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J Bacteriol. Nov 2005; 187(21): 7292–7308.
PMCID: PMC1272970

Whole-Genome Sequencing of Staphylococcus haemolyticus Uncovers the Extreme Plasticity of Its Genome and the Evolution of Human-Colonizing Staphylococcal Species

Abstract

Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of S.haemolyticus to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of S.haemolyticus, Staphylococcus aureus, and Staphylococcus epidermidis were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the “oriC environ,” likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of S.haemolyticus, S.aureus, and S.epidermidis elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the S.haemolyticus chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.

As a part of the normal bacterial flora, staphylococci colonize the skin and mucosal membranes of humans. In addition, staphylococci frequently cause opportunistic infections in patients with underlying disease, such as those with prosthetic devices, surgical patients, individuals undergoing dialysis, or patients with diabetes. Since antibiotic chemotherapy was introduced in the last century, staphylococci have successfully persisted by altering their genetic traits to avoid being killed. Multidrug-resistant staphylococcal strains, exemplified by methicillin-resistant staphylococci, are now prevalent worldwide.

Among 40 staphylococcal species described to date, Staphylococcus aureus is the most virulent species and poses the greatest threat in hospitals worldwide. In addition to its nosocomial spread, S.aureus has also become problematic in community settings, where individuals without predisposing factors have acquired methicillin-resistant staphylococcal infections. Although most community-acquired staphylococcal infections involve the skin and soft tissues, some otherwise healthy children have acquired potentially lethal S.aureus infections with severe symptoms, such as necrotizing pneumonia (5, 28, 29). To gain a better understanding of the overall pathogenesis of staphylococcal infections and identify novel targets for new chemotherapeutic agents, researchers have sequenced the genomes of seven S.aureus strains (3, 9, 13, 22). A comparative analysis of these strains has revealed that many genes involved in staphylococcal pathogenicity and drug resistance are carried on mobile genetic elements or genomic islands (GIs) (2, 3). The GIs are allelic among strains, conferring large variations in the combinations of virulence and resistance genes of individual S.aureus isolates, likely resulting in different clinical outcomes from infections caused by various S.aureus strains.

Though other staphylococcal species are less virulent than S.aureus, some of them colonize humans and may play important roles in hospital-acquired opportunistic infections. The most significant group of coagulase-negative staphylococci is the Staphylococcus epidermidis group, which includes S. epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, and Staphylococcus warneri. S. epidermidis colonizes the skin of virtually all humans and frequently causes foreign-body infections. The sequenced genomes of S. epidermidis ATCC 12228 (41) and RP62A (9) also carry GIs, but these strains lack many of the virulence genes characteristic of S. aureus, such as those encoding exotoxins, superantigens, and leukocidins.

Staphylococcus haemolyticus, also found among the normal skin flora, is commonly isolated from the axillae, perineum, and inguinal areas of humans (23). Among coagulase-negative staphylococci, S.haemolyticus is second only to S.epidermidis in its frequency of isolation from human blood cultures (14). S.haemolyticus may also cause septicemia, peritonitis, otitis, and urinary tract infections. It is notorious for its multidrug resistance and historically early acquisition of resistance to methicillin and glycopeptide antibiotics (7, 12). We observed that a highly glycopeptide-resistant S.haemolyticus strain JCSC1435 frequently generated mutants that lost antibiotic resistance during passage in drug-free medium. Moreover, difficulty or failure in species identification of S.haemolyticus by biochemical methods has been documented (33). Therefore, we determined the whole-genome sequence of S.haemolyticus strain JCSC1435 in order to evaluate its multidrug-resistant phenotype and features of its genetic background that allow for frequent phenotypic alterations. Whole-genome sequence determination of S.haemolyticus strain JCSC1435 revealed not only the presence of many antibiotic resistance genes but also a surprising number of insertion sequences (ISs), many of which were highly homologous. We suggest that the presence of many IS elements confers the frequent genomic rearrangement characteristic of this organism.

In this study, we also performed comparative genomic analyses of the three clinically important staphylococcal species, S.aureus, S.epidermidis, and S.haemolyticus. This investigation revealed that a large proportion of the open reading frames (orfs) in the three species were conserved in their sequence and order on the chromosome. However, we identified a region of the chromosome just downstream of the origin of replication that showed little homology among the species. We designated this region the “oriC environ,” and it seems important to the evolution and differentiation of each staphylococcal species. We also discuss the differences in biological characteristics and virulence potentials of each staphylococcal species.

MATERIALS AND METHODS

Genome sequencing and annotation.

Staphylococcus haemolyticus strain JCSC1435 was isolated from a Japanese inpatient at Juntendo Hospital, Tokyo, in 2000. Genome sequencing and annotation were performed as described previously (22). We prepared a genomic DNA library with an insert size of 1.3 to 2.5kbp, sequenced approximately 43,000 clones from both ends, and assembled them into 69 contigs. To fill the remaining gaps, we performed 6,532 PCRs between contigs, primer-walking sequencing, and in vitro transposition, followed by sequencing (TGSTM Template Generation System F-700; Finnzymes, Espoo, Finland). The finalized sequence achieved a base call accuracy of ≥99.9%. The sequence was confirmed experimentally by a series of PCRs, pulsed-field gel electrophoresis (PFGE), and Southern blotting analyses.

Identification and phylogenetic classification of homologs.

For interspecies comparative analysis, we grouped the orfs from S.aureus, S.epidermidis, S.haemolyticus, and other related bacterial species into “homologs” based on their mutual homology values. In order to save computation time, we roughly grouped together the orfs with a BLASTN (1) e-value of <10−6. Within each rough group, the orfs with a ≥70% quality ratio by the gap alignment program (8) were grouped together to form a homolog. Accordingly, orthologous (or paralogous) orfs would belong to the same homolog. In order to determine which homologs were common to staphylococcal species and which homologs were unique, we classified the homologs by their pattern of appearance in the three staphylococcal species. We analyzed 10 strains from staphylococci and 5 strains from the related Firmicutes (gram-positive bacterial) phylum. The strains included S.aureus strains COL, MRSA252 (EMRSA-16), MSSA476, Mu50, MW2, N315, and NCTC8325; S.epidermidis strains ATCC 12228 and RP62A; S.haemolyticus strain JCSC1435; and five strains from other species: Bacillus subtilis 168, Streptococcus pyogenes M1 GAS, Lactococcus lactis Il1403, Clostridium perfringens 13, and Deinococcus radiodurans R1. For the strains with draft sequences only, we extracted orfs by using GLIMMER (6) and RBS finder (35).

