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J Bacteriol. Aug 2006; 188(15): 5578–5585.
PMCID: PMC1540015

Sau1: a Novel Lineage-Specific Type I Restriction-Modification System That Blocks Horizontal Gene Transfer into Staphylococcus aureus and between S. aureus Isolates of Different Lineages

Abstract

The Sau1 type I restriction-modification system is found on the chromosome of all nine sequenced strains of Staphylococcus aureus and includes a single hsdR (restriction) gene and two copies of hsdM (modification) and hsdS (sequence specificity) genes. The strain S. aureus RN4220 is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from Escherichia coli. We show that it carries a mutation in the sau1hsdR gene and that complementation restored a nontransformable phenotype. Sau1 was also responsible for reduced conjugative transfer from enterococci, a model of vancomycin resistance transfer. This may explain why only four vancomycin-resistant S. aureus strains have been identified despite substantial selective pressure in the clinical setting. Using a multistrain S. aureus microarray, we show that the two copies of sequence specificity genes (sau1hsdS1 and sau1hsdS2) vary substantially between isolates and that the variation corresponds to the 10 dominant S. aureus lineages. Thus, RN4220 complemented with sau1hsdR was resistant to bacteriophage lysis but only if the phage was grown on S. aureus of a different lineage. Similarly, it could be transduced with DNA from its own lineage but not with the phage grown on different S. aureus lineages. Therefore, we propose that Sau1 is the major mechanism for blocking transfer of resistance genes and other mobile genetic elements into S. aureus isolates from other species, as well as for controlling the spread of resistance genes between isolates of different S. aureus lineages. Blocking Sau1 should also allow genetic manipulation of clinical strains of S. aureus.

Staphylococcus aureus is a commensal of the human nose and a common cause of both hospital- and community-acquired infection. It is becoming increasingly virulent and resistant to antibiotics due to the horizontal transfer of mobile genetic elements (MGE) encoding virulence and resistance genes (17). S. aureus can be classified into approximately 10 dominant lineages, each with unique surface protein profiles and each capable of causing disease (18). In addition, approximately 15% of any S. aureus genome consists of MGE, such as bacteriophage, transposons, plasmids, and pathogenicity islands (17, 18). These elements may be transferred horizontally between isolates at high frequency, both in the laboratory and in vivo (19, 21). However, this is not reflected in the epidemiological spread of resistance and virulence genes among naturally occurring S. aureus strains. For example, the transfer of vanA to S. aureus from vancomycin-resistant enterococci has been exceedingly slow (only four cases) given the high incidence of patients harboring both vancomycin-resistant enterococci and S. aureus and treated with vancomycin (30). Another example is that over more than 40 years, only some S. aureus lineages have acquired SCCmec elements (12, 25) despite the widespread use of methicillin-type antibiotics. Another example is that virulence genes in S. aureus carried on MGE include the Panton-Valentine leukocidin genes (lukS-PV and lukF-PV) found in approximately 2% of all S. aureus strains. Only very recently has this toxin been found in isolates that also carry SCCmec. These novel methicillin-resistant S. aureus (MRSA) isolates are unrelated to hospital-acquired MRSA and are responsible for emerging cases of community-acquired (CA) MRSA causing severe skin and soft tissue infections (5, 6).

In the laboratory, S. aureus is notoriously difficult to genetically manipulate and only one available strain, RN4220 (15), can accept Escherichia coli-propagated plasmids by electroporation. Consequently, most genetic studies of S. aureus are highly dependent on this strain, despite its limited clinical significance. RN4220 is a chemical mutagen of strain 8325-4, which is itself a phage-deficient variant of the clinical isolate 8325 (also known as RN1 or PS47). Neither 8325 nor 8325-4 can accept E. coli DNA, suggesting that they carry a specific, active mechanism that blocks uptake. S. aureus MGE can be moved between these laboratory strains at a high frequency via transduction, but transfer to clinical isolates is blocked. This prevents the genetic manipulation of representative clinical isolates, such as hospital-acquired MRSA and CA MRSA isolates, and hinders research. We hypothesized that S. aureus has specific mechanisms that control the ability of MGE to spread between strains and species and that this has an impact on the evolution of S. aureus and our ability to genetically manipulate clinical isolates.

