BACKGROUND
the genomic heterogeneity within a bacterial species reflects its lifestyle, the niche it occupies, and its exposure to mobile elements, such as bacteriophages and plasmids  <cit> .
even though organisms belonging to the same genus/species have a common gene set , individual organisms differ  in ways representative of the physiological and virulence properties of an organism  <cit> .
although not all genetic differences between strains are important for niche adaptation of the bacteria, strain-specific genes are thought to be responsible for the survival of an organism in its chosen niche.
this variation can be due to genetic noise   <cit> , gene loss  <cit> , gene duplication  <cit>  or modification of some of the existing genes  <cit> .
acquisition of new genes by lateral gene transfer is a predominant force in bacterial evolution.
laterally acquired genes provide a readily available novel pool of genes for developing physiological properties that are helpful for exploiting a new niche.
a recent study suggested that the total known genome content  of all contemporary streptococcus agalactiae strains will increases as hundreds of genomes are sequenced  <cit> .
although s. pyogenes belongs to the same genus, it has a smaller pan-genome and greater level of recombination in its core genome  <cit> .
these organisms provide a good model for identifying the causes of genome plasticity in human pathogens.
it has long been recognized that serological, genetic, and biochemical variations exist within the species s. mutans  <cit> .
s. mutans has been classified into four serotypes  based on the chemical composition of its cell surface rhamnose-glucose polymers  <cit> .
we previously developed a multilocus sequence typing  method using eight house-keeping genes.
ninety-two sequence types  were identified from  <dig> clinical isolates, indicating that s. mutans is a diverse population  <cit> .
in the mlst analysis, serotype c strains were widely distributed in the dendrogram, while serotype e, f, and k strains were differentiated into clonal complexes.
this suggests that serotype c, the dominant serotype among s. mutans clinical isolates , is the ancestral phenotype of this organism and that serotype e and f strains have evolved strain-specific genes.
although differences in modification of cell surface polymers reflect evolutionary trends, differences in cariogenicity have not been observed, and the relationship between serotype and clinical condition remains unclear.
studies of individual s. mutans genes have revealed sequence variations, resulting in altered function of the encoded proteins  <cit> .
for example, variation has been demonstrated in the occurrence of plasmids  <cit> , and in mutacin operons  <cit> , serotype antigens  <cit> , competence  <cit> , and the msm, bgl, cel, and gftbc loci  <cit> .
waterhouse and russell recently showed a mosaic of loci such as the msm, gbl, cel, and gftbc, which they called "dispensable genes," distributed among s. mutans strains  <cit> .
they also demonstrated that 20% of the s. mutans ua <dig> open reading frames  were absent from one or more of the nine test strains, and dispensable orf blocks  were identified by microarray analysis based on the ua <dig> genome  <cit> .
given the wide distribution and diversities of genotypes and genetic loci in s. mutans, it seems likely that other strains of s. mutans have both unique and common genetic loci not present on the ua <dig> genome  <cit> .
this is useful for charting s. mutans evolutionary history.
however, these analyses are based on only one genome, s. mutans ua <dig> even though extensive genomic variation between s. mutans strains has been predicted  <cit> .
genome sequence data are now available for numerous species of bacteria and comparative evolutionary approaches show positive selection pressure and lateral gene transfer in the evolution of many bacterial species.
these analyses have been performed for pathogenic bacteria such as helicobacter pylori  <cit> , mycobacterium species  <cit> , chlamydia species  <cit> , escherichia coli  <cit> , and salmonella species  <cit> .
the pathogenic streptococcus species include important human and agricultural pathogens  <cit> .
more than  <dig> whole genomes of streptococcus sp.
belonging to nine different species including s. pyogenes, s. pneumoniae, s. agalactiae, s. thermophilus, s. suis, s. sanguinis, s. gordonii, s. equi, and s. mutans are publicly available.
these organisms colonize diverse habitats including tooth, oral mucosal, pharyngeal, respiratory, intestinal, and urinogenital surfaces.
these species have acquired various genes for a specific niche mainly by lateral gene transfer.
for example, s. pyogenes acquires or tolerates bacteriophages that are important for new virulence determinants and that induce genomic rearrangement  <cit> .
s. agalactiae, the main cause of neonatal infection in humans, also tolerates bacteriophages  <cit> .
some of these organisms gain counterattack systems such as restriction modification or clustered regularly interspaced short palindromic repeats   <cit> .
multiple sequences of genomes from closely related species that inhabit different niches lead not only to an understanding of the pattern of gene movement but also to insights into the role of species-specific genes, and genome plasticity.
in this context, we determined the whole genome sequence of an s. mutans serotype c strain nn <dig> isolated from japan in  <dig>  and we compared the genome sequence, genome structure, and gene variation with the genomes of serotype c ua <dig> strain isolated in  <dig> from the united states, and with  <dig> clinical isolates from japan and finland, and other closely related streptococcal genera to provide useful information about the evolutionary events associated with s. mutans strains and streptococcus sp., and to provide new insights into streptococcal species-specific survival strategies.
RESULTS
general features of the strain nn2025
the genome of nn <dig> is a single, circular chromosome of  <dig> , <dig> bp .
the genome is almost identical in size to that of ua <dig> but is  <dig> kb shorter .
neither nn <dig> nor ua <dig> contains plasmids or prophages.
the genome start point for nn <dig> was assigned to the putative location of the dnaa gene, similar to s. mutans ua <dig> and other genomic sequences  <cit> .
the average gc content is  <dig> %, which is similar to that of ua <dig>  there are five rrna operons containing 5s, 16s, and 23s rrna genes.
the  <dig> predicted trna genes encode all  <dig> amino acids.
most trna genes are clustered near the rrna operons,  <dig> of  <dig> of these genes are less than  <dig> kb from an rrna operon , as in other streptococcal strains  <cit> .
the genome contains  <dig>  predicted proteins with an average size of  <dig> bp that cover  <dig> % of the whole sequence, which is similar to other streptococci.
both the sequence and the annotation have been deposited in the dna data bank of japan  .
genes and regions conserved between s. mutans nn <dig> and ua <dig> genomes
nn <dig> contains  <dig>  orfs, of which  <dig>   are predicted by reciprocal blast search analysis  to be common to ua <dig>  s. mutans strains nn <dig> and ua <dig> are classified into the same serological group c, and show the same biochemical properties , adhesive properties to glass surfaces, and cariogenic properties in rat infection models .
waterhouse et al.
reported that 80% of the s. mutans orfs are conserved among ten strains with different serotypes by microarray hybridization based on the ua <dig> genome, suggesting that 80% of orfs are conserved within s. mutans strains  <cit> .
this indicates that the core genome of s. mutans is more stable than that of other streptococcus species, where the core genome represents only about 60% of the genome  <cit> .
most vegetative growth genes are highly conserved.
carbohydrate metabolism is a key survival strategy for s. mutans  <cit> , and genes for transport and metabolism of various sugars, and the fermentation of carbohydrates are completely conserved between ua <dig> and nn <dig>  s. mutans is suggested to be capable of metabolizing a wider variety of carbohydrates than many other gram-positive organisms that have been sequenced.
s. mutans resides in the oral cavity, and this varied sugar metabolism is an important survival strategy for this organism.
s. mutans is predicted to possess at least five sugar abc transport systems and at least  <dig> sugar pts systems, and can use at least  <dig> sugars for glycolysis.
nine ptss were confirmed to be transcribed in the presence of  <dig> different sugars  <cit> .
in addition, orfs predicted as virulence factors of s. mutans including adhesins, glucan-producing and binding exoenzymes, were conserved.
adhesins of s. mutans are also important for protecting the bacterium against possible host defenses and for maintaining its ecological niche in the oral cavity.
comparative genome analysis revealed that the major s. mutans surface receptors spap   and wall-associated antigen a   are completely conserved.
an uncharacterized wall-associated protein, wape , and a dextranase precursor   <cit>  were determined from blast analysis to be s. mutans-specific genes within the sequenced streptococcus species , suggesting that these might be important for colonization.
strain-specific genes and regions in nn <dig> and ua159
strain-specific regions were determined using mauve software, where the locally collinear blocks  represent the landmarks  in chromosomes  <cit> .
an lcb is defined as a collinear  set of multi-mums .
