Characterization of a sar Homolog of Staphylococcus epidermidis

Abstract

Coagulase-negative staphylococci are common nosocomial pathogens. A regulatory element, designated sar, partially controls exoprotein synthesis in coagulase-positive Staphylococcus aureus by modulating the expression of another regulatory locus, called agr. We report here the cloning of a sar homolog in S. epidermidis. The major open reading frame within sar in S. epidermidis is highly homologous (84%) to the S. aureus SarA protein. Primer extension studies revealed three sar transcripts (0.64, 0.76, and 0.85 kb) initiated from three distinct promoters. The interpromoter region in S. epidermidis differs from its S. aureus counterpart, possibly suggesting target gene differences and a disparate pattern for sar activation. Remarkably, the S. epidermidis sar homolog interacts with an agr promoter fragment of S. aureus in gel shift assays. Additionally, S. epidermidis sar fragments could restore hemolysin production in an S. aureus sar mutant. As typical virulence determinants controlled by sar in S. aureus are not present in S. epidermidis, an examination of functional and structural similarities and divergence of sar in staphylococci will be of major interest.

Staphylococcus epidermidis and other coagulase-negative staphylococci, previously regarded as harmless contaminants, are increasingly being recognized as important pathogens (13). Coagulase-negative staphylococci, the predominant species being S. epidermidis, are common pathogens in nosocomial bacteremia (18). Infections caused by these organisms generally occur in the presence of indwelling catheters and other implanted devices. As implanted devices are becoming more common, infections due to S. epidermidis are apt to increase. More importantly, isolates of S. epidermidis are frequently resistant to multiple antibiotics. In many cases, the infections cannot be cured unless the offending intravascular devices are removed. Despite their clinical significance, very little is known about the virulence factors of coagulase-negative staphylococci and their mode of regulatory control.

By comparison, regulation of virulence factors in S. aureus has been shown to depend on at least two global regulatory systems, agr and sar (1, 17). The agr locus, consisting of five genes (agrA, agrC, agrB, agrD, and hld), has been shown to control the synthesis of both extracellular and cell wall proteins at the transcriptional level (17, 27). Inactivation of the agr locus leads to decreased production of exoproteins, while the synthesis of some surface proteins is increased (17). Transcriptional analysis indicated that the agr locus is composed of two divergent transcripts, designated RNAII and RNAIII, initiated from the P2 and P3 promoters, respectively. The RNAIII molecule has been implicated as being directly responsible for agr-mediated control of hemolysin production (16, 20, 23).

More recently, we described another pleiotropic regulatory locus, designated sar, that is also involved in the expression of extracellular (e.g., hemolysins) and cell wall virulence determinants (e.g., fibronectin binding proteins). Sequence analysis indicated that the sar locus in strain RN6390 contains a major open reading frame (ORF) (sarA) of 372 bp as well as two smaller ORFs upstream (1, 10). The sarA gene together with an additional 800-bp upstream sequence (≈1.3 kb) is necessary for the optimal transcription of RNAIII of agr to activate hemolysin production (1, 15, 23). Transcriptional analysis of the 1.3-kb sequence revealed three overlapping sar transcripts (designated sarA, sarC, and sarB, with sizes of 0.56, 0.8, and 1.15 kb, respectively) with a common 3′ end but originating from three distinct promoters in a parallel array (1). Gel shift studies demonstrated that the sar gene products bind to an agr P2 promoter fragment, thus indicating protein-DNA interaction between two components. As the P2 promoter potentiates the transcription of RNAII and subsequently RNAIII, these data are consistent with the hypothesis that the sar locus likely controls exoprotein synthesis via the RNAIII-mediated pathway of agr (15).