One way in which bacteria may control the uptake of foreign DNA is through the use of restriction-modification (R-M) systems (22). R-M systems are widespread in many types of bacteria. R-M enzymes are complexes that identify specific DNA sequences and modify them, usually by adding a methyl group. If they detect DNA with the same specific sequence that is unmodified, such as that from a foreign bacteriophage, the DNA is digested. The role of R-M is to prevent the uptake of potentially harmful or lethal DNA, such as bacteriophage which lyses and kills bacteria, or prevent the acquisition of superfluous genes that may compromise fitness due to the metabolic demand associated with their expression (7).

There are four types of R-M systems described, including the type II restriction enzymes widely used in genetic laboratories for nucleic acid digestion at specific sequences. Type I R-M systems require three genes, hsdR (restriction), hsdM (modification), and hsdS (specificity). Each gene product forms a subunit that combines as a complex of M2S or R2M2S. M2S catalyzes the transfer of a methyl group from S-adenosylmethionine to adenine residues within a target sequence and protects the DNA from restriction. The R2M2S complex is a restriction endonuclease that recognizes DNA unmethylated at the same target sequence and cleaves at a nonspecific distant site to the target sequence. The S subunit determines the specific sequence to be methylated or restricted (22).

Mutagenised S. aureus isolates with enhanced susceptibility to bacteriophage lysis were identified many years ago, and it was proposed that R-M systems were responsible for this phenotype (11, 29). The first S. aureus R-M system to be characterized in detail was Sau3A, a type II system that specifically digests DNA at GATC sites (28). This system is uncommon in typical S. aureus isolates and probably encoded on a MGE. The serotype F bacteriophage-encoded R-M system, Sau42I, was described more recently (4). Sau42I is also relatively rare in S. aureus (16) and prevents further phage infection of lysogenized host cells, thereby protecting the interests of the phage rather than the bacterium.

In this study, we aimed to identify the major mechanisms controlling horizontal gene transfer in all S. aureus strains. Analysis of the nine available genome sequences of S. aureus strains reveals the presence of conserved open reading frames in all strains with high homology to the type 1 R-M systems of other bacteria (10). We have identified a stop mutation in the sau1hsdR gene of strain RN4220. Cloning and complementation experiments indicate that this mutation is responsible for the enhanced ability of RN4220 to accept foreign DNA. Furthermore, we show that there is substantial variation in the sau1hsdS genes in different strains. These differences correspond to the major lineages of S. aureus and suggest that horizontal gene transfer within lineages occurs at a higher frequency than between lineages.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Bacterial strains, plasmids, and bacteriophage used in this study are listed in Table Table1.1. E. coli strains were routinely grown in Luria-Bertani (LB) broth or on LB plates. Unless otherwise stated, S. aureus isolates were grown in brain heart infusion (BHI) broth or on BHI agar plates. Enterococcus faecalis isolates were routinely grown in Todd-Hewitt broth (THB) or on THB agar. Media were supplemented where appropriate with chloramphenicol (10 μg/ml), ampicillin (50 μg/ml), tetracycline (20 μg/ml), fusidic acid (25 μg/ml), or erythromycin (20 μg/ml).

TABLE 1.
Bacterial strains and plasmids used in this study

Sequencing.

PCR products were amplified using Platinum Pfx DNA polymerase (Invitrogen) with conditions of 94°C for 5 min, followed by 25 cycles of 94°C for 30 s, 52°C for 30 s, and 68°C for 210 s. PCR products were cleaned on QIAquick spin columns (QIAGEN), and both strands of the products were sequenced by Lark Technologies. PCR primers to amplify and sequence the hsd genes and their promoters were as follows: sau1hsdR, 5′-CGTTTGCGTTGATTGTATTCGG-3′ and 5′-ATGCGGATCCTACACTAATCTAGCGAGG-3′; sau1hsdM1, 5′-TCAAAATTAGCTTGAAAGATGG-3′ and 5′-AACCCTGGGAACCTCAATTCTGG-3′; sau1hsdM, 25′-CCTGAAATTAAGAAATTCATTGC-3′ and 5′-CCTGGGAATCTCAACTCTGGC-3′; sau1hsdS1, 5′-AATGCATACCTGAAAGAACTTGG-3′ and 5′-GACACTGCGCTTTCACAGTGCC-3′; and sau1hsdS2, 5′-ATGCATACCTGAAAGAACTTGG-3′ and 5′-CAATTAATAGGTTGTTATCAGG-3′.

