Abstract
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.
ACKNOWLEDGMENTS
This work was supported by a grant under the category “Research Projects for Emerging and Re-emerging Infectious Diseases” from the Ministry of Health, Labor and Welfare of Japan (grant H22-013). This work was also supported by Grants-in-Aid for Young Scientists (B), Challenging Exploratory Research, and Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS) (grants 22790412, 25670469, and 25460545, respectively).
Footnotes
Citation Yoshida H, Ishigaki Y, Takizawa A, Moro K, Kishi Y, Takahashi T, Matsui H. 2015. Comparative genomics of the mucoid and nonmucoid strains of Streptococcus pyogenes, isolated from the same patient with streptococcal meningitis. Genome Announc 3(2):e00221-15. doi:10.1128/genomeA.00221-15.
REFERENCES
- 1.Sumby P, Porcella SF, Madrigal AG, Barbian KD, Virtaneva K, Ricklefs SM, Sturdevant DE, Graham MR, Vuopio-Varkila J, Hoe NP, Musser JM. 2005. Evolutionary origin and emergence of a highly successful clone of serotype M1 group A Streptococcus involved multiple horizontal gene transfer events. J Infect Dis 192:771–782. doi: 10.1086/432514. [DOI] [PubMed] [Google Scholar]
- 2.Ferretti JJ, McShan WM, Ajdic D, Savic DJ, Savic G, Lyon K, Primeaux C, Sezate S, Suvorov AN, Kenton S, Lai HS, Lin SP, Qian Y, Jia HG, Najar FZ, Ren Q, Zhu H, Song L, White J, Yuan X, Clifton SW, Roe BA, McLaughlin R. 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci U S A 98:4658–4663. doi: 10.1073/pnas.071559398. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Zheng PX, Chung KT, Chiang-Ni C, Wang SY, Tsai PJ, Chuang WJ, Lin YS, Liu CC, Wu JJ. 2013. Complete genome sequence of emm1 Streptococcus pyogenes A20, a strain with an intact two-component system, CovRS, isolated from a patient with necrotizing fasciitis. Genome Announc 1(1):e00149-12. doi: 10.1128/genomeA.00149-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Miyoshi-Akiyama T, Watanabe S, Kirikae T. 2012. Complete genome sequence of Streptococcus pyogenes M1 476, isolated from a patient with streptococcal toxic shock syndrome. J Bacteriol 194:5466. doi: 10.1128/JB.01265-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bisno AL, Brito MO, Collins CM. 2003. Molecular basis of group A streptococcal virulence. Lancet Infect Dis 3:191–200. doi: 10.1016/S1473-3099(03)00576-0. [DOI] [PubMed] [Google Scholar]
- 6.Graham MR, Smoot LM, Migliaccio CA, Virtaneva K, Sturdevant DE, Porcella SF, Federle MJ, Adams GJ, Scott JR, Musser JM. 2002. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc Natl Acad Sci U S A 99:13855–13860. doi: 10.1073/pnas.202353699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Cole JN, Barnett TC, Nizet V, Walker MJ. 2011. Molecular insight into invasive group A streptococcal disease. Nat Rev Microbiol 9:724–736. doi: 10.1038/nrmicro2648. [DOI] [PubMed] [Google Scholar]
- 8.Biswas I, Scott JR. 2003. Identification of rocA, a positive regulator of covR expression in the group A streptococcus. J Bacteriol 185:3081–3090. doi: 10.1128/JB.185.10.3081-3090.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Lynskey NN, Goulding D, Gierula M, Turner CE, Dougan G, Edwards RJ, Sriskandan S. 2013. RocA truncation underpins hyper-encapsulation, carriage longevity and transmissibility of serotype M18 group A streptococci. PLoS Pathog 9:e1003842. doi: 10.1371/journal.ppat.1003842. [DOI] [PMC free article] [PubMed] [Google Scholar]