An agr-like locus in coagulase-negative species such as S. epidermidis and S. lugdunensis has been described previously (25, 26). In S. lugdunensis, it has been shown that the agr homolog, like that of S. aureus, is actively transcribed during the postexponential phase (25). On the basis of the interactions between agr and sar in S. aureus, we hypothesize that sar may exist in coagulase-negative staphylococci. Here, we report the cloning and sequencing of a 372-bp sarA homolog in S. epidermidis. Surprisingly, the sarA gene is highly conserved between S. aureus and S. epidermidis (≈85% homology). Functional assays confirmed that the sar locus of S. epidermidis was able to complement sar-related phenotypes, including agr and alpha-hemolysin expression in an S. aureus sar mutant. Transcriptional analysis of a wild-type S. epidermidis strain revealed a multipromoter organization preceding the sarA gene. However, the promoter arrangement, a lack of smaller ORFs upstream, and the compactness of the multipromoter region are features not found in the S. aureus counterpart. These structural differences may reflect functional divergence in sar activation among staphylococcal species, since S. epidermidis lacks the typical virulence determinants (e.g., alpha-hemolysin and protein A) controlled by sar in S. aureus. In view of the fact that the sarA gene is structurally and functionally conserved in S. aureus and S. epidermidis and conceivably across the staphylococcal genus, it may be prudent to consider the sarA gene product, a putative virulence determinant in S. aureus (7, 11), as a candidate for the development of novel antistaphylococcal therapeutic agents.

MATERIALS AND METHODS

Media and antibiotics.

CYGP and 0.3GL media (22) were used to grow S. aureus and S. epidermidis, while Luria-Bertani broth was used to grow Escherichia coli. Antibiotics were used at the following concentrations: 10 μg/ml (erythromycin), 5 μg/ml (tetracycline), and 50 μg/ml (ampicillin).

Bacteria, plasmids, and phage.

The bacterial strains and plasmids used in this study are listed in Table 1. Phage φ11 was used as the transducing phage for S. aureus strains.

TABLE 1.

Bacterial strains and plasmids

Strain or plasmid	Reference or source	Comments
S. epidermidis 6937	9	Clinical isolate
S. aureus
RN4220	22	A mutant of strain 8325-4 that accepts foreign DNA
RN6390	17	Parental strain that maintains its hemolytic pattern when propagated on sheep RBC
ALC136	15	Isogenic mutant of RN6390 carrying sar::Tn917LTV1 mutation
ALC475	This work	ALC136 carrying the shuttle vector pSPT181
ALC853	This work	ALC136 with pALC853
ALC854	This work	ALC136 with pALC854
ALC70	1, 15	ALC136 with pALC70
ALC103	1, 15	ALC136 with pALC103
ALC955	This work	ALC136 with pALC955
E. coli
XL1-Blue	24	Highly transformable strain
INVαF′	Invitrogen	Host strain for the TA cloning vector (pCRII)
Plasmids
pCRII	Invitrogen	E. coli cloning vector for direct cloning of PCR fragments
pALC631	This work	pCRII with S. epidermidis sar fragment nt 916–1152 (Fig. 2)
pALC858	This work	pUC19 with an 8.5-kb HindIII fragment of S. epidermidis 6937
pSPT181	16	Shuttle vector
pALC103	10	pSPT181 with a sar fragment encompassing the S. aureus sarA transcript (nt 620–1349)
pALC70	1, 15	pSPT181 with a sar fragment encoding the S. aureus sarB transcript (nt 1–1349 plus additional sequence 300 bp upstream)
pALC853	This work	pSPT181 with S. epidermidis sar fragment nt 1–1597 (Fig. 2)
pALC854	This work	pSPT181 with S. epidermidis sar fragment nt 727–1597 (Fig. 2)
pALC955	This work	pSPT181 with S. epidermidis fragment nt 436–1597 (Fig. 2)

Southern blot hybridization.

Chromosomal DNAs of S. aureus 6390 and S. epidermidis 6937 (9) were extracted from lysostaphin-lysed cells as previously described (8). Southern blot hybridization was performed with random-primed samples of gel-purified DNA fragment as probes (8). The membrane (Hybond-N⁺; Amersham, Arlington Heights, Ill.) was allowed to hybridize with an α-³²P-labeled DNA probe at 65°C overnight, washed under high-stringency conditions, and autoradiographed as described previously (1).

