Genetic basis for the new pneumococcal serotype, 6C

Abstract

We have recently reported a new pneumococcal serotype (6C), which is closely related to serotype 6A (I. H. Park et al., J. Clin. Microbiol. 45:1225-1233, 2007). To investigate the genetic basis for serotype 6C, we studied the capsule gene loci of 14 6C isolates from three different continents, including one isolated in Alabama 27 years ago. The wciN region of all 6C isolates has a 1,029-bp-long sequence that replaces the 1,222-bp-long sequence of the 6A wciN region. This recombination event has created a new 1,125-bp-long open reading frame which encodes a product that is also homologous to glycosyl transferases. Flanking this introduced gene is 300 bp upstream and 100 bp downstream with only about 90% homology with 6A and which is identical in all 6C isolates. Transfer of the wciN region converts 6A to 6C. Determination of the DNA sequence of the entire capsule gene locus of one 6C isolate showed that the 6C capsule gene locus is almost identical (>98% homologous) to that of 6A except for the wciN region. These findings indicate that the 6C capsule type originated more than 27 years ago by a single recombination event in a 6A locus in which 6A wciN was replaced by a gene of unknown origin.

FIG. 1.

wciN region exchange experiment diagram. In step A, the wchA/wciN_6A/wciO-P region of TIGR6A4 was replaced with cassette 1. Cassette 1 has three parts (central core and two flanking regions), and each part is about 1 kb long. The central core has antibiotic susceptibility genes kanA and rpsL⁺. The two flanking regions were made with wchA and wciO-P regions from the AAU-33 strain. In step B, cassette 1 in TIGR6AX is replaced with cassette 2. Cassette 2 has wciN_6C, wchA, and wciO-P regions from a 6C strain (CHPA388). TIGR6C4 shows the final product that is obtained after cassette 2 is inserted. XbaI and BamHI sites in the PCR primers, which were introduced to simplify genetic manipulations, are shown.

FIG. 2.

Electrophoretic patterns of the PCR products of the wciN region of 6A and 6C isolates. The primers used were 5106 and 3101, which are located in wchA and wciO, respectively. M, DNA ladder. Standard markers with 2,000 and 1,650 bp are indicated on the left. Lanes 1 to 13 contain PCR products of 6C isolates CHPA37 (lane 1), CHPA388 (lane 2), BG2197 (lane 3), BZ17 (lane 4), BZ39 (lane 5), BZ86 (lane 6), BZ650 (lane 7), KK177 (lane 8), CH66 (lane 9), CH158 (lane 10), CH199 (lane 11), MX-67 (lane 12), and ACA-C21 (lane 13). Lanes 14 to 18 contain PCR products of 6A isolates CHPA67 (lane 14), CHPA78 (lane 15), BZ652 (lane 16), KK58 (lane 17), and AAU-33 (lane 18).

FIG. 3.

A. The nucleotide sequence of the wciN_6C ORF is shown along with the nucleotide sequences of the 3′ end of wchA and the 5′ end of wciO. The derived amino acid sequence of the wciN_6C ORF is shown below the nucleotide sequence. Also shown are putative termination sites of wchA and wciN_6C as well as putative initiation sites of wciN_6C and wciO. wciO has two potential initiation sites. B. DNA sequences of wciN_6A and wciN_6C regions (shown in boldface) of a 6A strain (GenBank accession no. CR931638) and a 6C strain (CHPA388). The sequence of the nonhomologous mid-region of wciN (about 900 to 1,110 bases) is not shown. Sites of PCR primers (5106, 3101, 5118, and 3113) are shown. Also shown are potential termination sites of wchA and wciN_6C and potential initiation sites of wciN_6C and wciO. C. Genetic map of the capsule loci surrounding wciN of 6A and 6C isolates. The map shows wchA (hatched), wciN (horizontal bars or black), wciO (checkered), and wciP (wavy lines) genes. The 6A locus has two unexpressed DNA fragments (indicated by arrows) upstream (95 bases long) or downstream (312 bases long) of wciN_6A. An alternative initiation site for wciO is located 32 bases upstream of the initiation site shown (position 2721 for 6A). For 6C isolates, the native DNA (1,222 bases; indicated by horizontal bars) in the wciN_6A locus is replaced with new DNA (1,029 bases; black). The replacement creates a new ORF (named wciN_6C) that has 1,125 bases. Nucleotide position 1 in this figure corresponds to nucleotide position 4902 of the 6A capsule genome sequence (GenBank accession no. CR931638).

FIG. 3.

A. The nucleotide sequence of the wciN_6C ORF is shown along with the nucleotide sequences of the 3′ end of wchA and the 5′ end of wciO. The derived amino acid sequence of the wciN_6C ORF is shown below the nucleotide sequence. Also shown are putative termination sites of wchA and wciN_6C as well as putative initiation sites of wciN_6C and wciO. wciO has two potential initiation sites. B. DNA sequences of wciN_6A and wciN_6C regions (shown in boldface) of a 6A strain (GenBank accession no. CR931638) and a 6C strain (CHPA388). The sequence of the nonhomologous mid-region of wciN (about 900 to 1,110 bases) is not shown. Sites of PCR primers (5106, 3101, 5118, and 3113) are shown. Also shown are potential termination sites of wchA and wciN_6C and potential initiation sites of wciN_6C and wciO. C. Genetic map of the capsule loci surrounding wciN of 6A and 6C isolates. The map shows wchA (hatched), wciN (horizontal bars or black), wciO (checkered), and wciP (wavy lines) genes. The 6A locus has two unexpressed DNA fragments (indicated by arrows) upstream (95 bases long) or downstream (312 bases long) of wciN_6A. An alternative initiation site for wciO is located 32 bases upstream of the initiation site shown (position 2721 for 6A). For 6C isolates, the native DNA (1,222 bases; indicated by horizontal bars) in the wciN_6A locus is replaced with new DNA (1,029 bases; black). The replacement creates a new ORF (named wciN_6C) that has 1,125 bases. Nucleotide position 1 in this figure corresponds to nucleotide position 4902 of the 6A capsule genome sequence (GenBank accession no. CR931638).

FIG. 4.

Capsule locus of a 6A strain (GenBank accession no. CR931638) and a 6C strain (CHPA388). All ORFs involved in capsule synthesis are shown as horizontal arrows, and their direction indicates the transcriptional orientation. For both 6A and 6C loci, the putative transcription initiation sites (bent arrows) and putative termination sites (vertical lines with a solid circle) were identified using fgenesB, BPROM, and FindTerm (Softberry Inc.), available at www.molquest.com. “Transposase” sequences (black boxes, labeled “tnp”) are found at either end of the capsule gene locus.