A Plasmid-Borne System To Assess the Excision and Integration of Staphylococcal Cassette Chromosome mec Mediated by CcrA and CcrB.

UNLABELLED: Resistance to methicillin and other ß-lactam antibiotics in staphylococci is due to mecA, which is carried on a genomic island, staphylococcal cassette chromosome mec (SCCmec). The chromosomal excision and integration of SCCmec are mediated by the site-specific recombinase CcrAB or CcrC, encoded within this element. A plasmid-borne system was constructed to assess the activities of CcrA and CcrB in the excision and integration of SCCmec in Escherichia coli and Staphylococcus aureus. The excision frequency in E. coli mediated by CcrAB from methicillin-resistant S. aureus (MRSA) strain N315 was only 9.2%, while the integration frequency was 31.4%. In S. aureus the excision and integration frequencies were 11.0% and 18.7%, respectively. Truncated mutants identified the N-terminal domain of either CcrB or CcrA to be necessary for both integration and excision, while the C-terminal domain was important for recombination efficiency. Site-directed mutagenesis of the N-terminal domain identified S11 and R79 of CcrA and S16, R89, T149, and R151 of CcrB to be residues essential for catalytic activities, and the critical location of these residues was consistent with a model of the tertiary structure of the N terminus of CcrA and CcrB. Furthermore, CcrAB and CcrC, cloned from a panel of 6 methicillin-resistant S. aureus strains and 2 methicillin-resistant Staphylococcus epidermidis strains carrying SCCmec types II, IV, and V, also catalyzed integration at rates 1.3 to 10 times higher than the rates at which they catalyzed excision, similar to the results from N315. The tendency of SCCmec integration to be favored over excision may explain the low spontaneous excision frequency seen among MRSA strains. IMPORTANCE: Spontaneous excision of the genomic island (SCCmec) that encodes resistance to beta-lactam antibiotics (methicillin resistance) in staphylococci would convert a methicillin-resistant strain to a methicillin-susceptible strain, improving therapy of difficult-to-treat infections. This study characterizes a model system by which the relative frequencies of excision and integration can be compared. Using a plasmid-based model for excision and integration mediated by the recombinases CcrA and CcrB, integration occurred at a higher frequency than excision, consistent with the low baseline excision frequency seen in most strains. This model system can now be used to study conditions and drugs that may raise the SCCmec excision frequency and generate strains that are beta-lactam susceptible.

Characterization of the Staphylococcus aureus rRNA methyltransferase encoded by orfX, the gene containing the staphylococcal chromosome Cassette mec (SCCmec) insertion site.

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to ß-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/ß-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 µm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/ß-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.

Characterization of DNA sequences required for the CcrAB-mediated integration of staphylococcal cassette chromosome mec, a Staphylococcus aureus genomic island.

The mobile element staphylococcal cassette chromosome mec (SCCmec), which carries mecA, the gene responsible for methicillin resistance in staphylococci, inserts into the chromosome at a specific site, attB, mediated by serine recombinases, CcrAB and CcrC, encoded on the element. This study sought to determine the sequence specificity for CcrB DNA binding in vitro and for CcrAB-mediated SCCmec insertion in vivo. CcrB DNA binding, as assessed in vitro by electrophoretic mobility shift assay (EMSA), revealed that a 14-bp sequence (CGTATCATAAGTAA; the terminal sequence of the orfX gene) was the minimal requirement for binding, containing an invariant sequence (TATCATAA) found in all chromosomal (attB) and SCCmec (attS) integration sites. The sequences flanking the minimal attB and attS binding sites required for insertion in vivo were next determined. A plasmid containing only 37 bp of attS and flanking sequences was required for integration into the attB site at 92% efficiency. In contrast, at least 200 bp of sequence within orfX, 5' to the attB core, and 120 bp of specific sequence 3' to the orfX stop site and attB core were required for the highest insertion frequency. Finally, an attS-containing plasmid was inserted into wild-type Staphylococcus aureus strains without integrated SCCmec (methicillin susceptible) at various frequencies which were determined both by sequences flanking the att site and by the presence of more than one att site on either the chromosome or the integration plasmid. This sequence specificity may play a role in the epidemiology of SCCmec acquisition.

Spontaneous staphylococcal cassette chromosome mec element excision in Staphylococcus aureus nasal carriers.