the weight  of an lcb serves as a measure of confidence that it is a true homologous region instead of a random match.
therefore, the orfs or sequences between the lcbs and any regions with low similarity  are identified as strain specific regions.
the nn <dig> genome contains eight strain-specific regions  containing  <dig> orfs that were unique in comparison with ua <dig>  other nn2025-specific orfs are randomly inserted into the nn <dig> genome, and these orfs are classified into transposase fragments, ribosomal proteins, hypothetical proteins and disrupted gene fragments.
only two orfs  are predicted to be functional genes from an unknown foreign source.
in contrast, the ua <dig> genome contains nine strain-specific regions  containing  <dig> unique orfs; another  <dig> ua159-specific orfs are distributed in the ua <dig> genome .
of the  <dig> regions that are unique between the nn <dig> and ua <dig> genomes, eight regions  exist in both strains, but the contents of the orfs are highly diverse.
these regions are designated "variable regions".
region  <dig> contains genes of the purine nucleotide biosynthesis pathway  and is thought to be important for bacterial growth, but genetic variation is found within this region.
region  <dig> in nn <dig> includes two transposase fragments, five hypothetical proteins, methyltransferase , gapc , transcriptional regulator gene , and luxs .
luxs plays an important role in the production of autoinducer- <dig>  in many bacterial species, and 30% of the genes of s. mutans are affected by luxs  <cit> .
region  <dig> in nn <dig> includes hypothetical proteins and abc transporters; however, the contents of this region in ua <dig> are quite different and include hypothetical proteins that appear to have been horizontally transferred from other species.
region  <dig> in ua <dig> and regions  <dig> and  <dig> in nn <dig> include putative restriction/modification  system genes.
the r/m system is composed of genes that encode a restriction enzyme and a modification methylase, and they defend against invaders by attacking non-self dna, or by killing cells that have eliminated them  <cit> .
waterhouse et al.
reported that 20% of ua <dig> orfs did not hybridize with one or more of the nine s. mutans genomes in a ua159-based microarray analysis  <cit> .
they also showed that  <dig> genomic blocks involving more than one orf were found within the test strains.
in the case of ua <dig>  comparison of the regions identified in this study by pcr with previously reported 'genomic blocks'  <cit>  and 'genomic island'  <cit>  showed a good correlation, confirming the accuracy of both our results and the earlier reports, even though these were derived from different experimental approaches .
the slight differences between the results, therefore, may have resulted from the characteristics of each method, because pcr primers target short sequences with high similarity, while microarray probes overall similarity to detect the target sequence.
this second s. mutans genome sequence  identifies new strain-specific content that could not be identified from only one genome.
based on their correspondence with the 'genomic islands', region  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> appear to be horizontally transferred.
the gene order of region  <dig> in ua <dig> is different from that in nn <dig>  which may suggest the existence of an unknown mechanism to reorder the genomic island .
these regions may reflect the phenotypic and biochemical differences of s. mutans strains.
although this comparative study was based on the ua <dig> genome, unique orfs or regions in the genomes of other strains were not completely analyzed.
therefore, we also determined the differences of these regions among  <dig> test strains using long-pcr against  <dig> regions  and clustering analysis .
however, no characteristic differences in serotype specificity, geographical distribution, or the ability for sucrose-dependent adhesion to a glass surface were found .
we have previously demonstrated that  <dig> clinical isolates from japan and finland were resolved into  <dig> sts by mlst  <cit> ,  <dig> of which were identified only once.
we could not determine any distribution or lineage differences between japanese isolates and finnish isolates.
we, therefore, conclude that the s. mutans population diversity is not caused by variation in gene content but probably with genetic recombination.
in some s. mutans strains, similar insertion/deletion events appear in the genomes of strains with very different origins based on pcr determination of  <dig> loci  <cit> .
in our analysis,  <dig> characteristic regions in the whole genome comparison were diverse, although they were isolated from only two geographical areas.
this indicates that insertions/deletions in these specific regions are acquired by unknown mechanisms, probably by multiple acquisition events and the spread of an ancestral acquisition through the species by recombination from other bacterial strains.
various genes appear to be moved by lateral gene transfer in s. mutans, resulting in strain-specific regions.
nn <dig> possesses  <dig> transposase orfs, and  <dig> is elements in its genome, whereas ua <dig> possesses  <dig> transposon-like elements   <cit> .
these mobile genetic elements are located near the strain-specific regions.
thus, either laterally acquired genes account for the emergence of strain-specific orfs or the comparator strain has lost these orfs.
tnsmu <dig> was found to be a conjugative transfer element in ua <dig>  <cit> .
this region in ua <dig> is large , involving smu.191- <dig>  one helicase gene , two transposase-like genes , and several hypothetical proteins.
this region in nn <dig> contains only seven short orfs, including two transposase fragments .
among  <dig> test strains,  <dig> isolates, including all the finnish isolates, show the same pattern as nn <dig> , and  <dig> %  of strains do not have this tnsmu <dig> region.
some isolates in japan possess the same tnsmu <dig> as found in ua <dig> in a  genomic island, along with  <dig> orfs predicted to be involved in biosynthesis and secretion of the antimicrobial peptide, bacitracin .
however, tnsmu <dig> has a high level of variation, including degenerated orfs, and is not found in all s. mutans strains.
in previous work, tnsmu <dig> was found only in ua <dig> and gs <dig>  <cit> .
the variation in the tnsmu <dig> region has already been reported in strains ingritt and lml <dig>  <cit> , whose these sequences are almost identical to that of nn <dig> .
therefore, we constructed various pcr primer sets based on the sequence of ua <dig> and determined whether tnsmu <dig> was found in our test strains.
in our experiments, 85%  of clinical strains did not contain this region, and regional bias was not observed .
these observations indicate that the existence of tnsmu <dig> and tnsmu <dig> may not be common or necessary for the virulence of s. mutans strains in rats.
putative bacitracin synthesis regions
the bacitracin synthesis gene cluster in tnsmu <dig> did not exist in most of the clinical strains, and two other bacitracin or polypeptide antibiotic synthesis gene clusters were found in nn <dig> .
bacitracin is a mixture of related cyclic polypeptide antibiotics produced by some strains of bacillus licheniformis and b. subtilis.
its primary mode of action is to bind to undecaprenyl pyrophosphate  in the presence of a bound divalent metal cation, sequestering upp and preventing its interaction with phosphatase  <cit> .
this in turn prevents the return of the undecaprenyl monophosphate lipid carrier, inhibiting peptidoglycan and teichoic acid biosynthesis  <cit> .
region  <dig> is a large  transposon-like region containing bacitracin synthesis genes , peptide synthetase , abc transporters , and an is <dig> fragment  .
smu2025_ <dig> and smu2025_ <dig> are multiple-domain proteins similar to nonribosomal peptide synthetases such as gramicidin or bacitracin, but the sequence identity is lower  than that of the known peptide synthetases .
region  <dig>  includes a nonribosomal peptide synthetase , a putative polyketide synthase , and a putative permease ; these genes are related to the synthesis of bacitracin or other peptide antibiotics .
s. mutans strains have bacitracin resistance  <cit> .
the existence of bacitracin resistant genes  in this organism may reflect bacitracin production for protection.
regions  <dig> and  <dig> are distributed among  <dig> %  and  <dig> % , respectively .
interestingly, all of the s. mutans strains used in this study possess at least one of these bacitracin or peptide antibiotic synthetases genes such as mutacins.
in fact, region  <dig> in nn <dig> includes mutacin iii  production genes   <cit> .
smunn2025_ <dig> and _ <dig> are similar to the scnm and scnt of s. pyogenes, respectively, but these two genes were not found and an apparent fragmentation of the polyketide genes has been shown in ua <dig>  <cit> .
although bacitracin synthesis associated with tnsmu <dig> should be experimentally confirmed, nn <dig> has a bactericidal effect , indicating that the bacitracin production and mutiii cluster might be active in this strain, and that s. mutans has acquired peptide antibiotic synthetase genes to exclude other bacteria, perhaps for niche acquisition.
crispr associated regions
numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats , composed of 25– <dig> bp repeats separated by unique sequence spacers of similar length  <cit> .
it is well known that bacteria have a plethora of mechanisms to counterattack a diverse phage population  <cit> .
