Analysis of the 5' portion of the type 19A capsule locus identifies two classes of cpsC, cpsD, and cpsE genes in Streptococcus pneumoniae

Abstract

Analysis of the sequence data obtained from the 5' portion of the Streptococcus pneumoniae type 19A capsular polysaccharide biosynthesis locus (cps19a) revealed that the first seven genes are homologous to the first seven genes in the type 19F (cps19f) locus. The former genes were designated cps19aA to -G and were 70 to 90% identical to their cps19f counterparts. Southern hybridization analysis of the cps loci from various S. pneumoniae serotypes with probes specific for the cps19aC, cps19aD, and cps19aE genes indicated a hybridization pattern complementary to that previously reported for cps19fC, cps19fD, and cps19fE. That is, all serotypes tested contained high-stringency homologues of either the cps19aC to -E genes or the cps19fC to -E genes, but not both. On this basis S. pneumoniae cps loci can be divided into two distinct classes. Long-range PCR was used to amplify the cps regions between cpsB and aliA from a variety of pneumococcal serotypes. Direct sequencing of the 5' end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes of cpsC. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.

FIG. 1

The dexB-to-aliA region of the S. pneumoniae type 19F chromosome. The regions conserved among all pneumococcal serotypes are indicated in black, the cps19G and -H genes are shaded grey to indicate a higher degree of identity between cps19f and cps19a in this region. (A) Arrangement of the cps19a locus between the CPS5′ and J22 primers. CPS5′ (5′-TGATGTTCAAGGTATAGGTGTTAATCA) is homologous to nucleotides 146 to 169 of the cps19f sequence (17), immediately preceding the cps19fA gene, and J22 (5′-AATTGAATTCTTTTATAGATTTAACACAAG) is complementary to nucleotides 6743 to 6772 of the cps19f sequence, in the 5′ region of cps19fH. (B) The portion of the cps locus between cpsB and aliA from various pneumococcal serotypes was amplified by using the two primers J39 and J36. The positions of the two primers J39 (5′-TAGTTCATGTAGTTGCAAGTGACATGCACAA, homologous to nucleotides 2190 to 2220 of the cps19f sequence, in the 3′ region of cps19fB) and J36 (5′-CAATAATGTCACGCCCGCAAGGGCAAGT, complementary to nucleotides 16463 to 16490 of the cps19f sequence, located just after the start of aliA) are indicated with half arrows. Abbreviations for restriction sites are as follows: B, BamHI; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; Nc, NcoI; Nr, NruI; P, PstI; S, SphI.

FIG. 2

Diagrammatic representation of the similarity of the cps19aAB genes to the cps19fAB genes. There are several possible recombination points in this region of the locus. Increasing similarity is represented by progressively darker shades of grey, and the percent identity is shown under the individual shaded regions. The arrows indicate the points of divergence, and the number below each arrow corresponds to the nucleotide number of the cps19a sequence.

FIG. 3

Long-range PCR products. PCR products, not digested (A) or digested with ClaI (B), were electrophoresed on a 1% agarose gel in the presence of ethidium bromide. ClaI-restricted PCR product was subjected to Southern hybridization analysis using DIG-labelled probes specific for cps19fC (C) or cps19fL (D). The probes specific for cps19fC and cps19fL correspond to nucleotides 2380 to 2998 and 11539 to 12493 of the cps19f sequence (17). The molecular size standards are shown on the right-hand side of the figure and correspond to HindIII-digested λ phage DNA.

FIG. 4

Comparison of class I and class II cps sequences. The first 500 nucleotides of the sequence obtained are shown (100 nucleotides per line). Dots indicate nucleotides which are identical to that for cps19f. The stop codon of the cpsB gene is indicated with an asterisk. The start codon of cpsC is underlined. (T) denotes an extra nucleotide, and - denotes the absence of a nucleotide in the cps14 DNA sequence. The vertical arrows indicate the region where the crossover between class I and class II sequences has occurred in cps4.

FIG. 5

Phylogenetic tree of cpsC sequences. The cpsC gene sequences were aligned by using CLUSTAL W (24), and the phylogenetic tree was generated by using MEGA (15), as described in the text. The numbers associated with the branches are bootstrapping confidence limits, resulting from 500 replications, as defined in MEGA. The scale represents the number of nucleotide substitutions per site.