Among 23 patients carrying methicillin-resistant Staphylococcus aureus (MRSA) in their anterior nares, 6 (26%) also carried methicillin-susceptible S. aureus (MSSA) as less prevalent flora. In 4 of the 6 patients, the MSSA was unrelated to prevalent MRSA, as determined by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal protein A (spa) typing. However, in two patients, the strains were identical except for the absence of spontaneous staphylococcal cassette chromosome mec (SCCmec). We consider this evidence of spontaneous SCCmec excision in vivo.

Clinical significance of Staphylococcus lugdunensis isolated from routine cultures.

Over 1 year, 42 Staphylococcus lugdunensis isolates, identified by phenotypic and genotypic testing, were recovered from clinical specimens. Thirty-six (86%) were clinically significant pathogens, mostly from healthy outpatients; 16 (44%) of 36 were isolated in pure culture; and 30 (83%) of 36 were from skin and soft-tissue infections.

Roles of CcrA and CcrB in excision and integration of staphylococcal cassette chromosome mec, a Staphylococcus aureus genomic island.

The gene encoding resistance to methicillin and other beta-lactam antibiotics in staphylococci, mecA, is carried on a genomic island, SCCmec (for staphylococcal cassette chromosome mec). The chromosomal excision and integration of types I to IV SCCmec are catalyzed by the site-specific recombinases CcrA and CcrB, the genes for which are encoded on each element. We sought to identify the relative contributions of CcrA and CcrB in the excision and integration of SCCmec. Purified CcrB but not CcrA was shown to mediate the gel shift of chromosomal target integration sequences (attB) in electrophoretic mobility shift assays. However, preincubation of CcrB-DNA complexes with increasing concentrations of CcrA blocked gel shift. The interaction of CcrB and CcrA was confirmed by Escherichia coli two-hybrid analysis. SCCmec excision mediated by plasmid-encoded and inducible ccrA, ccrB, or both genes was assessed by PCR in Staphylococcus aureus. CcrB alone could mediate excision but excision was at an alternate att site (attR2) within the right extremity of SCCmec. In contrast, both CcrB and CcrA were required to mediate excision at the chromosomal attB site (called attR when SCCmec is integrated). Insertion of a plasmid containing the SCCmec att site (attS) into the chromosome required both CcrA and CcrB, but CcrA overexpression lowered integration frequency. Thus, while CcrB binds DNA, interaction between CcrA and CcrB, in a precise ratio, is required for attB site-specific excision and SCCmec chromosomal insertion.

Differential expression of ccrA in methicillin-resistant Staphylococcus aureus strains carrying staphylococcal cassette chromosome mec type II and IVa elements.

Excision of staphylococcal cassette chromosome mec (SCCmec) is mediated through the ccrA- and -B-encoded recombinases. We investigated the effects of different antimicrobial agents on ccrA expression by using a ccrA::lacZ fusion and reverse transcription-PCR with methicillin (meticillin)-resistant Staphylococcus aureus strains MW2 (SCCmec IVa) and N315 (SCCmec II). Upregulation of ccrA was observed upon exposure to beta-lactam antibiotics. Vancomycin increased ccrA expression in MW2 but had no effect on N315. Vancomycin may contribute to the transfer of SCCmec IVa but have no effect in SCCmec II.

Analysis of the genotype and virulence of Staphylococcus epidermidis isolates from patients with infective endocarditis.

Staphylococcus epidermidis is one of the most common causes of infections of prosthetic heart valves (prosthetic valve endocarditis [PVE]) and an increasingly common cause of infections of native heart valves (native valve endocarditis [NVE]). While S. epidermidis typically causes indolent infections of prosthetic devices, including prosthetic valves and intravascular catheters, S. epidermidis NVE is a virulent infection associated with valve destruction and high mortality. In order to see if the differences in the course of infection were due to characteristics of the infecting organisms, we examined 31 S. epidermidis NVE and 65 PVE isolates, as well as 21 isolates from blood cultures (representing bloodstream infections [BSI]) and 28 isolates from nasal specimens or cultures considered to indicate skin carriage. Multilocus sequence typing showed both NVE and PVE isolates to have more unique sequence types (types not shared by the other groups; 74 and 71%, respectively) than either BSI isolates (10%) or skin isolates (42%). Thirty NVE, 16 PVE, and a total of 9 of the nasal, skin, and BSI isolates were tested for virulence in Caenorhabditis elegans. Twenty-one (70%) of the 30 NVE isolates killed at least 50% of the worms by day 5, compared to 1 (6%) of 16 PVE isolates and 1 (11%) of 9 nasal, skin, or BSI isolates. In addition, the C. elegans survival rate as assessed by log rank analyses of Kaplan-Meier survival curves was significantly lower for NVE isolates than for each other group of isolates (P < 0.0001). There was no correlation between the production of poly-beta(1-6)-N-acetylglucosamine exopolysaccharide and virulence in worms. This study is the first analysis suggesting that S. epidermidis isolates from patients with NVE constitute a more virulent subset within this species.

