Predicted functions and linkage specificities of the products of the Streptococcus pneumoniae capsular biosynthetic loci

Abstract

The sequences of the capsular biosynthetic (cps) loci of 90 serotypes of Streptococcus pneumoniae have recently been determined. Bioinformatic procedures were used to predict the general functions of 1,973 of the 1,999 gene products and to identify proteins within the same homology group, Pfam family, and CAZy glycosyltransferase family. Correlating cps gene content with the 54 known capsular polysaccharide (CPS) structures provided tentative assignments of the specific functions of the different homology groups of each functional class (regulatory proteins, enzymes for synthesis of CPS constituents, polymerases, flippases, initial sugar transferases, glycosyltransferases [GTs], phosphotransferases, acetyltransferases, and pyruvyltransferases). Assignment of the glycosidic linkages catalyzed by the 342 GTs (92 homology groups) is problematic, but tentative assignments could be made by using this large set of cps loci and CPS structures to correlate the presence of particular GTs with specific glycosidic linkages, by correlating inverting or retaining linkages in CPS repeat units with the inverting or retaining mechanisms of the GTs predicted from their CAZy family membership, and by comparing the CPS structures of serotypes that have very similar cps gene contents. These large-scale comparisons between structure and gene content assigned the linkages catalyzed by 72% of the GTs, and all linkages were assigned in 32 of the serotypes with known repeat unit structures. Clear examples where very similar initial sugar transferases or glycosyltransferases catalyze different linkages in different serotypes were also identified. These assignments should provide a stimulus for biochemical studies to evaluate the reactions that are proposed.

FIG. 1.

Schematic representation of the biosynthesis of CPS by the Wzy-dependent pathway. The biosynthesis of the CPS of serotype 23F is represented. UDP-linked components of the repeat CPS unit are synthesized by genes encoded within the cps locus or are available from central metabolism (1). Repeat unit biosynthesis is initiated by the transfer of glucose phosphate to the lipid carrier by the IT WchA (2), followed by sequential addition of the other components of the repeat unit, catalyzed by the GTs (3), and the lipid-linked repeat unit is transferred across the membrane by the Wzx flippase (4) and polymerized by Wzy to result in lipid-linked CPS (5). Finally, the lipid-linked CPS is linked to the cell wall by a poorly understood process involving the Wzd/Wze complex, with release of the undecaprenyl phosphate carrier (6). The genes in the representation of the cps locus and their gene products are color coded; a key is given for the repeat unit constituents (modified from reference 12).

FIG. 2.

Biosynthetic pathways of housekeeping components of S. pneumoniae CPS. Putative pathways are denoted by a dotted line, and the constituents of the repeat units are underlined. Glk, glucokinase; Pgi, glucose-6-phosphate isomerase; Pgm, phosphoglucomutase; GlmS, l-glutamine-d-fructose-6-phoshate amidotransferase; GlmM, phosphoglucosamine mutase; GlmU, UDP-N-acetyl-glucosamine pyrophosphorylase; GalU, UTP-glucose-1P uridylyltransferase; GalE, UDP-N-acetylglucosamine-4-epimerase/UDP-glucose-4-epimerase; RblA, ribitol-5-phosphate dehydrogenase; RblB, d-ribitol-5-phosphate cytidylyltransferase; AatA, UDP-GlcNAc 4,6-dehydratase; AatB, UDP-4-keto-6-deoxy-d-glucose 4-transaminase; ChoT (also known as LicB), choline transporter; ChoA (also known as LicA), choline kinase; ChoB (also known as LicC), cholinephosphate cytidylyltransferase. Spr numbers refer to the annotation of the S. pneumoniae R6 genome sequence.

FIG. 3.

Biosynthetic pathways of nonhousekeeping (CPS-specific) components of S. pneumoniae CPS. Putative pathways are denoted by a dotted line, and the constituents of the repeat units are underlined. All gene products are encoded within the cps loci, excepting ribulose phosphate 3-epimerase (Rpe), which is chromosomally encoded and marked by an asterisk. Ugd, UDP-glucose 6-dehydrogenase; Gla, UDP-galacturonate 4-epimerase; Glf, UDP-galactopyranose mutase; MnaA, UDP-N-acetylglucosamine-2-epimerase; MnaB, UDP-N-acetylmannosamine dehydrogenase; FnlA, steps 1 and 2 of UDP-FucNAc synthesis; FnlB, steps 3 and 4 of UDP-FucNAc synthesis; FnlC, step 5 of UDP-FucNAc synthesis; RmlA, glucose-1-phosphate thymidylyltransferase; RmlB, dTDP-d-glucose 4,6-dehydratase; RmlC, dTDP-4-keto-6-deoxy-d-glucose 3,5-epimerase; RmlD, dTDP-4-keto-l-rhamnose reductase; Mnp1, putative nucleotidyltransferase (NDP-mannitol pathway); Mnp2, putative reductase (NDP-mannitol pathway); RbsF, putative epimerase/dehydratase (NDP-ribose biosynthesis); Gct, CDP-glycerol biosynthetic protein; Gtp1-3, NDP-2-glycerol pathway; Abp1, putative nucleotidyltransferase (NDP-arabinitol pathway); Abp2, putative reductase (NDP-arabinitol pathway).

FIG. 4.

Assignment of Wzy polymerase linkage and IT specificity. (A) Circularized representation of CPS repeat unit structures for serotypes 12F and 12A, where the polymerase linkage and the initial sugar are not indicated. Each sugar is represented symbolically as in the report by Bentley et al. (12), and the nature of the linkage is represented by red if it is retaining and blue if it is inverting. Each circular structure contains five retaining linkages and a single inverting linkage. (B) Excepting remnant transposases, the cps clusters of serotypes 12F and 12A are syntenic, and both encode five GTs, all of which are retaining based on CAZy GT family membership (colored red). Shading between the two loci represents TBLASTX amino acid sequence similarity viewed by using ACT. (C) We can infer that the single inverting linkage in the circular structure (A) corresponds to the linkage made by the polymerase, which also allows us to define the initial sugar and depict the structures biologically, with the lipid-linked initial sugar depicted to the right of the structure. ///, lipid carrier.

FIG. 5.

Assignment of GT specificity. (A) cps loci of serotypes 10A and 10F are represented linearly with genes encoding GTs colored according to their CAZy membership—red for a GT with a predicted retaining mechanism, blue for an inverting mechanism, and yellow if the GT has not been assigned to a CAZy family. Shading between the two loci represents TBLASTX amino acid sequence similarity viewed by using ACT. (B) Structural representation of CPS repeat units. Glycosidic linkages are colored according to whether they require a retaining mechanism (red) or an inverting mechanism (blue). ///, lipid carrier. See the text for methods of GT assignment.