boulnois
G
G
Vann
W F
WF
Roberts
I S
IS
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D000216:N-Acylneuraminate Cytidylyltransferase / Q000235:genetics* / Q000302:isolation & purification*; D009345:Neisseria meningitidis / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012794:Sialic Acids / Q000096:biosynthesis*; D012799:Sialyltransferases / Q000235:genetics
D011994:Recombinant Proteins; D012794:Sialic Acids; D012799:Sialyltransferases; D000216:N-Acylneuraminate Cytidylyltransferase; D019158:N-Acetylneuraminic Acid