Cloning and sequencing strategies.

With S. epidermidis DNA as the template, a series of primer pairs derived from the sar sequence of S. aureus RN6390 were used for PCR amplification (1). Initial PCR amplifications with primer pairs flanking the sarA gene of S. aureus were unsuccessful. To explore the possibility that the sarA gene may be conserved across staphylococcal species, several primer pairs internal to the S. aureus sarA gene were employed. One of these primer pairs (upper primer, 5′-TGAGTTGTTATCAATGGTCACTT-3′, and lower primer, 5′-TCATCATGCTCATTACGTTTTTT-3′) was able to amplify a 237-bp sar-like gene fragment. This DNA fragment was cloned into pCRII (Invitrogen, San Diego, Calif.) to yield pALC631 and sequenced with ³⁵S-labeled α-dATP sequencing mix and Sequenase (U.S. Biochemicals, Cleveland, Ohio). Based on restriction analysis of this PCR fragment, chromosomal DNA of S. epidermidis 6937 was digested with HindIII, ligated to pUC19, and transformed into XL1-Blue. Transformants were screened by colony hybridization with an α-³²P-labeled 237-bp PCR probe. A clone containing a recombinant plasmid with an 8.5-kb HindIII insert was obtained. The insert was sequenced by primer walking and analyzed with Genetics Computer Group software (GCG package; University of Wisconsin, Madison).

Based on sequence analysis, three S. epidermidis PCR fragments (872, 1,162, and 1,596 bp) in pCRII were cleaved with XhoI/SacI, recloned into shuttle vector pSPT181 (16), and reintroduced into S. aureus sar mutant strain ALC136 as described previously (8).

Genetic manipulation of S. aureus.

Shuttle plasmids were transformed into S. aureus RN4220 by protoplast transformation as previously described (8). Transformants were selected at 32°C on DM3 agar containing tetracycline. For transduction, phage φ11 was used to produce a phage lysate of strain RN4220 containing the recombinant shuttle plasmid pSPT181 with S. epidermidis DNA fragments. The phage lysate was then used to infect the sar transposon mutant as described previously (ALC136) (8). Transductants were selected at 32°C on tetracycline-containing agar.

Isolation of RNA and Northern analysis.

RNA from S. aureus and S. epidermidis was obtained from bacterial cultures grown at 33°C as described previously (5). This temperature was chosen to accommodate the temperature-sensitive replicon of the shuttle plasmid without undue effects on agr-related transcription (1, 15). Total cellular RNA was isolated by the FastPrep system (BIO101, Vista, Calif.) as previously described (5). Ten micrograms of RNA was electrophoresed through a 1.2% agarose–0.66 M formaldehyde gel in morpholinepropanesulfonic acid (MOPS) running buffer (20 mM MOPS, 10 mM sodium acetate, 2 mM EDTA [pH 7.0]). RNA was transferred onto a Hybond N membrane (Amersham) under mildly alkaline conditions by using a Turboblotter system (Schleicher and Schuell, Keene, N.H.), fixed to the membrane by baking (at 80°C for 1 h), hybridized under aqueous conditions at 65°C with α-³²P-labeled gel-purified DNA fragments, washed, and autoradiographed (1). Band intensities were quantitated by densitometric scanning with SigmaGel software (Jandel Scientific, San Rafael, Calif.); these values are presented as integrated area units.

Primer extension analysis.