consequently, many bacterial species have developed a variety of natural defense mechanisms that target diverse steps of the phage life cycle, notably blocking adsorption, preventing dna injection, restricting the incoming dna, and abortive infection systems.
in the genus streptococcus, three distinct crispr loci have been identified within the s. thermophilus chromosome; namely, crispr- <dig>  crispr- <dig> and crispr- <dig>  <cit> .
crispr- <dig> and crispr- <dig> loci have been experimentally demonstrated to integrate novel spacers in response to bacteriophage  <cit> .
comparative analysis of crispr- <dig> sequences between various s. thermophilus strains has revealed polymorphisms  <cit> .
in addition, it was recently reported that crispr provides acquired resistance against viruses in prokaryotes, notably in s. thermophilus  <cit> .
phage tolerance capacity is known to be diverse  <cit> .
for example, phage integration in s. pyogenes is an important source for new virulence factors as well as for the genomic rearrangement of the prophage regions  <cit> .
natural phage resistance mechanisms in s. mutans have not previously described, because phage donor and acceptor strains are quite rare in s. mutans.
based on in silico and in vivo analyses, several hypotheses have been put forward proposing roles for crispr and cas genes, including providing immunity against foreign genetic elements via a mechanism based on rna interference  <cit> .
strain nn <dig> possesses two characteristic crispr regions, regions  <dig> and  <dig> .
region  <dig> contains nine genes highly similar to the cas and cse genes  in lactobacillus casei  .
the gene order of this crispr region is similar to the "ecoli" subtype  <cit> , or to the lcas <dig> of the ldbu <dig> family recently named by horvath  <cit> , indicating that region is unique for s. mutans.
it has been designated crispr- <dig> .
the crispr- <dig> locus was present in 30/ <dig> clinical isolates .
no variation of the length of repeat-spacer regions is found in these strains, indicating that this region might have been acquired in recent years or might be degenerate.
the crispr repeat, direct repeat , sequence length is  <dig> bp , and  <dig> spacer sequences are found adjacent to cse <dig>  in nn <dig>  of these  <dig> spacer sequences, six are identical to the sequence of the recently identified s. mutans phage m <dig>   <cit> .
in contrast, another crispr region  is found in both ua <dig> and nn <dig>  and has been designated as crispr- <dig>  in s. mutans.
this crispr- <dig> locus consists of four cas and csn genes  and two hypothetical proteins , and the gene order of the crispr- <dig> locus is almost completely conserved among s. mutans strains .
though the crispr subtype  in nn <dig> is the same as in ua <dig>  with the gene order, the lengths of the repeat sequences are quite different not only in these two strains but also among clinical isolates, ranging from  <dig> to  <dig> kb .
to determine the spacer variability in the clinical isolates, the crispr- <dig> loci of an additional six strains were sequenced for comparison .
strain nn <dig> possesses a  <dig> bp dr sequence , typical of the length of sthe <dig> family, and  <dig> spacer sequences were found just downstream of csn <dig>  <cit> .
this is the second example of a crispr locus with more than  <dig> repeats found in firmicutes and it contains the highest number of repeats found in the genus streptococcus.
the five sequenced strains and ua <dig> all have the same dr sequence as nn <dig>  and only strain mt <dig> has another  <dig> bp dr sequence  with the lowest repeat number in any tested strains.
this is the first example of a repeat length other than from  <dig> bp in this sthe <dig> family.
however, the crispr- <dig> locus gene order was completely conserved among strains.
the number of the spacers is quite divergent among strains, ranging from  <dig> to  <dig> .
we next determined the sequence similarity of spacer sequence with s. mutans phage m <dig> .
of the  <dig> spacers of nn <dig>   <dig>  are highly similar to the sequence of s. mutans phage m <dig>   <cit> , as are 20–30% of spacers among the sequenced strains  with the exception of strain mt <dig>  interestingly, strain lj <dig> possesses two spacers similar to m <dig>  and two spacers similar to s. thermophilus phages sfi <dig> and  <dig>  indicating that s. mutans strains might have been attacked by these streptococcal phages, and protected by the crispr- <dig> locus.
the biological significance of crispr against phage infection has recently been reported in s. thermophilus.
the presence of a crispr spacer identical to a phage sequence adjacent to cas provides resistance against phages containing that particular sequence.
limited information is available regarding the ecological role of bacteriophages in the oral cavity, although bacteriophages have been isolated from human saliva or dental plaque  <cit> .
only  <dig> lytic bacteriophages were isolated from more than  <dig> plaque samples tested for lytic activity against indicator strains of s. mutans and s. sobrinus  <cit> .
therefore, the absence in both strain ua <dig> and nn <dig> genomes of prophage or their remnants was not surprising.
the long spacer sequences found in the crispr- <dig> and - <dig> loci in nn <dig> , and their sequence similarity to the lytic phage, suggest that s. mutans might resist phage infection via crispr.
this observation is in contrast with the phage-permissive s. pyogenes, because s. pyogenes possesses two to eight prophage regions within its genome , and the number of crispr-associated direct repeats and spacers is less than that of other streptococcal species .
the average number of prophages in s. pyogenes that lack the cas genes found in s. mutans is six , while that in the other eight s. pyogenes strains carry the same cas gene set is  <dig>  .
the number of prophages in an s. pyogenes genome may be affected by the crispr gene set present, and further examination is needed, especially considering that the many sequenced genomes of streptococci carrying prophages have rarely demonstrated natural transformation  <cit> .
therefore, in the genus streptococcus, specific species may have evolved to function in a particular way to acquire foreign genes via natural transformation or bacteriophages, and the acquisition of new foreign genes via phage infection may not be favorable for the lifestyles of s. mutans in the oral cavity.
elucidation of the mechanism by which s. mutans acquires new genes will be interesting to clarify further the species-specific evolutionary strategies in the streptococci.
genomic analysis of the rearrangement in the ua <dig> and nn <dig> genomes
the physical structure of the bacterial genome is highly conserved during evolution in escherichia coli  <cit> .
however, x-shaped chromosomal inversion has been found between pyrococcus horikoshii and p. abyssi  <cit> , and between chlamydia pneumoniae and c. trachomatis  <cit> .
interestingly, a typical x-shaped chromosomal inversion is found in s. pyogenes  <cit> .
such rearrangements leading to genome plasticity in these bacteria might reflect or contribute to evolutionary processes in the creation of new pathogens.
the recombination sites near the ori region in nn <dig> are found within the 16s rrna, trna-ala, 23s rrna, trna regions, and the comx <dig> homologue in both regions .
the genome of s. mutans nn <dig> shows that the rearrangement breakpoint is located  <dig> bp downstream from the 23s rrna, and this rearrangement breakpoint is conserved within the other s. mutans strains isolated in taiwan in which huang et al.
firstly observed chromosomal inversion in 25/ <dig> strains  <cit> .
these observations indicate that the chromosomal recombination may frequently occur in this genomic site because isolates from japan, finland and taiwan shows the same inversion site though the frequency differs between our strains  and those reported by huang et al.
, and might occur by reca-dependent recombination between two ribosomal operons  <cit> .
in gram-negative salmonella typhimurium, inversion between large inverted repeats  separated by large intervals  has been shown to be reca- and recb-dependent  <cit> .
in addition, chromosomal inversion between the ribosomal operons was reported between s. typhimurium and s. paratyphi a  <cit> .
our sequencing analysis showed that long repeated sequences across the replication axis can also induce large-scale chromosomal rearrangements within a species of gram-positive bacteria.
although the role of homologous recombination in genomic rearrangements across the replication axis has not been elucidated, tillier and collins proposed an alternative model for the observed pattern of rearrangement  <cit> .
gene translocation across the replication axis may result in close physical proximity during the process of genome replication because homologous recombination equidistant from the ori region occurs between two replication forks and single- or double-stranded dna breaks  <cit> .
in fact, two rrn-comx regions were found equidistant from the ori region in the s. mutans genomes; thus, this model is in good agreement with the genomic rearrangement mechanism of s. mutans and s. pyogenes.
this sequence specificity may affect site-specific recombination during homologous recombination.
alignments and dot plots of the genomes of s. mutans nn <dig> and ua <dig> revealed extensive genomic rearrangements .
the origin of dna replication   <cit>  and dif-like termination sequence   <cit>  were conserved.