Identification of GIs.

In order to detect possible regions accumulating foreign genes, we extracted local regions on the chromosome that contain few orfs belonging to the homologs common to staphylococci; specifically, we screened regions containing at least 16 consecutive noncommon orfs. This method was designed to distinguish chromosomal regions acquired by horizontal transfer, such as GIs or transposons, from those vertically transmitted from a common ancestor. When an extracted region included an integrase or its remnant, we judged it to be a GI. If there were orfs characteristic of phages, we annotated the region as a prophage, and if not, classified it as a ν island (such as νSh1, νSh2, and νSh3). The precise boundary of each GI was determined by identifying a pair of direct repeats of 14 bp or longer flanking the region. We annotated extracted regions as integrated plasmids when they contained plasmid replication genes.

Identification and annotation of ISs.

We extracted ISs from the genome and classified them computationally (Michael Chandler, personal communication; see reference 26). For each orf identified as a transposase, we computed its homologous regions in the whole genome with BLASTN (1) and extracted the hits together with their 1-kbp upstream and 1-kbp downstream regions. We then aligned the sequences extracted from the genome and determined the matching region within them as an entire IS. Finally, such IS elements were searched throughout the genome again to extract partial ISs. For classification, we regarded IS elements with ≥80% mutual identity as copies of the same IS. We regarded those with ≥50% identity as related to known IS and designated ISknown-like. We judged an IS intact if its transposase did not include frameshifts or stop codons, if the terminal inverted repeats were preserved, and if its entire sequence aligned to other ISs of the same family; otherwise, the IS was regarded as a remnant.

Isolation of mutant strains and their characterization.

We isolated mutant strain WP12 from an overnight culture of S.haemolyticus JCSC1435 showing no aggregation, which turned out to have an altered PFGE banding pattern. Additional mutant strains were obtained by screening for antibiotic-susceptible cells in serial passages of a JCSC1435 culture. For each passage, we inoculated 103 CFU in 10 ml of brain heart infusion (BHI) broth (Becton Dickinson Co., Ltd., New Jersey) that was incubated at 37°C for 2 or 3 days. After 11 or 18 days, dilutions of the culture were spread on BHI agar plates (Becton Dickinson Co., Ltd., New Jersey), and teicoplanin- and ceftizoxime-susceptible mutants were selected by replica plating on BHI agar plates containing 10 mg/liter of the antibiotics.

Changes in the structure of the S.haemolyticus chromosome were analyzed by PFGE using the CHEFF MAPPER PFGE system (Bio-Rad, Hercules, CA). The procedure for PFGE was as described previously (40) except for employing four restriction enzymes, SacII, SmaI, BssHII, and BglI (Takara Bio Inc., Shiga, Japan). BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) was utilized to compare PFGE patterns among strains.

The locations of 14 genes on the PFGE fragments were confirmed by Southern hybridization analysis. Briefly, DNA fragments in agarose gels were transferred to Biodyne A nylon membranes (Pall Biosupport, NY) before hybridization with individual digoxigenin-labeled probes prepared from the genes to be analyzed (DIG DNA labeling and detection kit; Roche Molecular Systems Inc., CA). A series of PCR analyses was carried out in order to identify chromosomal changes caused by deletions and/or recombinations, and the exact locations of the chromosomal changes were then specified by nucleotide sequencing. For the PCR, DNA was extracted from the bacterial cells using IsoplantII (NIPPON GENE Co., Ltd., Tokyo, Japan) with slight modifications as described previously (38), and the amplification reactions were carried out using TaKaRa Ex Taq and TaKaRa LA Taq (Takara Bio Inc., Shiga, Japan) according to the manufacturer's specifications.

The biochemical characteristics of the strains were examined using the Staphyogram kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and antibiotic MICs were determined by the agar dilution method recommended by the CLSI (formerly NCCLS).

URLs.

Comprehensive data describing the S.haemolyticus JCSC1435 genome are available from http://www.bio.nite.go.jp/dogan/Top. Genome data for comparative analysis were obtained from http://www.ncbi.nlm.nih.gov/Genomes/. NCTC 8325 data are available from http://www.genome.ou.edu/staph.html. ISSha1 is registered at the ISfinder site (http://www-is.biotoul.fr).

RESULTS

Staphylococcal chromosomes.

The S. haemolyticus strain JCSC1435 genome was composed of a chromosome of 2,685,015 bp and three plasmids of 2,300 bp, 2,366 bp, and 8,180 bp in length. Tables Tables11 and and22 show the features of the S.haemolyticus JCSC1435 genome in comparison with those of S.aureus N315 and S.epidermidis ATCC 12228. The S.haemolyticus chromosome was comparable in size to those of S.aureus and S.epidermidis. The G+C content and the numbers of orfs and ribosomal and tRNA genes were also similar among the three species.

TABLE 1.
General features of S. haemolyticus JCSC1435 chromosome in comparison with those of other staphylococci
TABLE 2.
General features of S. haemolyticus JCSC1435 plasmids in comparison with those of other staphylococci

BLASTN analyses of the three chromosomes (Fig. 1a to c) revealed short segments of similarity forming the diagonal line in each plot, indicating overall homology of the staphylococcal chromosomes. Thus, a large proportion of the orfs in the three species were conserved in their sequences and their gene order on the chromosome. Indeed, the orfs found as orthologues (transferred from a common ancestral orf) in the three staphylococcal species showed an average of 78% identity. However, chromosome regions unique to each species were also found which distributed near oriC (the origin of chromosomal DNA replication), depicted in the bottom left of each plot in Fig. Fig.1,1, and in short gaps on the diagonal line that corresponded to GIs.

