The type III capsular polysaccharide of group B streptococci (GBS) consists of a linear backbone with short side chains ending in residues of N-acetylneuraminic acid, or sialic acid. The presence of sialic acid on the surface of the organism inhibits activation of the alternative pathway of complement and is thought to be an important element in the virulence function of the capsule. We showed previously that a mutant strain of GBS that expressed a sialic acid-deficient, or asialo, form of the type III polysaccharide was avirulent, supporting a virulence function for capsular sialic acid. We now report the derivation of an asialo capsule mutant from a highly encapsulated wild-type strain of type III GBS, strain COH1, by insertional mutagenesis with transposon Tn916AE. In contrast to the wild-type strain, the asialo mutant strain COHl-11 was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat model of GBS infection. The asialo mutant accumulated free intracellular sialic acid, suggesting a defect subsequent to sialic acid synthesis in the biosynthetic pathway leading to capsule sialylation. The specific biosynthetic defect in mutant strain COHl-11 was found to be in the activation of free sialic acid to CMP-sialic acid: CMP-sialic acid synthetase activity was present in the wild-type strain COH1 but was not detected in the asialo mutant strain COHl-11.One of the two transposon insertions in the asialo mutant COHl-11 mapped to the same chromosomal location as one of the two Tn916 insertions in the previously reported asialo mutant COH31-21, identifying this site as a genetic locus necessary for expression of CMP-sialic acid synthetase activity. These studies demonstrate that the enzymatic synthesis of CMP-sialic acid by GBS is an essential step in sialylation of the type III capsular polysaccharide.Group B streptococci (GBS) are the leading cause of neonatal sepsis and meningitis in the United States (4, 13). Several lines of evidence indicate that the GBS capsular polysaccharide is a critical factor in the ability of the organism to produce invasive infection. While the four major capsular types are found with similar frequencies among isolates cultured as commensals from the genital tracts of colonized women, type III strains predominate among isolates from the blood or cerebrospinal fluid of infants with GBS sepsis. Organisms of capsular type III account for approximately 60% of GBS isolated from infants with neonatal sepsis and 80 to 90% of those with meningitis (1, 6). That type III strains are disproportionately prevalent among GBS associated with invasive disease suggested that the type III capsule serves as a virulence factor. This hypothesis was supported by the observation that type III isolates from the blood or cerebrospinal fluid of ill infants were more highly encapsulated than were colonizing strains (16). More direct evidence for the role of the type III capsule in virulence came from our studies of an unencapsulated mutant strain of type III G...