A functional analysis of the Streptococcus pneumoniae genes involved in the synthesis of type 1 and type 3 capsular polysaccharides

Abstract

Type 3 pneumococci produce a capsule composed of cellobiuronic acid units connected in a beta (1-->3) linkage. Cellobiuronic acid is a disaccharide consisting of D-glucuronic acid (GlcA) beta (1-->4) linked to D-glucose (Glc). The genes implicated in the biosynthesis of the type 3 capsule have been cloned, expressed, and biochemically characterized. The three type 3-specific genes--designated as cap3ABC--are transcribed together. However, the two complete open reading frames located upstream of cap3A are not transcribed and, consequently, are not required for capsule formation. The promoter of the cap3 operon was localized by primer extension analysis. The products of cap3A, cap3B, and cap3C were biochemically characterized as a UDP-Glc dehydrogenase, the type 3 polysaccharide synthase, and a Glc-1-P uridyltransferase, respectively. The Cap3B synthase was expressed in Escherichia coli, and pneumococcal type 3 polysaccharide was synthesized in this heterologous system. When a recombinant plasmid (pLSE3B) containing cap3B was introduced by transformation into encapsulated pneumococci of types 1, 2, 5, or 8, the lincomycin-resistant transformants displayed a binary type of capsule, this is, they showed a type 3 capsule in addition to that of the recipient type. Unencapsulated (S2) laboratory strains of S. pneumoniae also synthesized a type 3 capsule when transformed with pLSE3B. On the other hand, we have cloned and sequenced seven type 1-specific genes (designated as cap1A-G), and their functions have been preliminarily assigned based on sequence similarities.