FIG. 1.
Homologous regions between pairs of staphylococcal chromosomes computed with BLASTN. DNA sequence homology between two staphylococcal species was computed with BLASTN and plotted as segmented lines. Regions with little homology between a pair of chromosomes ...

Taking a closer look, we identified a short diagonal line that extends from the bottom left corner in each panel of Fig. 1a to c, indicating that the sequences of the three chromosomes were well conserved (>71% identity) in the region from oriC to orfX (an orf of unknown function) located at 30 kbp. The high nucleotide homology in this region was expected, since this region contains the origin of replication and genes essential for replication. However, the region beyond orfX down to 0.4 to 0.6 Mbp (gray in Fig. Fig.1),1), which contained the staphylococcal cassette chromosomes (SCCs) (15), showed few segments of similarity in the plot, indicating that the region has little homology among species. In other words, this region was characterized by gene compositions that varied markedly from one species to another. We therefore designated this species-specific region the “oriC environ.” Since the downstream boundary of this region was not clearly seen in the BLASTN plot, we employed comparative analysis to formally define and characterize this region (described below).

Gene distribution.

We compared the three genomes by chromosomal distribution patterns of common and unique orfs (Fig. (Fig.2).2). For this purpose, we first grouped all orfs from the staphylococcal and related species into sets of homologous orfs (homologs; see Materials and Methods for the definition). Each homolog was classified phylogenetically according to the inclusion of its orfs in the staphylococcal genomes, and a colored bar code was assigned as indicated in Fig. Fig.2a.2a. Figure Figure2b2b illustrates the distribution of the homologs of various phylogenetic classes across each chromosome. This analysis was intended to reveal the evolutionary makeup of the chromosome on the assumption that the chromosomal regions vertically transmitted from a common ancestor comprise homologs common among the three staphylococcal species whereas those acquired horizontally by lateral gene transfer should comprise homologs unique to a species or some of its strains.

FIG.2.
Distribution of homologs across the chromosomes of three staphylococcal species. (a) Left: fine phylogenetic classification and coloration. Homologs are classified phylogenetically into 17 classes in the Venn diagram according to their distribution among ...

The black bars in Fig. Fig.2b2b represent the staphylococcal homologs shared by all strains of the three staphylococcal species (SASESH in Fig. Fig.2a).2a). Approximately 46% of orfs in each species belonged to this class. As shown in Fig. Fig.3,3, the common homologs are predicted to function in membrane bioenergetics (functional category I-4), metabolism of nucleotides (II-3), synthesis of aminoacyl-tRNA (III-7-2), or as ribosomal proteins (III-7-1), all of which are essential for bacterial viability. Thus, the chromosomal regions dense with black bars were considered to be those transmitted vertically from an ancestral chromosome, forming the “genomic backbone” of the staphylococcal chromosome.

FIG. 3.
Correlation between phylogenetic and functional categories of the homologs distributed among three staphylococcal species and the functional properties of the oriC environ. The relative proportions of biological function (20) encoded by homologs of each ...

On the contrary, the chromosomal regions containing few black bars corresponded to GIs possibly acquired by lateral gene transfer. In accordance with this view, orfs with anomalous codon usage patterns and biased G+C content at the third codon (GC3) (22) were frequently observed in these regions (Fig. (Fig.2b).2b). The oriC environ (in gray) was the largest region scarce in black bars. However, as discussed below, a region with few black bars was also present on the other side (upstream region) of oriC.

Although 73 of 1,158 homologs common to staphylococci (SASESH in Fig. Fig.2a)2a) and 1 of 129 homologs common to S.epidermidis and S.haemolyticus (SESH) were shared with B. subtilis, the other species (Streptococcus pyogenes, Lactococcus lactis, Clostridium perfringens, and Deinococcus radiodurans) shared no homologs with staphylococci.

Characteristic biological features of each staphylococcal species found by comparative analysis.

Figure Figure33 illustrates the putative biological function of the orfs that constituted the genomes of S.aureus, S.epidermidis, and S.haemolyticus. Since most genes that were not commonly found in all three species have an unknown function, we cannot infer all of the physiological features of these species simply from the presence or absence of specific genes. Nevertheless, some of the characteristic biological features of each staphylococcal species can be described by this analysis.

As many as 6% of the orfs unique to S.aureus encode pathogenic factors (functional category IV-6), as has been described previously (3, 4, 9, 13, 22). In contrast, pathogenic factors constituted only 2% of the orfs unique to S.epidermidis and S.haemolyticus. Key biological traits that distinguish S.aureus from other staphylococcal species, such as the production of coagulase and DNase, were reconfirmed by the comparative genomic analyses, since the genomes of the other staphylococcal species lacked these genes.

As shown in Fig. Fig.3,3, the S.epidermidis genome included many orfs belonging to the detoxification category (IV-2), which protects the bacterium from toxic substances, as well as genes related to bacteriocin production (IV-3). Compared with the other species, a high proportion of the S.epidermidis orfs were dedicated to specific pathways (II-1-1) not associated with the Embden-Meyerhof glycolysis pathway but involving the metabolisms of glycerol and other sugars.

S.haemolyticus contained more orfs for the regulation of RNA synthesis (III-5-2) than the other species (Fig. (Fig.3).3). Genes involved in transport of ribose and ribitol, essential components of nucleic acid and cell wall teichoic acid biosynthesis, respectively, were also unique to S.haemolyticus. Such orfs encoding d-mannonate dehydrolase, β-glucuronidase, and l-ribulokinase were specific to S.haemolyticus, as well as orfs involved in amino acid and coenzyme biosynthesis (II-5). The latter finding is consistent with the observation that strain JCSC1435 required only arginine for growth in defined medium (11), in contrast to S.aureus N315, which requires alanine, glycine, isoleucine, arginine, valine, and proline for growth (22).