both alignment and dot plot analyses using mauve and mummer software revealed x-shaped chromosomal inversions that were symmetrical across the replication axis  <cit> .
the locations of the genes around the ori region  were almost completely conserved between nn <dig> and ua <dig>  and the other homologous genes were translocated to an inverted position on the chromosome.
these chromosomal segments are symmetrical except for small gaps encoding the genes of strain-specific regions .
as a consequence, the inversion in nn <dig> relative to ua <dig> did not change gene orientation relative to the replication axis and produced an x-shaped dna dot plot  <cit> .
for s. pyogenes manfredo and s. pyogenes ssi- <dig>  there is a large central inversion , which probably resulted from reciprocal recombination between rrn-comx regions that are a similar distance from the terminus of replication  <cit> , and there is an additional  inversion near the terminus of ssi- <dig> caused by reciprocal recombination between prophages across the replication axis, possibly generating novel recombinant prophages with alternative cargos  <cit> .
the comparison between s. mutans strains, therefore, could provide evidence of both intraspecies and interspecies genomic rearrangement in streptococci.
comparison of genome content and architecture of s. mutans species with other streptococcal species
extensive comparative genomic analyses of positive selection, recombination, and the genome composition of  <dig> streptococcus genomes from six different species were reported by lefébure and stanhope  <cit> .
in this study, the analyses concentrated on the comparison of s. mutans species-specific gene groups and, because limited information is available regarding overall symmetry between the genomes of various streptococcus species, on the genome-wide rearrangement within nine species .
to identify sets of gene groups shared between two s. mutans strains or unique to each species/strain, the predicted orfs from the  <dig> streptococcal strains were clustered into groups using reciprocal blastp analysis.
the set of shared gene groups  for each species was determined to compare the numbers of species/strain-specific gene groups and those shared with s. mutans .
a total of  <dig> and  <dig> gene groups, respectively, comprised the pan- and core-genomes of the genus streptococcus, based on  <dig> complete genomes .
eighty-one percent of gene groups were shared between the two s. mutans strains.
comparison of the  <dig> groups constituting the core genome of s. mutans with other bacterial species/strains showed that  <dig> % to  <dig> % of gene groups are conserved between s. mutans and other streptococcal genomes .
from the 16s rrna sequences, these species can be grouped into the pyogenic group , the mutans group , the mitis group , the salivarius group , and others   <cit> .
this leads to difficulties in distinguishing between the species based on the number of shared gene groups.
these difficulties result only from the analysis of complete extant genomes, and the availability of fewer complete genomes in a species significantly affects the number of species-specific genes identified.
sequencing of further genomes in each species will be useful for defining actual species-specific genes.
as hypothesized by lefébure and stanhope  <cit> , s. agalactiae has a larger pan-genome and less recombination than s. pyogenes as evidenced by the species having a larger habitat and gene pool diversity.
this is supported by the fact that the pan-genomes of ecologically restricted organisms are smaller than those of bacteria that adopt a more generalist lifestyle  <cit> .
although s. mutans may have a smaller pan-genome and more recombination than s. agalactiae because it is usually isolated only from dental caries, the analyses here concentrated on the s. mutans species-specific regions as discussed, comparing nn <dig> and ua <dig> to understand the species .
in addition, long-pcr analysis of the dissimilar regions in the genome reveals the genomic diversity of the species, as well as the results of mlst analysis previously reported by nakano et al <cit>  .
s. mutans, s. sanguinis and s. gordonii are classified as oral streptococci.
although s. mutans and the viridans streptococci  are further classified into another subgroup within the non-pyogenic streptococci  <cit> , they have evolved to adapt to the oral environment.
therefore, we compared the gene groups common to oral streptococci with the other streptococcal species to identify the features of oral streptococci.
in total,  <dig> gene groups of s. mutans nn <dig> were identified as common to oral streptococci but not to other streptococci .
among them, genes for histidine metabolism are conserved among oral streptococci .
in particular, histidine biosynthesis genes related to the conversion of 5-phosphoribosyl-α-1-pyrophosphate to l-histidine are completely conserved between oral streptococci.
the physiological importance of histidine biosynthesis for colonization in the oral cavity is unclear, but we speculate that the presence of histidine biogenesis in situ may be more beneficial in oral environment than importing it from outside.
to confirm the chromosomal inversion at the gene level, we further compared the order of orthologous genes in s. mutans and s. pyogenes species  and other streptococcus strains   <cit>  using mauve and mummer software.
as shown in figure  <dig>  an x-shaped symmetrical dna dot plot was observed when comparing s. mutans nn <dig> and s. pyogenes strains.
interestingly, an x-shaped symmetrical dna dot plot is also found for the comparison between the genomes of s. mutans ua <dig> and s. pyogenes strains, as well as for the comparison of the two s. mutans strains .
this x-shaped symmetrical dna dot plot was also found when comparing the two s. mutans strains with other streptococcal strains of different species .
these observations indicate that chromosomal inversion across the replication axis occurs frequently in a number of streptococcal species.
we also determined the sequence alignment for the orf order between s. mutans and other streptococcal strains using mauve software.
the genome structure of s. pyogenes is more highly conserved against s. mutans strains than against other streptococcal strains .
the ori and ter regions of these two species are highly conserved, and there are fewer short lcbs  than in other streptococcal strains.
this suggests that there are fewer genome rearrangements between s. mutans and s. pyogenes than between other streptococcal species.
we hypothesize that these two species occupy similar ecological niches or smaller habitats and gene pools than s. agalactiae .
the eight strains of yersinia pestis and y. pseudotuberculosis include  <dig> lcbs, although these strains are included within the same species  <cit> .
interestingly, the lcbs in s. mutans and s. pyogenes, except for strain-specific mobile genetic elements, seem to shuffle across the replication axis.
chromosomal inversions were also observed between several s. pyogenes strains , indicating that chromosomal shuffling of an array of orfs as a unit has occurred following branching from a common ancestor.
however, it is not clear why chromosomal rearrangements are so frequently found within the genus streptococcus, although the abundance of mobile elements in the genome might affect the rearrangement distance  <cit> .
in circular bacterial genomes, dna replication divides the genome into two domains called replichores.
in the case of s. pyogenes, an undesirable phage integration  into one replichore may cause an unstable genome structure, and this unbalanced genome structure might induce chromosomal inversion between highly conserved regions  <cit> .
furthermore, a survey of salmonella genomes in culture has provided evidence that genomes with equally sized replichores  may be under positive selective pressure  <cit> .
there is also evidence that chromosome organization might influence rates of rearrangement  <cit> , and that a genome structure unbalanced by artificial genomic inversion induces an instability, that is detrimental to cell fitness or led to cell death in lactococcus lactis  <cit> .
these observations suggest that rewinding an unstable genome  may be important for survival of this organism.
two large conjugative transposon-like regions, tnsmu <dig> and tnsmu <dig>  have been inserted in ua <dig>  with tnsmu <dig> located near the rrn <dig> region of ua <dig>  these regions are not found in nn <dig>  in addition, the similarity in genome structure between s. mutans and s. pyogenes suggests two possibilities regarding the chromosomal inversion in s. mutans.
one option is that chromosomal rearrangement of s. mutans nn <dig> and ua <dig> happened by chance.
no characteristic differences of serotype specificity, geographical distribution, sts, or topological pattern from long-pcr analysis  was observed in these strain-specific regions, indicating that these regions might be acquired by chance as suggested by waterhouse and russell  <cit> .
in l. casei, topological discrepancies between the mlst and pulse-field gel electrophoresis typing trees were observed, suggesting that intragenic point mutations have accumulated at a slower rate than indels and genome rearrangements  <cit> .
therefore, the species-specific position and rearrangement of these genes could have occurred faster than the evolution of each protein, resulting in interference from the ancestor in the long-pcr analysis.
the other option is that this inversion occurred in ua <dig> by integration of foreign dna into one replichore.
however, the fact that the frequency of clinical isolates with chromosomal inversion was  <dig> of  <dig> test strains by long-pcr analysis  may indicate that the chromosomal orientation found in nn <dig> is the basic genome architecture of s. mutans strains.
further examination should be carried out with strains isolated from regions in addition to japan and finland.
following the discussions in hendrickson and lawrence  <cit>  and song et al.
