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Comparative Study
. 2009 Aug 5:10:358.
doi: 10.1186/1471-2164-10-358.

Comparative genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content

Fumito Maruyama  1 , Mitsuhiko Kobata, Ken Kurokawa, Keishin Nishida, Atsuo Sakurai, Kazuhiko Nakano, Ryota Nomura, Shigetada Kawabata, Takashi Ooshima, Kenta Nakai, Masahira Hattori, Shigeyuki Hamada, Ichiro Nakagawa
Affiliations
Comparative Study

Comparative genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content

Fumito Maruyama et al. BMC Genomics. .

Abstract

Background: Streptococcus mutans is the major pathogen of dental caries, and it occasionally causes infective endocarditis. While the pathogenicity of this species is distinct from other human pathogenic streptococci, the species-specific evolution of the genus Streptococcus and its genomic diversity are poorly understood.

Results: We have sequenced the complete genome of S. mutans serotype c strain NN2025, and compared it with the genome of UA159. The NN2025 genome is composed of 2,013,587 bp, and the two strains show highly conserved core-genome. However, comparison of the two S. mutans strains showed a large genomic inversion across the replication axis producing an X-shaped symmetrical DNA dot plot. This phenomenon was also observed between other streptococcal species, indicating that streptococcal genetic rearrangements across the replication axis play an important role in Streptococcus genetic shuffling. We further confirmed the genomic diversity among 95 clinical isolates using long-PCR analysis. Genomic diversity in S. mutans appears to occur frequently between insertion sequence (IS) elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. S. mutans may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats (CRISPRs). In particular, the CRISPR-2 region, which is highly divergent between strains, in NN2025 has long repeated spacer sequences corresponding to the streptococcal phage genome.

Conclusion: These observations suggest that S. mutans strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. In contrast, S. pyogenes tolerates phage infection for acquisition of virulence determinants for niche adaptation.

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Figures

Figure 1
Figure 1
Circular map of S. mutans strain NN2025. The outer circle shows the scale (bp). From outside, rings 1 and 2 show the coding sequence (ORF) by strands (ring 1, clockwise; ring 2, counterclockwise). The predicted ORFs are distinguished by different colors in the BLAST analysis against the database (see Methods)(indicated as "BLAST classification"). Rings 3 and 4 show the ORF by different colors in the COG classification (indicated as "COG classification"). Ring 5 shows the location of transposase ORFs (including fragment), insertion sequence, and CRISPR associated ORFs. Rings 6 and 7 show the transfer RNA and ribosomal RNA genes identified in the genome. Rings 8 and 9 show the G + C content and GC skew, respectively. The red arrowheads indicate the origin of DNA replication (ori) and the putative region of replication terminus (ter).
Figure 2
Figure 2
Local collinear blocks (LCBs) between chromosomal sequences of the two strains of Streptococcus mutans. (a) Representation of the whole 32 local collinear blocks (LCBs) between chromosomal sequences of the two strains of Streptococcus mutans, UA159 and NN2025, was generated by MAUVE software at a minimum weight of 411. The S. mutans UA159 DNA sequence given on the forward strand is the reference against which the sequence of the NN2205 was aligned and compared. LCBs placed under the vertical bars represent the reverse complement of the reference DNA sequence. The 32 connecting lines between genomes identify the locations of each orthologous LCB in the two genomes. Unmatched regions within an LCB indicate the presence of strain-specific sequence. Each sequential block represents homologous backbone DNA sequence without rearrangements. Black horizontal bars indicate rearrangement regions and strain-specific regions analyzed in detail (see additional files 2 and 3). Blue region numbers and bars indicate NN2025-specific regions, pink region numbers and bars indicate UA159-specific regions, and black region numbers and bars indicate variable regions. Distributions of regions 1–25 are analyzed by long-PCR in 97 S. mutans strains including strains NN2025 and UA159 (see additional files 4 and 6). A dot plot to compare genome structure of these strains is also shown in Figure 6. (b) Alignment of the two genomes is generated by artificially correcting for the inversion of NN2025 at the rrn-comX region by the PROmer of MUMmer software and GenomeMatcher software (green bar). Similarity is shown by color code as represented in this figure. Blue region numbers and arrows indicate NN2025-specific regions, pink region numbers and arrows indicate UA159-specific regions, and black region numbers and arrows indicate variable regions.
Figure 3
Figure 3
Putative bacitracin synthesis clusters located in putative conjugative transposon in the different position of S. mutans UA159 and NN2025 genome. Putative bacitracin synthesis clusters in S. mutans UA159 (a) and S. mutans NN2025 (b, c). The ORFs colors indicate the BLAST classification as shown in Figure 1. The BLASTP analysis was carried out across a non-redundant protein database in GenBank. The pink areas indicate the specific regions in each strain. Black dotted lines indicate orthologous genes that are located in identical relative positions or that are located in the inverted chromosomal regions. The whole gene list of these regions for each strain is shown in additional files 2 and 3.
Figure 4
Figure 4
CRISPR regions of S. mutans UA159 and NN2025. CRISPR-1 region (Region 19) in NN2025 is a newly acquired region (a), and CRISPR-2 region (Region 20) is conserved between NN2025 and UA159 (b). The ORF colors indicate the BLAST classification as shown in Figure 1. The BLASTP analysis was carried out across a non-redundant protein database in the GenBank. HP; hypothetical protein. Blue lines indicate the palindromic repeat and spacer sequences. Pink areas indicate the specific regions in each strain. Black dotted lines indicate orthologous genes that are located in identical relative positions or that are located in the inverted chromosomal regions. The whole gene list of this region for each strain is shown in additional file 2 and additional file 3.
Figure 5
Figure 5
Number of spacer sequences and its similarities against known phage genome of CRISPR in S. mutans strains. (a) CRISPR-2 regions, widely distributed among S. mutans, in strain NN2025, UA159 and six selected strains (MT8148, LJ29, SA13, SA15, NN2207 and NN2138; see additional file 5 and 6). Blue rectangles indicate the direct repeat (DR). Orange triangles indicate spacer regions without homology to the phage M102 genome and red triangles are spacer regions corresponding to the sequence of the phage. Numbers of the spacer regions are determined by CRISPRfinder (see Methods for details). (b) Location of the spacer sequences corresponding to the M102 phage sequence (red arrowheads). The number of each arrowhead indicates the spacer number of each strain (additional file 9). The sequences of leader, spacer and repeat of CRISPR loci in the strain NN2025 and UA159 are listed in additional files 8, 9 and 10.
Figure 6
Figure 6
Genome comparison of two S. mutans strains, or each S. mutans strain against 13 S. pyogenes strains based on the chromosomal organization of strain NN2025 or UA159. Dot plots of S. mutans NN2025 vs 13 S. pyogenes strains and of S. mutans UA159 vs the same set of S. pyogenes strains are presented. These were generated by PROmer of MUMmer software and were visualized with the GenomeMatcher software (see Methods).
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