S.haemolyticus had some unique orfs that were likely involved in bacterial pathogenesis (IV-6). As the name of the species implies, S.haemolyticus is generally hemolytic on blood agar plates. At least three orfs (SH0871, SH1134, and SH2193) showed homology to known staphylococcal hemolysins. A conserved-domain search found that SH0871 shared a motif with the Bacillus cereus hemolytic protein, and it showed 49% amino acid identity to the streptococcal hemolysin III. SH1134 shared a motif with the Aeromonas hydrophila hemolytic protein, and it showed 60% identity to the Bacillus halodurans α-hemolysin. SH2193 also shared a motif for “hemolysin and related molecules,” and it was 39% identical to a Helicobacter pylori hemolysin. However, none of these genes were located in GIs as described below.

Among the orfs carried by S.epidermidis and S. haemolyticus but not by S.aureus (SESH in Fig. Fig.3)3) were enzymes for synthesis of the poly-gamma-glutamate capsule (II-2) that is produced by certain Bacillus species, as well as by a number of coagulase-negative staphylococci (19). The capsule protects S.epidermidis against cationic antimicrobial peptides and likely plays a role in osmoprotection, even though its production in S.epidermidis is ~1 million-fold lower than in Bacillus anthracis (19). In addition, more orfs involved in heavy metal resistance (I-2), biosynthesis, and phosphorylation of thiamine (II-5), polyphosphate kinase (II-6), and sulfur metabolism (II-7) were identified in S. epidermidis and S. haemolyticus than in S.aureus. Genes for biosynthesis of thiamine were located within operons SE2057 to SE2063 of ATCC 12228 and SH0559 to SH0565 of JCSC1435 but were lost in N315 by a 4-kbp deletion. On the other hand, the polyphosphate kinase gene, involved in response to stress, such as amino acid starvation (21), is widely distributed in gram-positive, gram-negative, and cyanobacterial species. Curiously, the gene was missing in streptococci, corynebacteria, and only S.aureus among the three staphylococcal species. This may be correlated with the more aggressive nature of S.aureus, which degrades human exudates or tissues with its hydrolyzing enzymes, thereby providing additional sources of nutrients and amino acids.

The urease operon (II-2), the aureolysin gene (IV-6), and the saeRS genes were located in conserved regions of the S.aureus and S.epidermidis genomes but were missing from the JCSC1435 genome (SASE in Fig. Fig.3).3). These results are consistent with the fact that both S.aureus and S.epidermidis produce urease, whereas S.haemolyticus does not. Aureolysin is a protease that may damage human tissue. S.aureus-derived aureolysin has been shown to degrade the human antibacterial peptide LL-37 (34), and therefore, it may be involved in evasion of the host immune system. saeRS genes encode a two-component regulatory system that controls the production of several virulence factors in S.aureus, including α- and β-hemolysin, coagulase, DNase, and protein A (10). The production of aureolysin and the regulation of genes in S.epidermidis by saeRS have not yet been documented.

oriC environ.

Through a comparison of the three staphylococcal chromosomes, the oriC environ emerged as a chromosomal region unique to each species. We defined the extent of the oriC environ as the sum of continuous stretches of 20-kbp windows (or segments) with few orfs common to staphylococci. The proportion of orfs representing staphylococcal common homologs within such windows was <45%. According to this definition, the oriC environ began just downstream of orfX and spanned bp 34153 to 400000 in S.aureus N315, bp 32492 to 280000 in S.epidermidis ATCC 12228, and bp 29763 to 660000 in S.haemolyticus JCSC1435 (gray regions in Fig. Fig.11 and and2b2b).

The oriC environ contained integrated copies of SCC in its left part. The SCC region was diverse at the strain level (see below), whereas the remaining oriC environ was similar within a species but diverse among the different species. In fact, the right part of the oriC environ was common to all seven sequenced S.aureus strains but diverse across species (as shown with cyan bars in the gray region of N315 in Fig. Fig.2b).2b). In the same manner, the right part of the oriC environ in S. epidermidis strain ATCC 12228 was conserved in S. epidermidis strain RP62A but not in the strains of other species (Fig. (Fig.1d1d and the magenta bars in the gray region of ATCC 12228 in Fig. Fig.2b2b).

The right part of the oriC environs encompassed only 6 to 20% of the entire chromosome but contained 15 to 30% of the species-specific homologs (SA, SE, and SH in Fig. Fig.2a).2a). Genes encoding protein A (spa), coagulase (coa), and the capsule operon (cap5[8]A to -P), important virulence determinants of S.aureus, were all contained in the right part of the oriC environ. In contrast, as mentioned above, S.epidermidis contained many homologs belonging to the detoxification category, specific pathways, and antibiotic production (such as iturin A and surfactin), which were clustered in the right part of the oriC environ (“ATCC12228 130-280 kbp” in Fig. Fig.33).

The right part of the S.haemolyticus oriC environ contained a putative operon for capsular polysaccharide biosynthesis comprised of 13 homologs (capA to -M), which explained the high proportion of genes involved in the “adaptation to atypical condition” category (IV-1) in that region (JCSC1435 120-660 kbp in Fig. Fig.3).3). Capsules are antiphagocytic polymers that protect microbes from uptake and killing by professional phagocytes. S.aureus produces a capsule encoded by cap5[8]A to -P (also located in the right part of the S.aureus oriC environ), but such homologs are missing from S.epidermidis strains. The S.haemolyticus capA to -G genes showed ≥76% amino acid similarity to the S. aureus cap5[8]A to -G genes. However, the capH to -M genes were unique to S. haemolyticus and included orfs with homology to those involved in capsule transport, glycosyl transferases, aminotransferases, and transcriptional regulators. We identified similar genes by PCR in four of six clinical isolates of S. haemolyticus. This observation suggests that the capsule operon confers a survival advantage within the human host and may have been introduced exogenously into certain S. haemolyticus strains.

