Comparative Genomics of the Mucoid and Nonmucoid Strains of Streptococcus pyogenes, Isolated from the Same Patient with Streptococcal Meningitis.

Mucoid (MTB313) and nonmucoid (MTB314) strains of group A streptococcus emm type 1 were simultaneously isolated from a single patient suffering from streptococcal meningitis. Whole-genome sequencing revealed that MTB313 carried a nucleotide substitution within rocA, which generated an amber termination codon.

GENOME ANNOUNCEMENT

We identified mucoid (MTB313) and nonmucoid (MTB314) strains of group A streptococcus (GAS) emm type 1 at the same time on the same blood agar plate with application of a cerebrospinal fluid specimen of a 70-year-old Japanese woman suffering from streptococcal meningitis. We performed comparative genomics based on whole-genome sequencing of both GAS strains to comprehensively identify the naturally occurring nucleotide substitutions.

Genomic DNA samples of MTB313 and MTB314 were subjected to shotgun pyrosequencing by using a 454 GS Junior titanium system (454 Life Sciences, Branford, CT). All operations were carried out according to the protocols provided by the manufacturer. A putative annotation of the genome sequence was made using the Microbial Genome Annotation Pipeline (MiGAP; an auto annotation pipeline of the DDBJ, http://www.migap.org/). The sequences from MTB313 and MTB314 were analyzed for the detection of local and structural variations compared to those of MGAS5005 (a virulent type strain of emm1 GAS) (1) by using the GS Reference Mapper application (analysis software included with the 454 system).

The genome sizes of MTB313 and MTB314 (1,745,332 bp and 1,744,827 bp, respectively) were smaller than the previously completed genomes (1.84 Mbp in average size) of four other emm1 GAS strains (SF370 [2], MGAS5005 [1], A20 [3], and 476 [4], GenBank accession numbers AE004092, CP000017, CP003901, and AP012491, respectively) because of the lack of phage genomes. The comparative genomic analysis between MGAS5005 and MTB313 or MTB314 revealed that MTB313 carried a nucleotide substitution within rocA (G464A), which generated an amber chain-termination codon (UAG) at the 155th amino acid position (accession no. AB737848). In contrast, the same nucleotide sequences of rocA were detected in both MTB314 (accession no. AB737849) and MGAS5005 (gene ID 3571595).

covR-covS (control of virulence; also called csrR-csrS, capsule synthesis regulator), a two-component regulatory system (covR is the transcriptional regulator and covS is the sensor kinase), influences the expression of chromosomal genes of GAS (5–7). covR phosphorylated by covS is believed to negatively regulate the expression of several virulence genes of GAS, including hasA (hyaluronic acid capsule) (7). rocA has been reported to be a positive regulator of covR (8). Recently, Lynskey et al. (9) demonstrated that the rocA of serotype M18 GAS exhibited structural homology to the catalytic domain of the Escherichia coli osmoregulator envZ. Although rocA was shown to positively enhance covR transcription, quantitative proteomics revealed that rocA was a metabolic regulator with activity beyond the covR-covS regulon. A naturally occurring truncation of rocA contributed to the hyperencapsulation phenotype, led to prolonged nasopharyngeal carriage of GAS in mice, and promoted bacterial airborne transmission (9). These results suggest that MTB313 is a highly encapsulated phenotype associated with hasA expression through the suppression of covR expression by the depression of rocA.

Nucleotide sequence accession numbers.

The complete whole-genome sequences of MTB313 and MTB314 were registered to the DDBJ/ENA/GenBank database under the genome project data accession numbers AP014572 and AP014585, respectively.