Subsequently, all of the sau1hsdS1 genes from each of the lineages were amplified and sequenced using 5′-CATACCGAGATATGTCGATAC-3′ and 5′-CACTGTGCTATCACAGTGCC-3′.

Complementation of sau1hsdR.

Plasmid DNA, prepared from bacterial cultures using the QIAGEN plasmid mini kit, was introduced to E. coli cells by CaCl2 transformation (26). S. aureus transformations were carried out by electroporation, as previously described (27). Primers hsdRF (5′-GATCAAGCTTCGTTTGCGTTGATTGTATTCG-3′) and hsdRR (5′-ATGCGGATCCTACACTAATCTAGCGAGG-3′) were used to amplify a 3,214-bp fragment encompassing the putative sau1hsdR gene and 500 bp of its regulatory region from the strain 8325-4. The resulting PCR product was cloned into the BamHI-HindIII site of pUC18 to give pUChsdR. The insert was then subcloned into the KpnI site of pSK5632 (9) to give the plasmid phsdR. Ligations were performed using the rapid DNA ligation kit (Roche) according to the manufacturer's instructions.

Horizontal gene transfer assays and bacteriophage susceptibility.

Electroporations were performed according to the method of Schenk and Laddaga (27). The plasmid transferred was pMAD (1), a suicide vector which carries an ermC selectable marker. Plasmid DNA was extracted from E. coli DH5α host using QIAGEN plasmid midi kits, and 140 ng total plasmid DNA was added to each electroporation cuvette. Cells were electroporated and recovered for 2 h at 30°C before the entire electroporation mixture was plated and incubated for 48 h at 37°C and erythromycin-resistant colonies were counted.

For conjugation, the method of Clewell et al. (3) was used. The donor bacterial strain was E. faecalis FA373 carrying the pheromone-responsive conjugative plasmid pAM378. pAM378 is pAM373 that also carries a copy of Tn918 encoding a tet selectable marker. pAM373 encodes a pheromone receptor expressed on the E. faecalis surface that responds to pheromone produced by S. aureus. Once triggered, the E. faecalis donor produces aggregation substance and binds to S. aureus, allowing conjugative transfer of the pAM373 plasmid. The plasmid does not have a functional replication mechanism in S. aureus and is lost. However, Tn918 carried on pAM378 can jump to the S. aureus chromosome prior to loss and can be detected by resistance to tetracycline (3). An overnight culture (0.5 ml) of E. faecalis donor was mixed thoroughly with 0.05 ml of overnight S. aureus recipient strain culture and pipetted onto the surface of a Millipore filter (0.2 mm) placed on THB agar. The matings were incubated at 37°C for 18 h. The cells were then resuspended in 1.0 ml of BHI by vortexing. Aliquots (100 μl) were plated onto mannitol salt agar supplemented with tetracycline to select for S. aureus transconjugants. Mannitol salt agar contains salt which inhibits the growth of E. faecalis, and the S. aureus isolates ferment mannitol, causing their colonies to appear yellow. To allow estimation of transfer frequency per donor, aliquots were also plated on THB agar supplemented with fusidic acid, which is selective for E. faecalis. All plates were incubated at 37°C for 48 h, and colonies were counted.

For bacteriophage susceptibility assays, recipient cells were lysed with phage [var phi]75 of the international typing set, grown on either S. aureus RN4220 or S. aureus 879R4RF (3). S. aureus 879R4RF is reported to be R-M negative (29), although the reason for this phenotype is unknown and we could find no mutation in its sau1hsdR gene. It belongs to lineage CC51, as determined by a microarray analysis (18; data not shown), and has a typical sau1hsdS variant profile for this lineage. In comparison, 8325 and its derivatives are from lineage CC8. Recipient bacteria were grown in BHI until mid-log phase and centrifuged, and the pellets were resuspended in 7 ml phage buffer (1 mM MgSO4, 4 mM CaCl2, 0.1 M NaCl, 50 mM Tris-HCl, 0.1% gelatin, pH 7.8) plus 7 ml BHI. Phage stock (100 μl) was added, and the mixture was incubated at room temperature for 10 min. The tubes were transferred to a shaking water bath and incubated at 32°C and 70 rpm for 4 h and/or overnight until the cells had visibly lysed. To prepare a phage stock, lysed cultures were centrifuged to pellet any cellular debris, filter sterilized through a 0.2-μm filter, and stored at 4°C.