S.haemolyticus JCSC1435, but not strains of other species, carried in the right part of the oriC environ a mannitol-specific phosphotransferase system (PTS) that showed 89% amino acid identity to that of S.aureus. Mannitol utilization and acid production are key metabolic properties that distinguish S.aureus from other staphylococcal species. However, some non-aureus staphylococcal clinical strains are known to ferment mannitol. S.haemolyticus strain JCSC1435 ferments mannitol, and the PTS in S.haemolyticus was located within the right part of the oriC environ 230 kbp downstream of oriC. In contrast, the PTS in S.aureus was located at a fixed locus in all seven sequenced strains—at 2,210 kbp on the N315 chromosome. Since the mannitol PTS was missing from half of the S.haemolyticus isolates (4) and was easily deleted from strain JCSC1435 (see below), it is likely that this locus was horizontally acquired in the oriC environ of this strain.

It is likely that the oriC environ is present in bacteria other than those of the genus Staphylococcus. For example, we identified a previously unrecognized species-specific region downstream of oriC in the genomes of three different Bacillus species (see supplementary Fig. Fig.11 in reference 32 and Fig. Fig.44 in reference 36.

FIG. 4.
Large-scale chromosomal inversion and the possible involvement of the oriC environ in the process as suggested by a comparison of the distribution of the homologs common to the three staphylococcal species. For each strain, the homologs common to 10 strains ...

SCC.

Between nucleotide positions 29763 and 120899 of the S.haemolyticus genome, we identified one intact SCC and a few remnants of SCC in tandem within the oriC environ. The region contained at least six copies of the integration site sequence for SCC (ISS) in addition to the one in orfX, which divided the left side of the oriC environ into six parts. Since integration of each SCC generates an additional ISS at the chromosome-SCC junction (16), this suggests that up to six different SCCs carrying different sets of foreign genes were integrated sequentially into the region. Only one SCC retained the cassette chromosome recombinase (ccrC) (16) gene that is essential for SCC movement. Other SCCs were devoid of the ccr gene and were thus considered remnants of SCC and designated ΨSCC. Therefore, the structure of the region was described as orfX-ΨSCCh1-ΨSCCh2-SCCh1435-ΨSCCh3-ΨSCCmec(h1435)-ΨSCCh4 from left to right in this order. ΨSCCmec carried the mecA gene encoding methicillin resistance and the ars operon encoding arsenical resistance. These systems likely contribute to the characteristic multiresistant phenotype of strain JCSC1435. ΨSCCh1 also encoded the potassium-transporting ATPase genes (kdp).

S.epidermidis ATCC 12228 also had a large (102-kbp) SCC cluster region in which eight copies of ISS were distributed. The region was composed of two SCCs carrying ccrAB genes and five ΨSCCs. The region contained many transporter genes, such as an arsenical pump, an arginine/ornithine antiporter, a cadmium efflux system, and mercuric transporters. Two orfs encoding cell wall synthesis enzymes, a penicillin-binding protein (similar to PBP4), and a teichoic acid biosynthesis protein (similar to TagF) were also identified within this region. Since ATCC 12228 is not methicillin resistant, SCCmec was not found in the SCC cluster. However, the strain seems to have accumulated a considerable range of resistance genes to counter exposure to toxic substances other than antibiotics.

In contrast to the complexity of the oriC environs in S.epidermidis and S.haemolyticus, only a few copies of ISS are found in the S.aureus oriC environ, and SCCmec is the unique copy in most methicillin-resistant S.aureus strains. The SCCmec is a multidrug resistance element carrying resistance genes for antibiotics such as methicillin, macrolides, aminoglycosides, tetracycline, and even bleomycin, an anticancer chemotherapeutic agent (22).

Among staphylococcal GIs, SCC carries the largest number of nonessential orfs that are shared across the three species (Fig. (Fig.2b).2b). This observation is consistent with the view that the SCC can transfer useful genes across boundaries of bacterial species (15). Acquisition of SCCmec, carrying multidrug resistance genes, converts S.aureus into methicillin-resistant S.aureus, a difficult-to-treat organism found in both hospitals and the community (29). Some SCCs in S.aureus also carry virulence genes, such as those encoding a superantigen (seh) (3) or capsule expression (cap1) (25).

GIs in other chromosomal regions.

Figure Figure2b2b highlights the S.haemolyticus chromosomal regions containing few black bars (staphylococcal common homologs). These unique regions were the oriC environ, two integrated plasmids, πSh1 (SH2296 to SH2325) and πSh2 (SH2598 to SH2621), two prophages, [var phi]Sh1 (SH1746 to SH1808 with integrase SH1808) and [var phi]Sh2 (SH2331 to SH2396 with integrase SH2396), and three other GIs, νSh1 (SH1003 to SH1016 with integrase SH1003), νSh2 (SH2076 to SH2103 with integrase SH2102), and νSh3 (SH2565 to SH2581 with integrase SH2581). Both of the integrated plasmids had truncated rep genes, indicating that they had lost replication capability and were maintained in the chromosome.

The integrated plasmid πSh1 carried macrolide resistance genes (msrSA and mphBM) and cadmium resistance genes (cadD and cadX). πSh2 harbored ABC transporter genes, possibly constituting a multidrug efflux pump. In addition to these integrated plasmids, we identified three novel S.haemolyticus plasmids (pSHaeA, pSHaeB, and pSHaeC) that carried antibiotic resistance markers (Tables (Tables22 and and3).3). pSHaeA encoded fosfomycin resistance (fosB), pSHaeB encoded macrolide-lincosamide-streptogramin B resistance (ermC), and pSHaeC carried two detoxification-related genes (a multidrug efflux transporter and a cytochrome P450 homologue). Therefore, accumulation of drug resistance plasmids in integrated or free forms contributed to the multidrug resistance of S. haemolyticus strain JCSC1435. In contrast, only one plasmid carrying a β-lactamase gene and a cadmium resistance gene was found in S. aureus N315 (22), and only one plasmid encoding tetracycline resistance was identified in S. epidermidis ATCC 12228 (41).