<cit> , we hypothesize that the asymmetry observed in s. mutans and s. pyogenes is under selection and will probably balance through amelioration  <cit> , which has not been experimentally or theoretically demonstrated in the genus streptococcus.
CONCLUSIONS
analysis of the s. mutans genome and its comparison with other streptococcal species revealed new insights into species-specific survival strategies.
the chromosomal inversion across the replication axis between s. mutans strains occurred between homologous ribosomal operons located almost the same distance from the ori region as the s. pyogenes chromosomal inversion.
this chromosomal inversion, a characteristic x-shaped symmetrical dna dot plot, is found not only between s. mutans and s. pyogenes strains but also between s. mutans strains and all the streptococcal species in which the complete genome sequence is known.
these observations indicate that the chromosomal inversion across the replication axis has occurred frequently within streptococci during evolution.
this would possibly maintain the distortion of the replichore induced by the insertion of foreign genetic elements such as phage integration and/or generate genetic shuffling to create a novel genetic pool.
the s. mutans genome may preferentially defend against phage integration using crispr and/or various restriction/modification systems.
these counterattack systems are heterogeneous in the genus streptococcus as some streptococcal species tolerate phage integration.
in the case of s. pyogenes and s. agalactiae, 10–20% of orfs consist of phage genomes, and counterattack systems for phage integration exist but seem not to work efficiently.
in contrast, s. mutans might have evolved to avoid acquiring genes via phage-mediated transduction in order to protect their genome from the physical distortion via phage integration.
alternatively, it may permit the integration of crispr via conjugative plasmid  <cit>  and of antimicrobial peptide synthesis systems  via conjugative transposons and/or may have evolved to use natural transformation as the preferred method system to acquire new genetic traits.
this notable feature of foreign gene acquisition in s. mutans suggests that the genomic variation of the species might contribute to maintaining its niche.
there remain more questions regarding how the oral environment affects gene acquisition in s. mutans.
we believe that these findings will lead to new insights into the mechanisms of evolution in other streptococcal species.

 the genomic heterogeneity within a bacterial species reflects its lifestyle, the niche it occupies, and its exposure to mobile elements, such as bacteriophages and plasmids. even though organisms belonging to the same genus/species have a common gene set, individual organisms differ in ways representative of the physiological and virulence properties of an organism. although not all genetic differences between strains are important for niche adaptation of the bacteria, strain-specific genes are thought to be responsible for the survival of an organism in its chosen niche. this variation can be due to genetic noise, gene loss, gene duplication or modification of some of the existing genes. acquisition of new genes by lateral gene transfer is a predominant force in bacterial evolution. laterally acquired genes provide a readily available novel pool of genes for developing physiological properties that are helpful for exploiting a new niche. a recent study suggested that the total known genome content of all contemporary streptococcus agalactiae strains will increases as hundreds of genomes are sequenced. although s. pyogenes belongs to the same genus, it has a smaller pan-genome and greater level of recombination in its core genome. these organisms provide a good model for identifying the causes of genome plasticity in human pathogens. it has long been recognized that serological, genetic, and biochemical variations exist within the species s. mutans. s. mutans has been classified into four serotypes based on the chemical composition of its cell surface rhamnose-glucose polymers. we previously developed a multilocus sequence typing method using eight house-keeping genes. ninety-two sequence types were identified from clinical isolates, indicating that s. mutans is a diverse population. in the mlst analysis, serotype c strains were widely distributed in the dendrogram, while serotype e, f, and k strains were differentiated into clonal complexes. this suggests that serotype c, the dominant serotype among s. mutans clinical isolates, is the ancestral phenotype of this organism and that serotype e and f strains have evolved strain-specific genes. although differences in modification of cell surface polymers reflect evolutionary trends, differences in cariogenicity have not been observed, and the relationship between serotype and clinical condition remains unclear. studies of individual s. mutans genes have revealed sequence variations, resulting in altered function of the encoded proteins. for example, variation has been demonstrated in the occurrence of plasmids, and in mutacin operons, serotype antigens, competence, and the msm, bgl, cel, and gftbc loci. waterhouse and russell recently showed a mosaic of loci such as the msm, gbl, cel, and gftbc, which they called "dispensable genes," distributed among s. mutans strains. they also demonstrated that 20% of the s. mutans ua open reading frames were absent from one or more of the nine test strains, and dispensable orf blocks were identified by microarray analysis based on the ua genome. given the wide distribution and diversities of genotypes and genetic loci in s. mutans, it seems likely that other strains of s. mutans have both unique and common genetic loci not present on the ua genome. this is useful for charting s. mutans evolutionary history. however, these analyses are based on only one genome, s. mutans ua even though extensive genomic variation between s. mutans strains has been predicted. genome sequence data are now available for numerous species of bacteria and comparative evolutionary approaches show positive selection pressure and lateral gene transfer in the evolution of many bacterial species. these analyses have been performed for pathogenic bacteria such as helicobacter pylori, mycobacterium species, chlamydia species, escherichia coli, and salmonella species. the pathogenic streptococcus species include important human and agricultural pathogens. more than whole genomes of streptococcus sp. belonging to nine different species including s. pyogenes, s. pneumoniae, s. agalactiae, s. thermophilus, s. suis, s. sanguinis, s. gordonii, s. equi, and s. mutans are publicly available. these organisms colonize diverse habitats including tooth, oral mucosal, pharyngeal, respiratory, intestinal, and urinogenital surfaces. these species have acquired various genes for a specific niche mainly by lateral gene transfer. for example, s. pyogenes acquires or tolerates bacteriophages that are important for new virulence determinants and that induce genomic rearrangement. s. agalactiae, the main cause of neonatal infection in humans, also tolerates bacteriophages. some of these organisms gain counterattack systems such as restriction modification or clustered regularly interspaced short palindromic repeats. multiple sequences of genomes from closely related species that inhabit different niches lead not only to an understanding of the pattern of gene movement but also to insights into the role of species-specific genes, and genome plasticity. in this context, we determined the whole genome sequence of an s. mutans serotype c strain nn isolated from japan in and we compared the genome sequence, genome structure, and gene variation with the genomes of serotype c ua strain isolated in from the united states, and with clinical isolates from japan and finland, and other closely related streptococcal genera to provide useful information about the evolutionary events associated with s. mutans strains and streptococcus sp., and to provide new insights into streptococcal species-specific survival strategies.  general features of the strain nn2025 the genome of nn is a single, circular chromosome of, bp. the genome is almost identical in size to that of ua but is kb shorter. neither nn nor ua contains plasmids or prophages. the genome start point for nn was assigned to the putative location of the dnaa gene, similar to s. mutans ua and other genomic sequences. the average gc content is %, which is similar to that of ua there are five rrna operons containing 5s, 16s, and 23s rrna genes. the predicted trna genes encode all amino acids. most trna genes are clustered near the rrna operons, of of these genes are less than kb from an rrna operon, as in other streptococcal strains. the genome contains predicted proteins with an average size of bp that cover % of the whole sequence, which is similar to other streptococci. both the sequence and the annotation have been deposited in the dna data bank of japan. genes and regions conserved between s. mutans nn and ua genomes nn contains orfs, of which are predicted by reciprocal blast search analysis to be common to ua s. mutans strains nn and ua are classified into the same serological group c, and show the same biochemical properties, adhesive properties to glass surfaces, and cariogenic properties in rat infection models. waterhouse et al. reported that 80% of the s. mutans orfs are conserved among ten strains with different serotypes by microarray hybridization based on the ua genome, suggesting that 80% of orfs are conserved within s. mutans strains. this indicates that the core genome of s. mutans is more stable than that of other streptococcus species, where the core genome represents only about 60% of the genome. most vegetative growth genes are highly conserved. carbohydrate metabolism is a key survival strategy for s. mutans, and genes for transport and metabolism of various sugars, and the fermentation of carbohydrates are completely conserved between ua and nn s. mutans is suggested to be capable of metabolizing a wider variety of carbohydrates than many other gram-positive organisms that have been sequenced. s. mutans resides in the oral cavity, and this varied sugar metabolism is an important survival strategy for this organism. s. mutans is predicted to possess at least five sugar abc transport systems and at least sugar pts systems, and can use at least sugars for glycolysis. nine ptss were confirmed to be transcribed in the presence of different sugars. in addition, orfs predicted as virulence factors of s. mutans including adhesins, glucan-producing and binding exoenzymes, were conserved. adhesins of s. mutans are also