Mapping of the 5′ ends of individual transcripts by primer extension was performed with synthesized oligonucleotides. The 23-mer primers used (see Fig. 4A and B) correspond to the complementary strand of the sequences given in Fig. 2 as follows: 5′-AGCCATTAATGAAACCTCCCTAT-3′ (positions 892 to 870) and 5′-AAATACGAAAGTGTCCGTCATAA-3′ (positions 743 to 721), respectively. For primer extension, 30 μg of RNA was coprecipitated with ∼100,000 cpm of γ-³²P-end-labeled primer and annealed at 35°C overnight. Following ethanol precipitation, reverse transcription was carried out with SuperscriptII (Gibco-BRL, Gaithersburg, Md.) at 42°C for 90 min. The reaction product was incubated with RNase H (2 U) for 15 min at 37°C, ethanol precipitated, resuspended in 10 μl of Sequenase stop solution, denatured, and applied onto a 6% sequencing gel. Sequencing reaction mixtures primed by an oligonucleotide identical to the one used for primer extension were applied in parallel lanes on the gel.

FIG. 4.

Mapping of the 5′ ends of three sar transcripts of S. epidermidis by primer extension analysis. Base mapping was done by comparing the migration of the extension product with a parallel sequencing reaction primed by an identical oligonucleotide. Putative transcription start sites (marked by asterisks ± 2 bp) are labeled P1, P2, and P3.

FIG. 2.

(A) Nucleotide sequence of the sar homolog region of S. epidermidis. Shown are the sequences encoding the sarA homolog and the upstream element encompassing three distinct promoters. The putative ribosome-binding site is underlined twice and labeled SD. Start and stop codons of the sar homolog in S. epidermidis are in bold-faced type. The mapped 5′ ends of the mRNAs (±2 bp) identified by primer extension are indicated by vertical lines. The putative −10 and −35 promoter boxes are denoted by lines over their respective sequences, and the respective promoter regions (P1, P2, and P3) are underlined. The predicted termination site as determined by the computer program TERMINATOR is shown by an asterisk, and the termination signal is labeled by a broken arrow. (B) Alignment of the SarA proteins between S. aureus RN450 (or strain DB) and S. epidermidis 6937. With the exception of a conservative substitution at residue 53 (phenylalanine for leucine), the SarA protein of strain RN6390 is highly homologous to those described for strains RN450 and DB of S. aureus (1). Identical amino acids are indicated by semicolons, and conserved substitutions are underlined. The deduced molecular size of the SarA homolog is 14.7 kDa, with a pI of 8.5 in contrast to a molecular weight of 14,718 and a pI of 8.52 for SarA of S. aureus.

Phenotypic characterization.

Strains were tested in duplicate for the production of hemolysins on plain and cross-streaked sheep and rabbit erythrocyte (RBC) agar plates, with specific indicator strains as standards (8).

Alpha-hemolysis was also assayed by a microtiter method. Briefly, the supernatant of an overnight culture was serially diluted in phosphate-buffered saline and incubated for 2 h with 4% rabbit RBC. Sodium dodecyl sulfate (2%) was used as a positive lysis control. The data were given as the reciprocal of the highest dilution that gave complete lysis.

Production of cell extracts and gel shift analysis.

Cell extracts were prepared as previously described (15, 19) from S. aureus RN6390, the isogenic S. aureus sar mutant containing the shuttle vector pSPT181 alone (ALC475), or the recombinant vectors carrying PCR-generated sar fragments of S. epidermidis (ALC853 and ALC854). For the gel shift assay, 12 μl of each cell extract was added to reaction mixtures containing 10 mM Tris HCl (pH 7.5) with EDTA (1 mM), dithiothreitol (1 mM), NaCl (50 mM), glycerol (5%), and 1 μg of poly(dI-dC) to a final volume of 25 μl. Approximately 1 × 10⁴ to 2 × 10⁴ cpm of the γ-³²P-end-labeled P2 promoter probe was then added. Unlabeled P2 promoter DNA and a 200-bp PCR fragment of the structural protein A gene were used as specific and nonspecific competitors, respectively. The reaction mixtures were incubated at room temperature for 5 min, iced for 5 min, and electrophoresed on a 6% polyacrylamide gel in 0.25× Tris-borate-EDTA for 2 h at 200 V. The gels were dried and exposed to film.