For transduction, the marker transferred was the small class I plasmid pT181 carried in the sequenced strain COL and carrying a tet selectable marker. The phage stock used was 80α grown on S. aureus 879R4RF transduced with the pT181 plasmid. The transduction method developed by Sean Watson in Simon Foster's laboratory was used, derived from Novick (23). Recipient S. aureus strains were grown in 50 ml LK broth (1% tryptone, 0.5% yeast extract, 0.7% KCl) overnight and resuspended in 3.5 ml of LK broth. Fifty microliters of 1 M CaCl2 and 1 ml of phage stock were added. The phage stock had a titer of 1.7 × 1011 PFU per ml when grown on RN4220. The mixture was incubated at 37°C statically for 25 min and then for 15 min in a shaking water bath at 37°C. One milliliter of ice-cold 0.02 M sodium citrate was added and centrifuged, and the pellet was resuspended in 1 ml ice-cold 0.02 M sodium citrate and left on ice for 2 h. The entire mixture was spread plated (100 ml per plate) onto LK with 1% agar plates containing 0.2 mg/ml tetracycline and incubated for 1 h at 37°C. An overlay agar (LK with 0.3% agar base and 20 mg/ml tetracycline) was carefully poured onto the plates, which were incubated for 48 h, and transductants were counted.

All bacteriophage susceptibility and horizontal transfer assays were performed in triplicate, and data are expressed as means and standard deviations. Statistical significance was determined using Student's t test.

Microarray analyses.

Microarray analyses were performed as reported previously (18). Briefly, 161 isolates of S. aureus from the noses of healthy donors and from patients with community-acquired S. aureus infection were hybridized to a seven-strain S. aureus PCR product microarray containing spots for every gene from the seven S. aureus sequencing projects (31). When designing the microarray, we specifically included spots corresponding to putative target recognition domain (TRD) regions of sau1hsdS that show major sequence variation. They were (with forward and reverse primers) as follows: N315sau1hsdS2TRD1 (5′-TGTTTTAACATCGTCAAGACAAG-3′ and 5′-ATCGTCCGACTTTTGAAGATTG-3′), MW2sau1hsdS1TRD1 (5′-CAAAGGCATACCATTTTTAAGGA-3′ and 5′-AGACCTTCTCGACTACCTCCA-3′), MW2sau1hsdS1TRD2 (5′-TTGAGAATAAGGGTGGCACTG-3′ and 5′-AAAACGCCTTTTTCCTCTCTTT-3′), MRSA252sau1hsdS2TRD2 (5′-CAAGTATATGGAGGCGGAACAC-3′ and 5′-TTTTGAAGTAATCCTTGTTTGAGA-3′), MRSA252sau1hsdS1TRD1 (5′-GGAAAAGAATATTTTGGCTCAGG-3′ and 5′-GAAACAGGGTATATGACCTTCA-3′), and MRSA252sau1hsdS1TRD2 (5′-AAAAGGCTATATGCAGAAAATC-3′ and 5′-TTAAATTTATGAATCATTCTATGTG-3′). The prefix refers to the sequenced strain used as the template in the PCR. Using GeneSpring version 7.0 (Silicon Genetics) analysis and a gene list containing 728 core variable genes (all genes minus core genes minus MGE genes), the isolates were clustered. The isolates fell into 10 dominant and some minor groups, and these groups correlated with clonal complexes determined by multilocus sequence typing (18). For this study, we focused on the microarray spots corresponding to the sau1hsdS gene variants. The presence or absence of an hsdS variant region in a test isolate is determined by its hybridization to the relevant spot (detected by Cy3 fluorescence) in comparison to hybridization of the reference strain MRSA252 (detected by Cy5 fluorescence) and is expressed as a ratio. The ratios can be visualized in GeneSpring by their color, with yellow indicating the presence of an hsdS variant region in both the test and reference strains, red in the test strain only, and blue in the reference strain only. Fully annotated microarray data have been deposited in BμG@Sbase (accession number E-BUGS-33; http://bugs.sgul.ac.uk/E-BUGS-33) and also ArrayExpress (accession number E-BUGS-33).