important for protecting the bacterium against possible host defenses and for maintaining its ecological niche in the oral cavity. comparative genome analysis revealed that the major s. mutans surface receptors spap and wall-associated antigen a are completely conserved. an uncharacterized wall-associated protein, wape, and a dextranase precursor were determined from blast analysis to be s. mutans-specific genes within the sequenced streptococcus species, suggesting that these might be important for colonization. strain-specific genes and regions in nn and ua159 strain-specific regions were determined using mauve software, where the locally collinear blocks represent the landmarks in chromosomes. an lcb is defined as a collinear set of multi-mums. the weight of an lcb serves as a measure of confidence that it is a true homologous region instead of a random match. therefore, the orfs or sequences between the lcbs and any regions with low similarity are identified as strain specific regions. the nn genome contains eight strain-specific regions containing orfs that were unique in comparison with ua other nn2025-specific orfs are randomly inserted into the nn genome, and these orfs are classified into transposase fragments, ribosomal proteins, hypothetical proteins and disrupted gene fragments. only two orfs are predicted to be functional genes from an unknown foreign source. in contrast, the ua genome contains nine strain-specific regions containing unique orfs; another ua159-specific orfs are distributed in the ua genome. of the regions that are unique between the nn and ua genomes, eight regions exist in both strains, but the contents of the orfs are highly diverse. these regions are designated "variable regions". region contains genes of the purine nucleotide biosynthesis pathway and is thought to be important for bacterial growth, but genetic variation is found within this region. region in nn includes two transposase fragments, five hypothetical proteins, methyltransferase, gapc, transcriptional regulator gene, and luxs. luxs plays an important role in the production of autoinducer- in many bacterial species, and 30% of the genes of s. mutans are affected by luxs. region in nn includes hypothetical proteins and abc transporters; however, the contents of this region in ua are quite different and include hypothetical proteins that appear to have been horizontally transferred from other species. region in ua and regions and in nn include putative restriction/modification system genes. the r/m system is composed of genes that encode a restriction enzyme and a modification methylase, and they defend against invaders by attacking non-self dna, or by killing cells that have eliminated them. waterhouse et al. reported that 20% of ua orfs did not hybridize with one or more of the nine s. mutans genomes in a ua159-based microarray analysis. they also showed that genomic blocks involving more than one orf were found within the test strains. in the case of ua comparison of the regions identified in this study by pcr with previously reported'genomic blocks' and'genomic island' showed a good correlation, confirming the accuracy of both our results and the earlier reports, even though these were derived from different experimental approaches. the slight differences between the results, therefore, may have resulted from the characteristics of each method, because pcr primers target short sequences with high similarity, while microarray probes overall similarity to detect the target sequence. this second s. mutans genome sequence identifies new strain-specific content that could not be identified from only one genome. based on their correspondence with the'genomic islands', region and appear to be horizontally transferred. the gene order of region in ua is different from that in nn which may suggest the existence of an unknown mechanism to reorder the genomic island. these regions may reflect the phenotypic and biochemical differences of s. mutans strains. although this comparative study was based on the ua genome, unique orfs or regions in the genomes of other strains were not completely analyzed. therefore, we also determined the differences of these regions among test strains using long-pcr against regions and clustering analysis. however, no characteristic differences in serotype specificity, geographical distribution, or the ability for sucrose-dependent adhesion to a glass surface were found. we have previously demonstrated that clinical isolates from japan and finland were resolved into sts by mlst, of which were identified only once. we could not determine any distribution or lineage differences between japanese isolates and finnish isolates. we, therefore, conclude that the s. mutans population diversity is not caused by variation in gene content but probably with genetic recombination. in some s. mutans strains, similar insertion/deletion events appear in the genomes of strains with very different origins based on pcr determination of loci. in our analysis, characteristic regions in the whole genome comparison were diverse, although they were isolated from only two geographical areas. this indicates that insertions/deletions in these specific regions are acquired by unknown mechanisms, probably by multiple acquisition events and the spread of an ancestral acquisition through the species by recombination from other bacterial strains. various genes appear to be moved by lateral gene transfer in s. mutans, resulting in strain-specific regions. nn possesses transposase orfs, and is elements in its genome, whereas ua possesses transposon-like elements. these mobile genetic elements are located near the strain-specific regions. thus, either laterally acquired genes account for the emergence of strain-specific orfs or the comparator strain has lost these orfs. tnsmu was found to be a conjugative transfer element in ua. this region in ua is large, involving smu.191- one helicase gene, two transposase-like genes, and several hypothetical proteins. this region in nn contains only seven short orfs, including two transposase fragments. among test strains, isolates, including all the finnish isolates, show the same pattern as nn, and % of strains do not have this tnsmu region. some isolates in japan possess the same tnsmu as found in ua in a genomic island, along with orfs predicted to be involved in biosynthesis and secretion of the antimicrobial peptide, bacitracin. however, tnsmu has a high level of variation, including degenerated orfs, and is not found in all s. mutans strains. in previous work, tnsmu was found only in ua and gs. the variation in the tnsmu region has already been reported in strains ingritt and lml, whose these sequences are almost identical to that of nn. therefore, we constructed various pcr primer sets based on the sequence of ua and determined whether tnsmu was found in our test strains. in our experiments, 85% of clinical strains did not contain this region, and regional bias was not observed. these observations indicate that the existence of tnsmu and tnsmu may not be common or necessary for the virulence of s. mutans strains in rats. putative bacitracin synthesis regions the bacitracin synthesis gene cluster in tnsmu did not exist in most of the clinical strains, and two other bacitracin or polypeptide antibiotic synthesis gene clusters were found in nn. bacitracin is a mixture of related cyclic polypeptide antibiotics produced by some strains of bacillus licheniformis and b. subtilis. its primary mode of action is to bind to undecaprenyl pyrophosphate in the presence of a bound divalent metal cation, sequestering upp and preventing its interaction with phosphatase. this in turn prevents the return of the undecaprenyl monophosphate lipid carrier, inhibiting peptidoglycan and teichoic acid biosynthesis. region is a large transposon-like region containing bacitracin synthesis genes, peptide synthetase, abc transporters, and an is fragment. smu2025_ and smu2025_ are multiple-domain proteins similar to nonribosomal peptide synthetases such as gramicidin or bacitracin, but the sequence identity is lower than that of the known peptide synthetases. region includes a nonribosomal peptide synthetase, a putative polyketide synthase, and a putative permease; these genes are related to the synthesis of bacitracin or other peptide antibiotics. s. mutans strains have bacitracin resistance. the existence of bacitracin resistant genes in this organism may reflect bacitracin production for protection. regions and are distributed among % and %, respectively. interestingly, all of the s. mutans strains used in this study possess at least one of these bacitracin or peptide antibiotic synthetases genes such as mutacins. in fact, region in nn includes mutacin iii production genes. smunn2025_ and _ are similar to the scnm and scnt of s. pyogenes, respectively, but these two genes were not found and an apparent fragmentation of the polyketide genes has been shown in ua. although bacitracin synthesis associated with tnsmu should be experimentally confirmed, nn has a bactericidal effect, indicating that the bacitracin production and mutiii cluster might be active in this strain, and that s. mutans has acquired peptide antibiotic synthetase genes to exclude other bacteria, perhaps for niche acquisition. crispr associated regions numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats, composed of 25– bp repeats separated by unique sequence spacers of similar length. it is well known that bacteria have a plethora of mechanisms to counterattack a diverse phage population. consequently, many bacterial species have developed a variety of natural defense mechanisms that target diverse steps of the phage life cycle, notably blocking adsorption, preventing dna injection, restricting the incoming dna, and abortive infection systems. in the genus streptococcus, three distinct crispr loci have been identified within the s. thermophilus chromosome; namely, crispr- crispr- and crispr-. crispr- and crispr- loci have been experimentally demonstrated to integrate novel spacers in response to bacteriophage. comparative analysis of crispr- sequences between various s. thermophilus strains has revealed polymorphisms. in addition, it was recently reported that crispr provides acquired resistance against viruses in prokaryotes, notably in s. thermophilus. phage tolerance capacity is known to be diverse. for example, phage integration in s. pyogenes is an important source for new virulence factors as well as for the genomic rearrangement of the prophage regions. natural phage resistance mechanisms in s. mutans have not previously described, because phage donor and acceptor strains are quite rare in s. mutans. based