Characterization of cpsF and its product CMP-N-acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli.
Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N-acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al., 1992). In this paper, we report the identification and characterization of cpsF, a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthase encoded by the Escherichia coli K1 gene, neuA. The enzymatic function of the protein encoded by cpsF was established by cloning the gene E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF, K1 antigen expression was restored. We concluded that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.
Haft
R F
RF
Channing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Wessels
M R
MR
Mebane
M F
MF
Conaty
N
N
Rubens
C E
CE
D000942:Antigens, Bacterial; D016667:Bacterial Capsules; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005779:Immunodiffusion; D008969:Molecular Sequence Data; D009154:Mutation; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D013292:Streptococcus agalactiae / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial
D000942:Antigens, Bacterial; D011135:Polysaccharides, Bacterial; C036962:capsular polysaccharide K1; C038687:capsular polysaccharide, streptococcal group B type II; D000216:N-Acylneuraminate Cytidylyltransferase