RESULTS

Identification of an hsdR mutation in RN4220 and complementation.

RN4220 was derived from 8325-4 by chemical mutagenesis and is widely used in the laboratory as a recipient for electroporated plasmids from E. coli (15). It seemed possible that RN4220's proficiency in accepting foreign DNA was due to some deficiency in its restriction-modification system. The S. aureus sequencing projects, including that of 8325 (accession number CP000253), identified a putative type I R-M system common to all nine sequenced S. aureus isolates. It consists of one hsdR gene found on the chromosome, an hsdMS operon on genomic island alpha (GIα), and a second hsdMS operon on GIβ. hsdR and both hsdM genes are highly homologous in the nine whole-genome sequences. They are 75% and 85% homologous to hsdR and an hsdM gene in Staphylococcus saprophyticus ATCC 15205 and 59% and 76% homologous to hsdR and an hsdM gene in Staphylococcus epidermidis RP62A. Otherwise, they do not belong to the type IA to IE families (2) but represent a new family. Following standard nomenclature (24), we propose that the genes be called sau1hsdR, sau1hsdM1, and sau1hsdM2 and that their products be called R.Sau1, M1.Sau1, and M2.Sau1, respectively. The two hsdS genes varied substantially within each isolate. In addition, there was substantial variation between hsdS genes in different sequenced isolates. They have been named according to their lineage, for example, the genes from S. aureus N315 of lineage CC5 are named sau1CC5hsdS1 and sau1CC5hsdS2 and their subunit products S1.Sau1CC5 and S2.Sau1CC5. While other putative R-M systems are identified in the whole-genome sequencing projects, they are all associated with MGE. Sau1 is the only chromosomal R-M system and the only one with widespread distribution in all S. aureus isolates.

All five hsd genes from RN4220 were sequenced and compared to those of parent strain 8325-4. The hsdR gene from RN4220 contained a G-to-A substitution, which introduces a premature TGA stop codon resulting in a truncated R.Sau1 product of 192 amino acids, which is about 20% of the wild-type length. The sequences of the other hsd genes were identical in RN4220 and 8325-4. A lack of functional R.Sau1 is predicted to prevent RN4220 from digesting foreign DNA that is unmodified at sites specified by RN4220 S1.Sau1 and S2.Sau1; however, normal modification of host DNA should be possible. The mutation would explain the ability of RN4220 to accept foreign DNA from E. coli, assuming that E. coli DNA is modified differently from RN4220 DNA. It also suggests that RN4220 should be more efficient at accepting foreign DNA from other hosts such as enterococci via conjugation. Furthermore, if there is functional variation in S1.Sau1 and S2.Sau1 in some S. aureus isolates, it suggests that RN4220 should be more susceptible to bacteriophage if that phage was grown on an S. aureus isolate with a modification or specificity system different from that of RN4220. Similarly, RN4220 should have enhanced ability to accept DNA from S. aureus isolates with different modification or specificity systems via transduction compared to 8325-4. To confirm these hypotheses, it was necessary to complement the sau1hsdR mutation and determine the ability of this derivative to accept foreign DNA. To facilitate expression of full-length sau1hsdR in RN4220, the intact sau1hsdR gene from 8325-4 was cloned into the shuttle vector pSK5632 (9) to create phsdR. RN4220 was transformed with phsdR or, as a control, pSK5632, and tested for its susceptibility to bacteriophage lysis and its ability to accept foreign DNA via electroporation, transduction, and conjugation from a variety of donors. Although pSK5632 is a multicopy plasmid, R.Sau1 must combine with M.Sau1 and S.Sau1 to form a functional enzyme complex, limiting the effect of multiple gene copies.

Horizontal gene transfer assays and bacteriophage susceptibility.