RESULTS

Cloning and sequencing of the sar locus of S. epidermidis.

To ascertain the existence of a sar homolog in S. epidermidis, we probed chromosomal HindIII, PstI, and HindIII/PstI digests with a 1.3-kb S. aureus probe encompassing the entire sar locus (nucleotide [nt] 1 to 1349 according to a published sequence) (15). As there are no HindIII or PstI sites within the sarA gene of S. aureus, we predicted from the Southern blot data that one copy of the sar-like gene(s) is likely to be present in S. epidermidis 6937 (Fig. 1). Using an internal primer pair derived from the sarA coding region of S. aureus (see Materials and Methods) and S. epidermidis genomic DNA as the template, we generated a 237-bp PCR fragment which hybridized with DNA of both S. epidermidis and S. aureus on a Southern blot (data not shown). Sequence analysis revealed that this PCR fragment encoded sequence homologous to sarA of S. aureus. This 237-bp PCR fragment was subsequently used as a probe to screen an E. coli plasmid library containing HindIII fragments of S. epidermidis genomic DNA ligated to pUC19. A positive clone yielding an 8.5-kb insert was found. The insert was subsequently sequenced.

FIG. 1.

Southern blot of S. aureus and S. epidermidis chromosomal digests probed with a 1.3-kb sar fragment encoding the entire sar locus (nt 1 to 1349) of S. aureus (1).

The sequence of a 1.6-kb fragment within the insert encompassing the sar region of S. epidermidis is shown in Fig. 2A (GenBank accession no. AF054173). Sequence analysis revealed one large (372-bp) ORF beginning at position 887 (ATG), preceded 8 bp upstream by a conserved Shine-Dalgarno sequence (GGGAGG). This ORF shows a high degree of homology to the sarA gene of S. aureus RN450 (10) as well as to strain RN6390 (1), with ≈85% identity at the nucleotide level (Fig. 2A). The nucleotide sequence flanking the sarA coding region, with ≈50% homology to its S. aureus counterpart, diverges significantly from that of S. aureus. As both species are AT rich, this degree of sequence similarity outside the sarA coding region is not highly significant. The SarA homolog of S. epidermidis is 84% identical to SarA of S. aureus (Fig. 2B). With the residues that are not identical, 59% contains conserved substitutions (Fig. 2B). The mature SarA homolog of S. epidermidis has a small predicted molecular size (14.7 kDa) and a deduced basic pI of 8.5.

Transcriptional analysis of sar transcripts in S. epidermidis.

In a Northern blot of S. epidermidis RNA probed with an ≈1.2-kb homologous fragment (nt 436 to 1597 in Fig. 2) encompassing the sar-like region, it can be observed that S. epidermidis 6937 yielded three sar-related transcripts with sizes of 0.64, 0.76, and 0.85 kb (1). With the exception of the smallest transcript, these transcripts are shorter than their S. aureus counterparts, which have sizes of 0.58, 0.84, and 1.12 kb, respectively (1). Based on sequence analysis of the transcription termination site (Fig. 2A), the size of the observed transcripts, and the predicted sar genetic organization as derived from S. aureus (1), we amplified two sar-like fragments, representing the sarA homolog alone or the entire sar locus of S. epidermidis together with their respective promoters, and introduced them into an S. aureus sar mutant (ALC136). Remarkably, the sar mutant clone containing the homologous sarA gene (ALC854 with nt 727 to 1597) produced one transcript of ≈0.64 kb in size, while the same strain carrying the entire putative sar locus (ALC853 and ALC955) expressed three transcripts analogous to those observed in the parental strain (Fig. 3), thus clearly implying that the smaller transcript did not arise from the processing of the larger, 0.85-kb transcript.

FIG. 3.