TABLE 3.
Susceptibility to antibiotics and the genes conferring resistance of S. haemolyticus JCSC1435

The size of prophage [var phi]Sh1 (49 kbp) in JCSC1435 was slightly greater than that of the staphylococcal phages so far identified (41 to 46 kbp). Integration of the 6-kbp Tn552 (conferring β-lactam resistance) into [var phi]Sh1 accounted for its large size. [var phi]Sh2 carried a truncated mercuric reductase homologue, and the other S.haemolyticus GIs encoded hypothetical proteins similar to sporulation control protein Spo0M (νSh1) and a Na+-transporting ATP synthase (νSh3), but most of the genes within the GIs had an unknown function. In contrast, antibiotic resistance genes in the S.aureus chromosome were confined to SCCmec, and only two orfs encoding proteins similar to β-lactamase are localized within νSaβ and νSa4 of the N315 chromosome (22).

There were 57 orfs associated with virulence in S. haemolyticus (Table (Table4)4) and 39 in S.epidermidis; these genes were scattered all over the chromosome rather than localized in GIs. Six GIs were identified in S.epidermidis ATCC 12228 (Fig. (Fig.2b):2b): SCCs (32,492 to 134,675 bp), a prophage, [var phi]Se1 (SE1474 to SE1509 with integrase SE1509, defined as νSe2 in reference 9), and four other GIs, νSe3 (SE0568 to SE0588 with integrase SE0568), νSe4 (SE0988 to SE0994 with integrase SE0988), νSe5 (SE1463 to SE1473 with integrase SE1472) and νSe6 (SE2339 to SE2346 with fragmented integrase SE2343 to SE2345). In addition, νSeγ and νSe1 were annotated previously (9) but were not detected by our criteria. Only two candidate genes for virulence were found in the GIs. Except in SCCmec, drug resistance genes were not identified in either GIs or prophages of the S.epidermidis chromosome.

TABLE 4.
Candidate orfs for virulence found in S. haemolyticus JCSC1435

The virulence genes in S.haemolyticus and S.epidermidis were rather “benign” in nature, as indicated by surface adhesins, secretory antigens, serine proteases, and exonucleases. In contrast, S.aureus had as many as 110 virulence orfs (Fig. (Fig.2b).2b). S.aureus carried homologs of the virulence genes identified in S.haemolyticus and S.epidermidis, as well as those encoding numerous toxic proteins, such as superantigens, exotoxins, and leukocidins. Thirty of these were located within three GIs and one prophage on the S.aureus N315 chromosome (22).

The integrase of νSh1 in S.haemolyticus was highly homologous to that of νSa4 in S.aureus (88% amino acid identity), and yet the GIs are integrated at separate sites in the chromosome. A similar level of homology was observed between the integrases νSh2 and νSe3 (75%) and νSh3 and νSe6 (84%). Though these pairs of GIs were each inserted in comparable chromosomal sites (Fig. (Fig.1c),1c), the attachment site sequences for these GIs (if any), recognized as direct repeats flanking the island, were different from one another, indicating that these islands were independently acquired and specialized for each species.

Chromosomal inversions.

All the seven S.aureus strains sequenced to date have colinear chromosomes (data not shown). In contrast, the chromosomes of S.epidermidis RP62A and ATCC 12228 are inverted around oriC (Fig. (Fig.1d).1d). The chromosome of S.haemolyticus was colinear to that of S.epidermidis RP62A (Fig. (Fig.1b)1b) but had an inversion compared with that of S.aureus N315 (Fig. (Fig.1a)1a) or S.epidermidis ATCC 12228 (Fig. (Fig.1c).1c). The inversion of the S.haemolyticus JCSC1435 chromosome against that of S.aureus N315 was confirmed experimentally by a series of PCRs and PFGE-Southern blotting analyses.

Lines in Fig. Fig.44 connect the staphylococcal common homologs present in a single orf on each chromosome to show the relative orientation of vertically transmitted chromosomal regions. For the most part, the homologs were arranged colinearly across the chromosomes (connected by blue lines between chromosomes in Fig. Fig.4).4). Large chromosomal inversions among the staphylococcal strains were clearly demonstrated by this “homolog distribution analysis” (Fig. (Fig.4).4). It was noted that one of the two breakpoints of the large inversions in each chromosome was always located within the oriC (JCSC1435 and RP62A) or at its right end (N315 and ATCC 12228).

Each circular chromosomal DNA strand is divided into two parts: replichore 1, spanning clockwise from oriC downstream to terC (the putative terminus of chromosome replication), and replichore 2, spanning the rest of the circular chromosome (24). Replichore 1 has a positive G-C skew, and replichore 2 a negative G-C skew (Fig. (Fig.4).4). Although the two replichores were of similar lengths in N315, replichore 1 was longer in JCSC1435 and RP62A, and replichore 2 was longer in ATCC 12228. These imbalances in the replichore lengths seem to reflect the accumulation of many exogenous genes in certain chromosomal regions, most notably within the oriC environ.

We hypothesize that the chromosomes of S.haemolyticus JCSC1435 and S.epidermidis RP62A are colinear to the common ancestor of the three staphylococcal species and that accumulation of foreign genes in their oriC environ caused elongation of replichore 1. On the other hand, S.aureus and S.epidermidis ATCC 12228 seemed to have individually undergone a chromosomal inversion around oriC, which resulted in resolution of the imbalance (S.aureus) or alternate elongation of replichore 2 (ATCC 12228). In accordance with the assumed inversion in either strain, the oriC environ ended at one of the breakpoints of inversion, and only a few staphylococcal common homologs were found immediately upstream of the other breakpoint, where the oriC environ should have continued prior to the inversion (highlighted in pink in Fig. Fig.44).