The electroporation results are presented in Fig. Fig.1A.1A. The two recipients with intact sau1hsdR, 8325-4 and RN4220 phsdR, were unable to accept DNA from E. coli. In contrast, RN4220 and the control RN4220 pSK5632, both with a mutated sau1hsdR gene, accepted plasmid pMAD from E. coli at high rates. This result shows that the complemented sau1hsdR gene is able to completely block foreign DNA uptake from E. coli by RN4220, accounting entirely for this phenotype.

FIG. 1.
Horizontal gene transfer assays with sau1hsdR-positive and sau1hsdR mutant strains. 8325-4 carries sau1hsdR, and RN4220 carries the sau1hsdR mutation. RN4220 pSK5632 carries the control plasmid, and RN4220 phsdR carries an intact copy of sau1hsdR. All ...

The conjugation transfer results are presented in Fig. Fig.1B.1B. The two recipients with a mutated sau1hsdR gene accepted Tn918 from enterococci at a rate of 80 and 73.3 transconjugants per 108 donors. In contrast, RN4220 phsdR accepted Tn918 at a frequency of 4.6 transconjugants per 108 donors, which is 16 times less efficient; this difference is statistically significant (P < 0.001). 8325-4 did not accept Tn918 in this series of experiments at a transfer rate of <2.3 transconjugants per 108 donors. These data show that RN4220 can accept foreign DNA via conjugation from enterococcal donors at a significantly higher rate than its parent 8325-4 and that this phenotype is due to sau1hsdR mutation.

RN4220 and 8325-4 (lineage CC8) differed in their susceptibilities to lysis by certain phages that have been prepared on other S. aureus strains. Cultures of RN4220, but not 8325-4, were readily lysed by particles of [var phi]75 that were produced by lytic infection of S. aureus 879R4RF (lineage CC51). Cultures of RN4220 pSK5632 were lysed by the same phage, but cultures of RN4220 phsdR were not lysed (Fig. (Fig.2).2). Thus, the mutation in sau1hsdR in RN4220 causes the bacterium to be susceptible to lysis, and the complemented sau1hsdR gene protects from lysis. Furthermore, our hypothesis predicts that phage grown on S. aureus of the same lineage as 8325-4 and RN4220 (CC8) will not be digested in 8325-4 with intact sau1hsdR. This is because the phage DNA will be modified and not digested by the Sau1 complex. When infected by [var phi]75 grown on RN4220 (using stock phage taken from the earlier experiment; RN4220 is prophage negative), all four recipients were lysed (Fig. (Fig.2).2). This shows that the modification and specificity subunits sau1hsdM and sau1hsdS are functional and sufficient to modify phage DNA as “self.” We could repeat this experiment with a range of different phages grown on 879R4RF, including [var phi]80 and [var phi]83C from the international phage typing set and the generalized transducing phages 80α and [var phi]11.

FIG. 2.
Illustration of bacteriophage susceptibility experiments. Bacteriophage [var phi]75 was grown on S. aureus 879R4RF, where we assume its DNA was modified by an R-M system. This phage could infect RN4220 pSK5632 but could not infect RN4220 phsdR as the ...

Results from the transduction experiments are presented in Fig. Fig.1C.1C. Plasmid pT181 could be transduced from 879R4RF by using generalized transducing phage 80α into RN4220 and RN4220 pSK5632. However, it could not be transferred into 8325-4 or RN4220 phsdR. pT181 could be transferred from RN4220 by using 80α into all four recipient strains (data not shown).

Distribution of hsdS types in 161 S. aureus isolates.

The seven whole-genome sequencing projects revealed that sau1hsdR and both copies of sau1hsdM are highly conserved in all strains but that significant variation occurs in the two copies of sau1hsdS genes (Fig. (Fig.3),3), both between the copies and between strains. For all strains, the two copies of sau1hsdS genes were different, and we refer to them as sau1hsdS1 and sau1hsdS2 according to the GIs they are carried on, GIα and GIβ, respectively. A short region of conserved sequence is found in the center of the gene in all cases, the central conserved region (CCR). Most sau1hsdS genes also have a conserved short 5′ end called the proximal conserved region (PrCR) as well as a 3′ end called the distal conserved region (DCR). The CCR is approximately twice the size of the PrCR or DCR. The majority of the gene variation occurs in two large regions, TRD1 and TRD2. Some strains carry the same TRD1 region in both sau1hsdS1 and sau1hsdS2. We also noted that both sau1hsdS genes were highly conserved in the sequenced isolates of the same lineage; 8325, COL, and USA300 of CC8 were the same, MW2 and MSSA479 of CC1 were the same, and N315 and Mu50 of CC5 were the same.

