Comparative genetics of capsular polysaccharide biosynthesis in Streptococcus pneumoniae types belonging to serogroup 19

Abstract

The genetic basis for the structural diversity of capsule polysaccharide (CPS) in Streptococcus pneumoniae serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19 cps loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (cps19aI and cps19fI). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two cps loci (cps19cS), which is likely to encode a glucosyl transferase. The arrangement of the genes within the cps19 loci is highly conserved, with 13 genes (cps19A to -H and cps19K to -O) common to all four serogroup 19 members. These cps genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19 cps loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the cps19 gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.

FIG. 1

Biological repeat units of pneumococcal type 19F, 19A, 19B, and 19C CPSs. The orders of the sugars in the repeat units have been altered compared to the published chemical structures for 19F (36), 19A (21), and 19B and 19C (6), reflecting the fact that glucose is the first sugar in the biological repeat unit. d-Glc, glucose; d-ManNAc, N-acetylmannosamine; l-Rha, rhamnose; d-Rib, ribose.

FIG. 2

Schematic representation of the PCR products amplified by using cps19f primers. The cps19f genes which hybridized (at high stringency) to type 19A chromosomal DNA are shown in black, and the remainder of the locus is shown in grey. (A) Primers used to amplify the cps19a locus. (B) The cps19a locus. Regions with >95% nucleotide sequence identity with the cps19f locus are shown in black. Regions with 90 to 95% identity are shown in dark grey, and regions with 70 to 90% identity are shown in light grey. Restriction sites are as follows: Nc, NcoI; C, ClaI; H, HindIII; B, BAMHI; Nr, NruI; S, SphI; P, PstI; K, KpnI; E, EcoRI.

FIG. 3

Diagrammatic representation of the crossover points in the Rx1-19A transformants. The cps19f locus is shown in solid colors, where black represents >90% identity between the cps19f and cps19a loci and grey indicates regions with 70 to 80% identity. The horizontally hatched region within cps19fI indicates the site of pVA891 insertion. The cps19a locus is vertically hatched. The arrows indicate the points from which the sequence becomes cps19a specific.

FIG. 4

Diagrammatic representation of the cps19aL-aliA regions of cps19a and cps19a₂. The black region of cps19a₂ is >90% identical to the same region in cps19a, and the grey region exhibits 75 to 80% identity to the equivalent regions in cps19a. The positions of the potential promoter sequences and the putative stem-loop terminator are also indicated.

FIG. 5

Physical map of part of the cps19c locus. Boxed arrows represent potential ORFs. Gene designations are indicated below the map; cps19cB to -S are abbreviated as B to S, respectively. Restriction sites are as follows: B, BamHI; C, ClaI; E, EcoRI; H, HindIII; Nd, NdeI; Ns, NsiI. The region of DNA subcloned into pBluescript KS(+) is shown below the map. The BamHI restriction site is bracketed because it is generated by the J27 primer and is not present in the chromosome of S. pneumoniae type 19C.

FIG. 6

Comparison of the cps loci of S. pneumoniae group 19.

FIG. 7

Putative biosynthetic pathways for 19F, 19A, 19B, and 19C.