As a result of the chromosomal inversions described above, remnants of the oriC environ of some strains were translocated upstream of oriC (to the left of oriC on the circular chromosome) in other strains. For example, the ica operon encoding the polysaccharide intercellular adhesin is found in the oriC environ of S.epidermidis strain RP62A. In contrast, the ica operon is found in a well-conserved upstream region of oriC in the S.aureus chromosomes (at kbp 2770 in strain N315). S.epidermidis strain ATCC 12228 does not carry the ica operon, but the loci flanking ica in S.epidermidis RP62A were found separated at kbp 280 and 2330, which exactly corresponded to the breakpoints of inversion in strain ATCC 12228 (Fig. (Fig.4).4). S.haemolyticus JCSC1435 did not possess an ica operon.

ISs and transposons.

A remarkable characteristic of the S.haemolyticus JCSC1435 chromosome was that it contains as many as 82 IS elements and, based on their nucleotide sequences, 60 of the ISs were intact (Table (Table5;5; Fig. Fig.5b).5b). The S.epidermidis ATCC 12228 chromosome also had many (64) IS copies, but only 18 were intact. The S.aureus chromosomes contained up to 29 IS copies with 13 intact.

FIG. 5.
Spontaneous phenotypic mutants of S.haemolyticus JCSC1435 arose due to oriC environ rearrangement. (a) PFGE banding patterns of mutant strains compared with JCSC1435. (b) Circular representation of JCSC1435 chromosome and the deletions in the mutant strains ...
TABLE 5.
Insertion sequences and transposons identified in S. haemolyticus JCSC1435 and other staphylococcal genomes

Two IS groups within S.haemolyticus, ISSha1 (a novel group) and IS1272-SH, comprised 68% of the IS elements. IS1272-SH of S.haemolyticus was a new subtype of the IS1272 family with 85% nucleotide identity to IS1272-SA of S.aureus and 81% identity to IS1272-SE of S.epidermidis. IS1272-SH in S.haemolyticus was almost identical to the IS fragment found on types I and IV SCCmec of methicillin-resistant S.aureus (MRSA) (17), suggesting that it was introduced into S.aureus from S.haemolyticus by SCCmec.

JCSC1435 carried only two types of antibiotic resistance transposons integrated in the bacterial chromosome—-two copies of Tn552 encoding β-lactamase (blaZ) and a composite transposon (Tn4001) encoding aminoglycoside resistance genes (aacA-aphD) (Table (Table5).5). S.haemolyticus did not carry Tn554, encoding macrolide resistance (ermA), which is often present in more than one copy in MRSA strains (Table (Table55).

Frequent rearrangement of S.haemolyticus chromosome.

As described above, our study revealed that S.haemolyticus JCSC1435 possessed many drug resistance genes, especially within the mobile genetic elements. The MICs of S.haemolyticus JCSC1435 with various antibiotics and the genes responsible for the resistance are summarized in Table Table3.3. S.haemolyticus was the first staphylococcal species that acquired resistance to the glycopeptide antibiotics teicoplanin and vancomycin (7, 12). In fact, JCSC1435 exhibited the highest teicoplanin resistance (MIC, 64 mg/liter) ever reported for clinical staphylococcal strains. During passage in drug-free medium, strain JCSC1435 frequently generated mutants that lost teicoplanin or methicillin resistance (Table (Table6).6). Moreover, the change in MIC was frequently accompanied by a change in the metabolic profile of the strain such that it might be misidentified by conventional typing methods and even by the criteria listed in Bergey's manual (Table 17.15 of reference 4) (Table (Table66).

TABLE 6.
Antibiotic resistance and biochemical characteristics of the mutant strains of S. haemolyticus JCSC1435

Figure Figure5a5a illustrates the PFGE patterns of the wild-type strain JCSC1435 and its mutants, showing that they are closely related strains with one or two genetic alterations in the chromosome. Subsequent analyses by PCR and sequencing revealed deletions of various sizes in the chromosomes of the mutants. As shown in Fig. Fig.5b,5b, the deleted regions of mutant strains WP12, 3DC1, 8HT4, 8GC1, and 4IA1 were all within the oriC environ, and this region was mostly deleted in mutant 3DC1.

Unlike the parental strain JCSC1435, overnight cultures of mutant WP12 did not show bacterial aggregation. Otherwise, no phenotypic change was observed with this mutant (Table (Table6).6). WP12 had a 31.7-kbp deletion flanked by two copies of ISSha1 (Fig. (Fig.5b).5b). The deleted region contained 21 orfs, including SH0326, which encodes a putative 3,608-amino-acid protein with 54% amino acid identity to a streptococcal hemagglutinin protein of S.epidermidis ATCC 12228 and a cell wall-anchored protein of Streptococcus pneumoniae TIGR4. These data suggested that loss of SH0326 resulted in the loss of bacterial cell agglutination in mutant WP12.

Mutant 3DC1 had a large deletion of 427 kbp in the oriC environ (Fig. (Fig.5b).5b). The deletion started at the end of ISSha1, located at bp 88354, but there was no IS element on the other side of the deletion. The genes mtlA and mtlF (PTS, mannitol specific factor II), argF (ornithine carbamoyltransferase), ptsG (PTS enzyme, maltose-and-glucose-specific factor II homologue), arcA (arginine deiminase), and SH0462 (conserved hypothetical protein, similar to alpha-acetolactate decarboxylase) were located in the deleted regions, which explained the inability of the mutant 3DC1 to produce acid from mannitol, to produce acetoin, or to hydrolyze arginine (Table (Table6).6). However, the mutant still utilized maltose, which suggested the presence of another maltose utilization system in the chromosome, although we could not identify it.