Spontaneous deletion of the methicillin resistance determinant, mecA, partially compensates for the fitness cost associated with high-level vancomycin resistance in Staphylococcus aureus.

Treatment of infections caused by Staphylococcus aureus is often confounded by the bacterium's ability to develop resistance to chemotherapeutic agents. Methicillin-resistant S. aureus (MRSA) arises through the acquisition of staphylococcal chromosomal cassette mec (SCCmec), a genomic island containing the methicillin resistance determinant, mecA. In contrast, resistance to vancomycin can result from exposure to the drug, a mechanism that is not dependent upon a gene acquisition event. Here we describe three MRSA strains that became resistant to vancomycin during passage in the presence of increasing concentrations of the drug. In each case two derivative strains were isolated, one that had lost mecA and one that retained mecA during passage. Strain 5836VR lost mecA by the site-specific chromosomal excision of SCCmec, while the other two strains (strains 3130VR and VP32) deleted portions of their SCCmec elements in a manner that appeared to involve IS431. Conversion to vancomycin resistance caused a decrease in the growth rate that was partially compensated for by the deletion of mecA. In mixed-culture competition experiments, vancomycin-resistant strains that lacked mecA readily outcompeted their mecA-containing counterparts, suggesting that the loss of mecA during conversion to vancomycin resistance was advantageous to the organism.

Gene acquisition at the insertion site for SCCmec, the genomic island conferring methicillin resistance in Staphylococcus aureus.

Staphylococcus aureus becomes resistant to methicillin by acquiring a genomic island, known as staphylococcal chromosome cassette mec (SCCmec), which contains the methicillin resistance determinant, mecA. SCCmec is site-specifically integrated into the staphylococcal chromosome at a locus known as the SCCmec attachment site (attB). In an effort to gain a better understanding of the potential that methicillin-sensitive S. aureus (MSSA) isolates have for acquiring SCCmec, the nucleotide sequences of attB and surrounding DNA regions were examined in a diverse collection of 42 MSSA isolates. The chromosomal region surrounding attB varied among the isolates studied and appears to be a common insertion point for acquired foreign DNA. Insertions of up to 15.1 kb were found containing open reading frames with homology to enterotoxin genes, restriction-modification systems, transposases, and several sequences that have not been previously described in staphylococci. Two groups, containing eight and four isolates, had sequences found in known SCCmec elements, suggesting SCCmec elements may have evolved through repeated DNA insertions at this locus. In addition, the attB sequences of the majority of MSSA isolates in this collection differ from the attB sequences of strains for which integrase-mediated SCCmec insertion or excision has been demonstrated, suggesting that some S. aureus isolates may lack the ability to site-specifically integrate SCCmec into their chromosomes.

Potential associations between hematogenous complications and bacterial genotype in Staphylococcus aureus infection.

BACKGROUND: The impact of bacterial clonality on infections caused by Staphylococcus aureus is unclear. METHODS: Three hundred seventy-nine S. aureus isolates (125 methicillin-resistant S. aureus [MRSA] and 254 methicillin-susceptible S. aureus [MSSA]) were genotyped by spa typing and multilocus sequence typing. For MRSA isolates, the staphylococcal chromosomal cassette mec (SCCmec) element was also typed. Three clinical categories were identified: nasal carriage only (n=118), uncomplicated infection (n=104), and bacteremia with hematogenous complications (n=157). RESULTS: By use of eBURST, 18 clonal complexes (CCs) were found in 371 isolates. Eight CCs accounted for 89% of isolates and occurred in all clinical categories. CC5 (P=.0025) and CC30 (P=.0308) exhibited a significant trend toward more frequent hematogenous complications. Isolates within spa types 2 and 16 showed the same significant trend and grouped within CC5 and CC30, respectively. SCCmec II isolates also showed the same significant trend compared with SCCmec IV; 96% were CC5 or CC30. CONCLUSIONS: Although most S. aureus genotypes exhibited the capacity to cause invasive disease, strains within CC5 and CC30 exhibited a significant trend toward increasing levels of hematogenous complications. Isolates within these CCs were also implicated by use of spa and SCCmec typing. The genetic determinants underlying these findings remain to be demonstrated.