FIG. 3.
Variation in hsdS genes. Artemis Comparison Tool comparison of hsdS1 nucleotide sequences of (in descending order) MRSA252, 8325, N315, and MW2. The major regions marked are according to those described by Kim et al. (14) and are TRD1 and TRD2, PrCR (labeled ...

We had previously built a seven-strain S. aureus microarray and specifically designed PCR product probes corresponding to TRD1 and TRD2 regions (31). In a study of 161 community isolates of S. aureus, the isolates clustered into 10 dominant lineages and several minor lineages based on “core variable” genes, including many surface protein variants (18). Figure Figure44 shows that each lineage also has a unique pattern of sau1hsdS TRD region carriage and that there is a strong similarity between isolates of the same lineage. The sau1hsdS genes are carried on genomic islands GIα and GIβ, and these regions are highly variable. However, we have previously reported that they are conserved within lineages and appear stable (18).

FIG. 4.
Distribution of sau1hsdS TRD variant regions in 161 community isolates of S. aureus by using microarrays. Each vertical line represents an isolate of S. aureus and has been clustered into the dominant lineages (marked on the bottom row) by using core ...

Since the microarray carries TRD sequences corresponding to the first seven sequenced strains which come from four lineages (CC1, CC5, CC8, and CC30), it is likely that isolates from nonsequenced lineages carry novel sau1hsdS variants and that these regions are not represented on the microarray. We therefore sequenced sau1hsdS genes from isolates of other lineages.

Sequencing of hsdS from other lineages.

Novel sau1hsdS1 gene sequences from isolates representing lineages CC8, CC12, CC15, CC22, CC25, CC45, CC39, and CC51 were sequenced. We have deposited them in GenBank with accession numbers DQ30949 to DQ30955. A Clustal W alignment of these sequences along with those from the sequencing projects is available from the authors on request. As expected, there is enormous variety in sau1hsdS gene sequences in the different lineages, and we have identified five types of TRD1 regions and nine types of TRD2 regions which can interchange such that each isolate (representing a lineage) has its own unique combination. Interestingly, sau1CC12hsdS1 on GIα has a stop mutation leading to a truncated S1.Sau1. To confirm that this was not a mutation in a single strain, a second CC12 isolate was sequenced, and it had exactly the same stop mutation.

DISCUSSION

We have described the major mechanism in S. aureus that controls the uptake of foreign DNA in all strains of S. aureus. It is a type 1 R-M system encoded by an sau1hsdR gene on the chromosome and by two copies of sau1hsdM and sau1hsdS on genomic islands GIα and GIβ. GIα and GIβ are stable in S. aureus chromosomes, and there is no evidence that they are mobilized (18). The R-M system serves to protect the bacterial cell from phage lysis as well as stringently controls all of the major mechanisms of foreign DNA acquisition, namely, transduction, conjugation, and transformation (via electroporation). Furthermore, we prove that RN4220 has a mutation in hsdR that allows it to accept foreign DNA and thus accounts for its usefulness as an intermediary in generating genetically modified S. aureus in the laboratory.

The Sau1 type 1 R-M system found in all S. aureus strains recognizes and digests “foreign” DNA. But what is foreign? We show here that the 10 dominant lineages of S. aureus all carry unique combinations of sau1hsdS genes that control the sequence specificity of the system. Thus, DNA from S. aureus strains of different lineages is foreign. This has implications for the evolution of S. aureus. Firstly, a significant proportion of the S. aureus genome consists of MGE carrying various virulence and resistance genes. We therefore predict that MGE present in one strain will transfer horizontally to an S. aureus strain of the same lineage at a higher frequency than to S. aureus strains of other lineages. This could therefore explain why methicillin resistance (mecA) encoded on a staphylococcal cassette chromosome (SCCmec) appears to have moved into only six of the dominant S. aureus lineages (CC1, CC5, CC8, CC22, CC30, and CC45). Similarly, we might predict that if vanA transfers again into S. aureus but is not identified and contained, it may not spread immediately to other S. aureus lineages. Sau1 likely delays the evolution of new strains carrying multiple virulence and resistance gene combinations.