Mutants 8HT4, 8GC1, and 4IA1 had similar deletions of 91.9 kbp, 91.9 kbp, and 86.0 kbp, respectively, and each deletion began at the end of ISSha1 located at bp 324358 (Fig. (Fig.5b).5b). Similar to our findings with the 3DC1 mutant, there was no IS element on the other side of the deletion. The arcA gene was located in the deleted regions of all three mutants, and this was consistent with their inability to hydrolyze arginine (Table (Table6).6). Mutant 8GC1 had a second deletion (47.6 kbp) involving the methicillin resistance gene mecA. The deletion started from the boundary of IS431 in one side (at bp 95476), but no IS element was identified on the other side. We have previously shown this type of IS431-mediated chromosomal deletion in MRSA strains (37). Likewise, mutant 4IA1 also had a second deletion (54.5 kbp) in the SCC cluster region (Fig. (Fig.5b).5b). The deletion involved ΨSCCh1, ΨSCCh2, and SCCh1435 but spared ΨSCCmec, thus leaving the methicillin resistance phenotype intact. The excised region was flanked by two copies of ISS, which suggests that the deletion was catalyzed by CcrC (cassette chromosome recombinase C).

DISCUSSION

Complete genome sequencing of S.haemolyticus JCSC1435 uncovered extreme genetic flexibility of the species and also revealed how the medically important staphylococcal species diversified themselves to successfully colonize or infect the human host. Our analysis highlighted the importance of the oriC environ as the most active diversifier of the staphylococcal chromosomes. The region did not contain genes essential for bacterial viability, since most of the region could be deleted without affecting growth, as exemplified by mutant strain 3DC1. However, the oriC environ dictates many biological features that characterize each staphylococcal species. Genomic evolution in staphylococci thus represents a new paradigm in speciation that is distinct from mechanisms reported for other bacterial genera (18, 27, 30).

To identify chromosomal regions acquired by horizontal transfer, we performed a phylogenetic classification of homologs and plotted their distribution on the genome. S. aureus, S. epidermidis, and S. haemolyticus each had a characteristic region downstream of oriC that harbored species-specific genes (Fig. (Fig.2b).2b). Spontaneous mutants of S. haemolyticus JCSC1435 all had deletions within the oriC environ, which caused phenotypic alterations resulting in species misidentification (Table (Table6).6). In fact, difficulty or failure in species identification of S. haemolyticus by biochemical methods has been well documented (31, 33). Mutant strain 3DC1 lost more than 16% of its entire chromosome, which is significant, since the definition of a bacterial species is based on nucleotide sequence differences that exceed 30% (39).

The diversifying power of the oriC environ was likely a driving force for the generation of staphylococcal species capable of survival within the human host. We propose that the SCCs served as efficient vehicles for the introduction of exogenous genes into the oriC environ and that abundant IS elements and other recombinases within the region served as the machinery for excision of genes that were not beneficial to the bacterium. Genes within the oriC environ that survived selection during the long speciation process are now recognized as “species-specific” genes.

G-C skew analysis showed that replichore 1 of the S. haemolyticus JCSC1435 chromosome was significantly longer than replichore 2. This size imbalance was resolved in mutant 3DC1 by a large deletion in the oriC environ. It seems that a disproportionately long replichore 1 resulting from the expansion of the oriC environ could be deleterious to DNA replication and cell multiplication in rapidly growing cultures. Chromosomal inversion could also resolve the replichore imbalance caused by a large oriC environ. Moreover, it was notable that all the chromosome inversions observed among staphylococcal genomes had one breakpoint in the oriC environ and another in the upstream region of oriC. The prior occurrence of at least one chromosomal inversion would explain why species-specific genes were localized upstream of oriC in some strains (pink region in Fig. Fig.4)4) and within the oriC environ in other strains. This might have served to preserve certain useful foreign genes (e.g., ica genes) by transferring them to the other side of oriC, protecting them from IS-mediated deletion occurring in the oriC environ.

S.haemolyticus strain JCSC1435 possessed as many as 82 ISs. This seems to be a common feature of the species, since the S.haemolyticus type strain, ATCC 29970, isolated in the United States in 1975, shared at least 7 of the 29 ISSha1-integrated loci of JCSC1435 (Y. Morimoto, unpublished observation). These IS elements may promote frequent rearrangements in the S.haemolyticus genome, as observed in the JCSC1435 mutants, which accelerate diversification of the species for better adaptation to humans exposed to a diverse array of chemicals, including antibiotics. Moreover, we suspect that the IS elements within S.haemolyticus may contribute to the innate ability of this organism to acquire drug resistance by a mechanism other than simply increasing genome plasticity. It is known that an IS can either inactivate a gene by direct integration into the orf or activate a gene next to its integration site by providing the gene with a potent promoter (26). The abundant IS copies in S.haemolyticus may thus contribute to the potent ability of the species to acquire antibiotic resistance by activating or inactivating the genes that either regulate or mediate antibiotic resistance (12). With a detailed chromosomal map of IS integration, JCSC1435 will provide an ideal tool for elucidating the genetic mechanisms for acquisition of glycopeptide and β-lactam antibiotic resistance that poses such a difficult medical problem in modern hospitals.

In this study, we revealed characteristic genetic and physiological differences among three important staphylococcal species and identified an oriC environ within each. The virulent features of S.aureus were highlighted by comparing its genome with the genomes of the more “benign” staphylococcal species. However, S.epidermidis and S.haemolyticus are opportunistic pathogens of humans that are difficult to eradicate because of their resistance to antibiotics. Sequence information from the genomes of these organisms will provide a better understanding of their bacterial lifestyle, as well as vital information necessary for the development of novel immunotherapeutic and chemotherapeutic approaches to control them.

Acknowledgments

We thank T. Oguri for providing the S.haemolyticus strain JCSC1435 and Y. Terui, Y. Fukuhara, A. Oguchi, Y. Hongo, S. Tanigawa, M. Yanagii, and K. Isono for assistance in genome sequencing. Preliminary sequence data of NCTC8325 were obtained from the University of Oklahoma.

This work was supported by a Grant-in-Aid for 21st Century COE, a Grant-in-Aid for Scientific Research on Priority Areas (13226114) and for Scientific Research B (14370097) from the Ministry of Education, Science, Sports, Culture and Technology of Japan, and NIH grant AI29040.

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