Use of outer surface protein repeat regions for improved genotyping of Staphylococcus epidermidis.

Staphylococcus epidermidis is an important nosocomial pathogen, but little is known of its epidemiology. Accurate, reproducible typing systems would greatly improve epidemiologic investigations of S. epidermidis. The sequence-based typing technique most recently evaluated, multilocus sequence typing (MLST), often lacks discrimination and can be expensive. PCR and sequence-based analyses of the serine-aspartate repeat region of sdrG (Fbe) and the repeat region of the accumulation-associated protein gene (aap) were evaluated for the ability to discriminate among previously well-characterized S. epidermidis clinical isolates. Forty-eight strains were investigated, with sdrG found in 100% and aap found in 79% of all strains tested. Both genes demonstrated PCR product size and nucleotide sequence variation. Each system by itself gave an index of discrimination similar in value to that of MLST (0.924 and 0.953 compared to 0.96), but discrimination was further improved when combinations of the three systems were used. We conclude that typing systems using amino acid and nucleotide repeat regions of the S. epidermidis surface proteins SdrG and Aap show promise as typing tools and should be investigated using a larger panel of clinically relevant isolates.

Lack of relationship between purine biosynthesis and vancomycin resistance in Staphylococcus aureus: a cautionary tale for microarray interpretation.

Previous microarray data (E. Mongodin, J. Finan, M. W. Climo, A. Rosato, S. Gill, and G. L. Archer, J. Bacteriol. 185:4638-4643, 2003) noted an association in two vancomycin-intermediate Staphylococcus aureus (VISA) strains between high-level, passage-induced vancomycin resistance, a marked increase in the transcription of purine biosynthetic genes, and mutation of the putative purine regulator purR. Initial studies to report on the possible association between vancomycin resistance and alterations in purine metabolism in one of these strains (VP-32) confirmed, by Western analysis, an increase in the translation of PurH and PurM, two purine pathway enzymes. In addition, PurR was identified, by knockout and complementation in a vancomycin-susceptible strain, as a repressor of the purine biosynthetic operon in S. aureus, and the PurR missense mutation was shown to inactivate the repressor. However, despite the apparent relationship between increased purine biosynthesis and increased vancomycin resistance in VP-32, neither the addition of exogenous purines to a defined growth medium nor the truncation or inactivation of purR improved the growth of vancomycin-susceptible S. aureus in the presence of vancomycin. Furthermore, the passage of additional vancomycin-susceptible and VISA strains to high-level vancomycin resistance occurred without changes in cellular purine metabolism or mutation of purR despite the development of thickened cell walls in passaged strains. Thus, we could confirm neither a role for altered purine metabolism in the development of vancomycin resistance nor its requirement for the maintenance of a thickened cell wall. The failure of biochemical and physiological studies to support the association between transcription and phenotype initially found in careful microarray studies emphasizes the importance of follow-up investigations to confirm microarray observations.

Improved multilocus sequence typing scheme for Staphylococcus epidermidis.

We evaluated three multilocus sequence typing (MLST) schemes for Staphylococcus epidermidis and selected the seven most discriminatory loci for the formation of a new, more powerful MLST scheme. This improved scheme gave 31 sequence types (STs) and 5 clonal complexes (CCs), whereas the other schemes delineate 16 to 24 STs and 1 to 3 CCs.

Vancomycin-intermediate Staphylococcus aureus strains have impaired acetate catabolism: implications for polysaccharide intercellular adhesin synthesis and autolysis.