on in silico and in vivo analyses, several hypotheses have been put forward proposing roles for crispr and cas genes, including providing immunity against foreign genetic elements via a mechanism based on rna interference. strain nn possesses two characteristic crispr regions, regions and. region contains nine genes highly similar to the cas and cse genes in lactobacillus casei. the gene order of this crispr region is similar to the "ecoli" subtype, or to the lcas of the ldbu family recently named by horvath, indicating that region is unique for s. mutans. it has been designated crispr-. the crispr- locus was present in 30/ clinical isolates. no variation of the length of repeat-spacer regions is found in these strains, indicating that this region might have been acquired in recent years or might be degenerate. the crispr repeat, direct repeat, sequence length is bp, and spacer sequences are found adjacent to cse in nn of these spacer sequences, six are identical to the sequence of the recently identified s. mutans phage m. in contrast, another crispr region is found in both ua and nn and has been designated as crispr- in s. mutans. this crispr- locus consists of four cas and csn genes and two hypothetical proteins, and the gene order of the crispr- locus is almost completely conserved among s. mutans strains. though the crispr subtype in nn is the same as in ua with the gene order, the lengths of the repeat sequences are quite different not only in these two strains but also among clinical isolates, ranging from to kb. to determine the spacer variability in the clinical isolates, the crispr- loci of an additional six strains were sequenced for comparison. strain nn possesses a bp dr sequence, typical of the length of sthe family, and spacer sequences were found just downstream of csn. this is the second example of a crispr locus with more than repeats found in firmicutes and it contains the highest number of repeats found in the genus streptococcus. the five sequenced strains and ua all have the same dr sequence as nn and only strain mt has another bp dr sequence with the lowest repeat number in any tested strains. this is the first example of a repeat length other than from bp in this sthe family. however, the crispr- locus gene order was completely conserved among strains. the number of the spacers is quite divergent among strains, ranging from to. we next determined the sequence similarity of spacer sequence with s. mutans phage m. of the spacers of nn are highly similar to the sequence of s. mutans phage m, as are 20–30% of spacers among the sequenced strains with the exception of strain mt interestingly, strain lj possesses two spacers similar to m and two spacers similar to s. thermophilus phages sfi and indicating that s. mutans strains might have been attacked by these streptococcal phages, and protected by the crispr- locus. the biological significance of crispr against phage infection has recently been reported in s. thermophilus. the presence of a crispr spacer identical to a phage sequence adjacent to cas provides resistance against phages containing that particular sequence. limited information is available regarding the ecological role of bacteriophages in the oral cavity, although bacteriophages have been isolated from human saliva or dental plaque. only lytic bacteriophages were isolated from more than plaque samples tested for lytic activity against indicator strains of s. mutans and s. sobrinus. therefore, the absence in both strain ua and nn genomes of prophage or their remnants was not surprising. the long spacer sequences found in the crispr- and - loci in nn, and their sequence similarity to the lytic phage, suggest that s. mutans might resist phage infection via crispr. this observation is in contrast with the phage-permissive s. pyogenes, because s. pyogenes possesses two to eight prophage regions within its genome, and the number of crispr-associated direct repeats and spacers is less than that of other streptococcal species. the average number of prophages in s. pyogenes that lack the cas genes found in s. mutans is six, while that in the other eight s. pyogenes strains carry the same cas gene set is. the number of prophages in an s. pyogenes genome may be affected by the crispr gene set present, and further examination is needed, especially considering that the many sequenced genomes of streptococci carrying prophages have rarely demonstrated natural transformation. therefore, in the genus streptococcus, specific species may have evolved to function in a particular way to acquire foreign genes via natural transformation or bacteriophages, and the acquisition of new foreign genes via phage infection may not be favorable for the lifestyles of s. mutans in the oral cavity. elucidation of the mechanism by which s. mutans acquires new genes will be interesting to clarify further the species-specific evolutionary strategies in the streptococci. genomic analysis of the rearrangement in the ua and nn genomes the physical structure of the bacterial genome is highly conserved during evolution in escherichia coli. however, x-shaped chromosomal inversion has been found between pyrococcus horikoshii and p. abyssi, and between chlamydia pneumoniae and c. trachomatis. interestingly, a typical x-shaped chromosomal inversion is found in s. pyogenes. such rearrangements leading to genome plasticity in these bacteria might reflect or contribute to evolutionary processes in the creation of new pathogens. the recombination sites near the ori region in nn are found within the 16s rrna, trna-ala, 23s rrna, trna regions, and the comx homologue in both regions. the genome of s. mutans nn shows that the rearrangement breakpoint is located bp downstream from the 23s rrna, and this rearrangement breakpoint is conserved within the other s. mutans strains isolated in taiwan in which huang et al. firstly observed chromosomal inversion in 25/ strains. these observations indicate that the chromosomal recombination may frequently occur in this genomic site because isolates from japan, finland and taiwan shows the same inversion site though the frequency differs between our strains and those reported by huang et al., and might occur by reca-dependent recombination between two ribosomal operons. in gram-negative salmonella typhimurium, inversion between large inverted repeats separated by large intervals has been shown to be reca- and recb-dependent. in addition, chromosomal inversion between the ribosomal operons was reported between s. typhimurium and s. paratyphi a. our sequencing analysis showed that long repeated sequences across the replication axis can also induce large-scale chromosomal rearrangements within a species of gram-positive bacteria. although the role of homologous recombination in genomic rearrangements across the replication axis has not been elucidated, tillier and collins proposed an alternative model for the observed pattern of rearrangement. gene translocation across the replication axis may result in close physical proximity during the process of genome replication because homologous recombination equidistant from the ori region occurs between two replication forks and single- or double-stranded dna breaks. in fact, two rrn-comx regions were found equidistant from the ori region in the s. mutans genomes; thus, this model is in good agreement with the genomic rearrangement mechanism of s. mutans and s. pyogenes. this sequence specificity may affect site-specific recombination during homologous recombination. alignments and dot plots of the genomes of s. mutans nn and ua revealed extensive genomic rearrangements. the origin of dna replication and dif-like termination sequence were conserved. both alignment and dot plot analyses using mauve and mummer software revealed x-shaped chromosomal inversions that were symmetrical across the replication axis. the locations of the genes around the ori region were almost completely conserved between nn and ua and the other homologous genes were translocated to an inverted position on the chromosome. these chromosomal segments are symmetrical except for small gaps encoding the genes of strain-specific regions. as a consequence, the inversion in nn relative to ua did not change gene orientation relative to the replication axis and produced an x-shaped dna dot plot. for s. pyogenes manfredo and s. pyogenes ssi- there is a large central inversion, which probably resulted from reciprocal recombination between rrn-comx regions that are a similar distance from the terminus of replication, and there is an additional inversion near the terminus of ssi- caused by reciprocal recombination between prophages across the replication axis, possibly generating novel recombinant prophages with alternative cargos. the comparison between s. mutans strains, therefore, could provide evidence of both intraspecies and interspecies genomic rearrangement in streptococci. comparison of genome content and architecture of s. mutans species with other streptococcal species extensive comparative genomic analyses of positive selection, recombination, and the genome composition of streptococcus genomes from six different species were reported by lefébure and stanhope. in this study, the analyses concentrated on the comparison of s. mutans species-specific gene groups and, because limited information is available regarding overall symmetry between the genomes of various streptococcus species, on the genome-wide rearrangement within nine species. to identify sets of gene groups shared between two s. mutans strains or unique to each species/strain, the predicted orfs from the streptococcal strains were clustered into groups using reciprocal blastp analysis. the set of shared gene groups for each species was determined to compare the numbers of species/strain-specific gene groups and those shared with s. mutans. a total of and gene groups, respectively, comprised the pan- and core-genomes of the genus streptococcus, based on complete genomes. eighty-one percent of gene groups were shared between the two s. mutans strains. comparison of the groups constituting the core genome of s. mutans with other bacterial species/strains showed that % to % of gene groups are conserved between s. mutans and other streptococcal genomes. from the 16s rrna sequences, these species can be grouped into the pyogenic group, the mutans group, the mitis group, the salivarius group, and others. this leads to difficulties in distinguishing between the species based on the number of shared gene groups. these difficulties result only from the analysis of complete extant genomes, and the availability of fewer complete genomes in a species significantly affects the number of species-specific genes