Secondly, each of the S. aureus lineages is very distinct from one another, differing in hundreds of gene and gene variant combinations. Within lineages, there is still some variation, particularly point mutations (10). Genetic exchange of nonmobile DNA between S. aureus isolates can occur via generalized transduction and is thought to contribute to the evolution of the different lineages (8, 18). The R-M model suggests that genetic exchange of any kind may be more efficient between isolates with the same sau1hsdS profile than between those with different profiles. Therefore, sau1hsdS variation could “define” each lineage, limiting the ability of isolates to exchange DNA with other lineages and contributing to the divergence of lineages. As S. aureus is known to be unusually clonal compared to other pathogens such as Neisseria meningitidis, the Sau1 system may provide a biological explanation for this clonality.

Recently, Kim et al. (14) determined the crystal structure of a type 1 specificity subunit from Methanococcus jannaschii and showed that the two TRDs bind independently to specific 3- to 5-base-pair DNA sequences. Both the CCR and DCR form alpha-helices that interact and form a “ruler,” separating the TRD regions. This results in a nonspecific gap in the target sequence of 6 to 8 base pairs. This structure agrees with the genomic sequence of the sau1hsdS variant genes, although we also notice a PrCR in sau1hsdS. Since both the PrCR and DCR are smaller than the CCR in S.Sau1, it is possible that they interact “end to end” to form a functional alpha-helix.

Type I R-M systems are uniquely suited for diversification of sequence specificity because only a single subunit is necessary for specificity (22). The option of two S.Sau1 specificity units in S. aureus, which presumably can interchange, adds further flexibility. Others have reported significant chromosomal hsdS gene variation between isolates of the same species (20, 22). We believe that this is the first report to link functional hsdS variation to stable and well-defined lineages of a bacterial species, suggesting a key role in controlling genetic exchange and evolution of an important bacterial pathogen.

This study also has important implications for the genetic manipulation of S. aureus in the laboratory. It is possible to devise a strategy for the manipulation of S. aureus from lineages other than CC8, including many hospital-acquired MRSA and CA MRSA isolates. Chemical mutagens of isolates from each of the dominant lineages could be screened for susceptibility to bacteriophage, and the resultant mutants would be novel “intermediate” strains for accepting foreign DNA. Bacteriophage grown on these strains would also serve as useful transducing phage for moving marked DNA into the parental clinical isolates. An alternative approach would be to artificially modify S. aureus DNA in E. coli or S. aureus carrying functional copies of the specific sau1hsdS and sau1hsdM genes before transfer.

In summary, the Sau1 R-M system is found in all S. aureus strains and controls the acquisition of DNA from other bacterial species and from S. aureus strains of other lineages. The results of this study suggest that Sau1 has contributed to the evolution of the distinct S. aureus lineages and controls the horizontal transfer of MGE that leads to the emergence of increasingly virulent and resistant strains. Manipulation of the R-M system will also enable the generation of molecular tools for the future study of clinical S. aureus, leading to greater understanding of pathogenesis and epidemiology and to new strategies for preventing and treating S. aureus infection.

Acknowledgments

This work was supported by grant G17474 from the Biotechnology and Biological Sciences Research Council, United Kingdom. The Bacterial Microarray Group at St. George's (BμG@S) is supported by the Wellcome Trust under its Functional Genomics Resources Initiative (grant number 062511).

For assistance with microarray studies, we are indebted to BμG@S (http://www.bugs.sgul.ac.uk) and particularly Jason Hinds, Adam Witney, Kate Gould, Lucy Brooks, and Philip Butcher. We thank Don Clewell, Ron Skurray, and Michel Débarbouillé for kindly donating strains and plasmids. We thank Julia Sung and Joshua Cockfield for helpful discussions.

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