The most common mechanism by which Staphylococcus aureus gains resistance to vancomycin is by adapting its physiology and metabolism to permit growth in the presence of vancomycin. Several studies have examined the adaptive changes occurring during the transition to vancomycin-intermediate resistance, leading to a model of vancomycin resistance in which decreased cell wall turnover and autolysis result in increased cell wall thickness and resistance to vancomycin. In the present study, we identified metabolic changes common to vancomycin-intermediate S. aureus (VISA) strains by assessing the metabolic and growth characteristics of two VISA strains (vancomycin MICs of 8 microg/ml) and two isogenic derivative strains with vancomycin MICs of 32 microg/ml. Interestingly, we observed the parental strains had impaired catabolism of nonpreferred carbon sources (i.e., acetate), and this impairment became more pronounced as vancomycin resistance increased. To determine if acetate catabolism impairment is common to VISA strains, we assessed the ability of VISA and vancomycin-sensitive S. aureus (VSSA) clinical isolates to catabolize acetate. As expected, a significantly greater percentage of VISA strains (71%) had impaired acetate catabolism relative to VSSA (8%). This is an important observation because staphylococcal acetate catabolism is implicated in growth yield and antibiotic tolerance and in regulating cell death and polysaccharide intercellular adhesin synthesis.

Successful therapy of experimental endocarditis caused by vancomycin-resistant Staphylococcus aureus with a combination of vancomycin and beta-lactam antibiotics.

VRS1 is the first isolated strain of vancomycin-resistant Staphylococcus aureus (VRSA) found to carry the vanA gene complex previously described in Enterococcus. Under vancomycin pressure, VRS1 makes aberrant cell walls consisting of stem tetrapeptide and depsipeptide that lack the terminal D-Ala-D-Ala residues targeted by vancomycin. Previous data have suggested that this aberrant cell wall is not cross-linked by PBP2a, the enzyme responsible for cell wall transpeptidation in the presence of beta-lactam antibiotics. We examined the efficacy of treating VRS1 with a combination of vancomycin and beta-lactam antibiotics in vitro and in vivo. We found that the MIC of oxacillin for VRS1 decreased from >256 microg/ml to <1 microg/ml in the presence of vancomycin. Using the rabbit model of endocarditis, we treated VRS1-infected rabbits with nafcillin alone, vancomycin alone, or a combination of nafcillin and vancomycin. Treatment with nafcillin in combination with vancomycin cleared bloodstream infections within 24 h and sterilized 12/13 spleens (92%), as well as 8/13 kidneys (62%), following 3 days of treatment. Mean aortic valve vegetation counts were reduced 3.48 log(10) CFU/g with the combination therapy (compared to untreated controls) and were significantly lower than with either vancomycin or nafcillin given alone. VRS1 was extremely virulent in this model, as no untreated rabbits survived the 3-day trial. Treatment of clinical infections due to VRSA with the combination of vancomycin and beta-lactams may be an option, based on these results.

A subset of Staphylococcus aureus strains harboring staphylococcal cassette chromosome mec (SCCmec) type IV is deficient in CcrAB-mediated SCCmec excision.

The gene encoding resistance to beta-lactam antibiotics in the staphylococci is found on the chromosome in a genomic island designated staphylococcal cassette chromosome mec, or SCCmec. In addition to the resistance gene mecA, SCCmec also contains site-specific recombinase genes that are capable of catalyzing the chromosomal excision and reintegration of SCCmec. SCCmec is found in five major isotypes partially defined by the recombinase genes present, either ccrAB or ccrC. Of these, SCCmec type IV is presumed to be mobile in the environment, and this mobility may be partially responsible for the rise in community-associated methicillin-resistant staphylococcal infections. In this study, we investigate the presumptive first step in type IV SCCmec mobility: chromosomal excision of the element. CcrAB from a panel of six Staphylococcus aureus and four Staphylococcus epidermidis strains were able to catalyze chromosomal excision of SCCmec types I and II, indicating that these proteins maintain recombinase activity despite varying by up to 3.7% at the amino acid level. Excision of type IV SCCmec was not universally seen, as a subset of S. aureus strains with type IV SCCmec did not excise their element. These strains are all highly related and represent a lineage of successful community-associated pathogens. In addition, the inability to excise SCCmec in these strains is associated with the insertion of a presumptive mobile element containing the gene for staphylococcal enterotoxin H (seh) immediately downstream of SCCmec on the chromosome. Acquisition of this mobile element, containing a known virulence gene, appears to have stabilized the chromosomal integration of the methicillin resistance gene in these strains.

Structure of the MecI repressor from Staphylococcus aureus in complex with the cognate DNA operator of mec.

The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of beta-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 A resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI-mec complex, but unlike the MecI-bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.

Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain.

Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the approximately 2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the approximately 2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis.

Crystal structures of the BlaI repressor from Staphylococcus aureus and its complex with DNA: insights into transcriptional regulation of the bla and mec operons.

The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding beta-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to beta-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when beta-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0- and 2.7-A resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix alpha3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.