identified. sequencing of further genomes in each species will be useful for defining actual species-specific genes. as hypothesized by lefébure and stanhope, s. agalactiae has a larger pan-genome and less recombination than s. pyogenes as evidenced by the species having a larger habitat and gene pool diversity. this is supported by the fact that the pan-genomes of ecologically restricted organisms are smaller than those of bacteria that adopt a more generalist lifestyle. although s. mutans may have a smaller pan-genome and more recombination than s. agalactiae because it is usually isolated only from dental caries, the analyses here concentrated on the s. mutans species-specific regions as discussed, comparing nn and ua to understand the species. in addition, long-pcr analysis of the dissimilar regions in the genome reveals the genomic diversity of the species, as well as the results of mlst analysis previously reported by nakano et al. s. mutans, s. sanguinis and s. gordonii are classified as oral streptococci. although s. mutans and the viridans streptococci are further classified into another subgroup within the non-pyogenic streptococci, they have evolved to adapt to the oral environment. therefore, we compared the gene groups common to oral streptococci with the other streptococcal species to identify the features of oral streptococci. in total, gene groups of s. mutans nn were identified as common to oral streptococci but not to other streptococci. among them, genes for histidine metabolism are conserved among oral streptococci. in particular, histidine biosynthesis genes related to the conversion of 5-phosphoribosyl-α-1-pyrophosphate to l-histidine are completely conserved between oral streptococci. the physiological importance of histidine biosynthesis for colonization in the oral cavity is unclear, but we speculate that the presence of histidine biogenesis in situ may be more beneficial in oral environment than importing it from outside. to confirm the chromosomal inversion at the gene level, we further compared the order of orthologous genes in s. mutans and s. pyogenes species and other streptococcus strains using mauve and mummer software. as shown in figure an x-shaped symmetrical dna dot plot was observed when comparing s. mutans nn and s. pyogenes strains. interestingly, an x-shaped symmetrical dna dot plot is also found for the comparison between the genomes of s. mutans ua and s. pyogenes strains, as well as for the comparison of the two s. mutans strains. this x-shaped symmetrical dna dot plot was also found when comparing the two s. mutans strains with other streptococcal strains of different species. these observations indicate that chromosomal inversion across the replication axis occurs frequently in a number of streptococcal species. we also determined the sequence alignment for the orf order between s. mutans and other streptococcal strains using mauve software. the genome structure of s. pyogenes is more highly conserved against s. mutans strains than against other streptococcal strains. the ori and ter regions of these two species are highly conserved, and there are fewer short lcbs than in other streptococcal strains. this suggests that there are fewer genome rearrangements between s. mutans and s. pyogenes than between other streptococcal species. we hypothesize that these two species occupy similar ecological niches or smaller habitats and gene pools than s. agalactiae. the eight strains of yersinia pestis and y. pseudotuberculosis include lcbs, although these strains are included within the same species. interestingly, the lcbs in s. mutans and s. pyogenes, except for strain-specific mobile genetic elements, seem to shuffle across the replication axis. chromosomal inversions were also observed between several s. pyogenes strains, indicating that chromosomal shuffling of an array of orfs as a unit has occurred following branching from a common ancestor. however, it is not clear why chromosomal rearrangements are so frequently found within the genus streptococcus, although the abundance of mobile elements in the genome might affect the rearrangement distance. in circular bacterial genomes, dna replication divides the genome into two domains called replichores. in the case of s. pyogenes, an undesirable phage integration into one replichore may cause an unstable genome structure, and this unbalanced genome structure might induce chromosomal inversion between highly conserved regions. furthermore, a survey of salmonella genomes in culture has provided evidence that genomes with equally sized replichores may be under positive selective pressure. there is also evidence that chromosome organization might influence rates of rearrangement, and that a genome structure unbalanced by artificial genomic inversion induces an instability, that is detrimental to cell fitness or led to cell death in lactococcus lactis. these observations suggest that rewinding an unstable genome may be important for survival of this organism. two large conjugative transposon-like regions, tnsmu and tnsmu have been inserted in ua with tnsmu located near the rrn region of ua these regions are not found in nn in addition, the similarity in genome structure between s. mutans and s. pyogenes suggests two possibilities regarding the chromosomal inversion in s. mutans. one option is that chromosomal rearrangement of s. mutans nn and ua happened by chance. no characteristic differences of serotype specificity, geographical distribution, sts, or topological pattern from long-pcr analysis was observed in these strain-specific regions, indicating that these regions might be acquired by chance as suggested by waterhouse and russell. in l. casei, topological discrepancies between the mlst and pulse-field gel electrophoresis typing trees were observed, suggesting that intragenic point mutations have accumulated at a slower rate than indels and genome rearrangements. therefore, the species-specific position and rearrangement of these genes could have occurred faster than the evolution of each protein, resulting in interference from the ancestor in the long-pcr analysis. the other option is that this inversion occurred in ua by integration of foreign dna into one replichore. however, the fact that the frequency of clinical isolates with chromosomal inversion was of test strains by long-pcr analysis may indicate that the chromosomal orientation found in nn is the basic genome architecture of s. mutans strains. further examination should be carried out with strains isolated from regions in addition to japan and finland. following the discussions in hendrickson and lawrence and song et al., we hypothesize that the asymmetry observed in s. mutans and s. pyogenes is under selection and will probably balance through amelioration, which has not been experimentally or theoretically demonstrated in the genus streptococcus.  analysis of the s. mutans genome and its comparison with other streptococcal species revealed new insights into species-specific survival strategies. the chromosomal inversion across the replication axis between s. mutans strains occurred between homologous ribosomal operons located almost the same distance from the ori region as the s. pyogenes chromosomal inversion. this chromosomal inversion, a characteristic x-shaped symmetrical dna dot plot, is found not only between s. mutans and s. pyogenes strains but also between s. mutans strains and all the streptococcal species in which the complete genome sequence is known. these observations indicate that the chromosomal inversion across the replication axis has occurred frequently within streptococci during evolution. this would possibly maintain the distortion of the replichore induced by the insertion of foreign genetic elements such as phage integration and/or generate genetic shuffling to create a novel genetic pool. the s. mutans genome may preferentially defend against phage integration using crispr and/or various restriction/modification systems. these counterattack systems are heterogeneous in the genus streptococcus as some streptococcal species tolerate phage integration. in the case of s. pyogenes and s. agalactiae, 10–20% of orfs consist of phage genomes, and counterattack systems for phage integration exist but seem not to work efficiently. in contrast, s. mutans might have evolved to avoid acquiring genes via phage-mediated transduction in order to protect their genome from the physical distortion via phage integration. alternatively, it may permit the integration of crispr via conjugative plasmid and of antimicrobial peptide synthesis systems via conjugative transposons and/or may have evolved to use natural transformation as the preferred method system to acquire new genetic traits. this notable feature of foreign gene acquisition in s. mutans suggests that the genomic variation of the species might contribute to maintaining its niche. there remain more questions regarding how the oral environment affects gene acquisition in s. mutans. we believe that these findings will lead to new insights into the mechanisms of evolution in other streptococcal species. 
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 clinical isolates using long-pcr analysis. genomic diversity in s. mutans appears to occur frequently between insertion sequence elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. 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. 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. in contrast, s. pyogenes tolerates phage infection for acquisition of virulence determinants for niche adaptation. s. mutans may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats. we have sequenced the complete genome of s. mutans serotype c strain nn and compared it with the genome of ua the nn genome is composed of, bp, and the two strains show highly conserved core-genome. these observations suggest that s. mutans strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. streptococcus mutans is the major pathogen of dental caries, and it occasionally causes infective endocarditis.
 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.  we have sequenced the complete genome of s. mutans serotype c strain nn and compared it with the genome of ua the nn genome is composed of, 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 clinical isolates using long-pcr analysis. genomic diversity in s. mutans appears to occur frequently between insertion sequence 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. in particular, the crispr- region, which is highly divergent between strains, in nn has long repeated spacer sequences corresponding to the streptococcal phage genome.  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. 