The capsule polysaccharide synthesis locus of Streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E

To identify a chromosomal region of Streptococcus pneumoniae serotype 14 involved in capsule polysaccharide synthesis, two strategies were used: (i) Tn916 mutagenesis, followed by the characterization of four unencapsulated mutants, and (ii) cross-hybridization with a capsule polysaccharide synthesis gene (cps) probe from S. agalactiae, which has a structurally similar capsule. The two approaches detected the same chromosomal region consisting of two adjacent EcoRI fragments, One of these EcoRI fragments was cloned and hybridized with a cosmid library. This resulted in clone cMK02. A similar cosmid clone was obtained from an unencapsulated Tn916 mutant, Spn14.H. Sequence analysis of the two cosmid clones revealed that in the Tn916 mutant, a gene, cps14E, which is homologous to other bacterial genes encoding glycosyl transferases, had been inactivated, An open reading frame immediately downstream of sps14E, designated cpsI4F, shows no significant homology with any known genes or proteins. A functional assay shelved that cpsl4E encodes a glycosyl transferase and that a gene-specific knockout mutant lacks this enzyme activity, whereas inactivation of cpsl4F does not have this effect.