Transcriptional analysis of S. aureus sar mutant clones carrying shuttle plasmids with homologous sar fragments from S. epidermidis. Ten micrograms of total cellular RNA derived from the clones was applied to each lane. The probe is a 1.2-kb sar fragment (nt 436 to 1597 [Fig. 2]) of S. epidermidis. Clones ALC854 and ALC853 carry the putative sarA transcript and the entire sar locus, respectively. Clone ALC955, carrying the entire homologous sar locus of S. epidermidis (nt 436 to 1597), yielded results similar to those for clone ALC853. The top panel represents the proposed transcriptional organization of the sar locus in S. epidermidis.

We also mapped the 5′ ends of these transcripts by primer extension (Fig. 4A and B). Three primer extension products with putative transcriptional start sites at positions 792 ± 2 bp (mean ± standard deviation), 672 ± 2 bp, and 581 ± 2 bp (designated P1 at −95, P2 at −215, and P3 at −308 relative to the sarA homolog start codon in Fig. 2) were found. To verify the primer extension data, Northern blot analyses were performed with RNA from parental strain 6937 with PCR fragments positioned within the multipromoter region. With a probe encompassing the region between the P1 and P2 promoters (see the sar organization in Fig. 3), two transcripts (0.76 and 0.85 kb) were detected, while a single transcript (0.85 kb) was found to hybridize with a probe comprising sequences between the P2 and P3 promoters (data not shown). As expected, a probe corresponding to the homologous sarA coding region yielded three transcripts. These data are consistent with three different sites of transcription initiation.

Using the Genetics Computer Group sequence analysis software, we ran the program TERMINATOR, which searched for prokaryotic RNA polymerase terminators independent of rho factors. A putative transcriptional termination signal was found, with a predicted termination site at position 1433.

Functional analysis of the sar homolog of S. epidermidis in the expression of RNAII and RNAIII in an S. aureus sar mutant (Fig. 5).

FIG. 5.

Northern blots of agr transcripts in S. aureus sar mutant clones carrying shuttle plasmids with homologous sar fragments from S. epidermidis. RNA (10 μg each) was obtained from cells harvested at late log phase. Equal intensities of the 23S and 16S ribosomal RNA bands among different lanes in the gel were ensured prior to transfer to Hybond-N⁺ membrane. The probes for RNAII and RNAIII were α-³²P-labeled fragments of agrA (nt 3830 to 4342) and hld (nt 999 to 1510) (17), respectively. ALC854 and ALC853 carry the homologous sarA transcript and the entire sar locus of S. epidermidis, respectively. RN6390 is the parental control; ALC475 is the sar mutant clone carrying the vector alone.

To determine if the sarA gene of S. epidermidis activates agr-related transcription in S. aureus, S. aureus sar mutant clones containing sar fragments from S. epidermidis (ALC853 and ALC854) were probed in Northern blots to assay for RNAII and RNAIII transcription. Using an agrA fragment (nt 3830 to 4342) (17) to detect RNAII transcription, we found that the RNAII level was greatly reduced in the sar mutant carrying the plasmid alone (ALC475) compared to that in the S. aureus parental strain RN6390 (integrated area units of 1,516 versus 8,182). Complementation of the S. aureus sar mutant with the sarA homolog of S. epidermidis (ALC854) partially restored RNAII transcription (integrated area unit of 2,952), while the sarA gene together with a sequence 790-bp upstream (ALC853) that encompassed all three S. epidermidis sar transcripts reestablished the expression of RNAII in the mutant to the parental level (integrated area unit of 9,796). These levels of RNAII expression are comparable to those found in S. aureus sar mutant clones complemented with homologous sar fragments from S. aureus (1, 15).

We also examined the level of RNAIII expression in these clones. Like that of RNAII, the level of RNAIII was diminished in the sar mutant ALC475 compared with that in the parent strain (integrated area units of 3,102 and 6,301, respectively) but was restored by the introduction of a plasmid containing sar fragments of S. epidermidis (ALC853 and ALC854). In contrast to the finding with RNAII, the sarA-like fragment (ALC854) as well as the one encompassing all three sar-like transcripts (ALC853) led to higher levels of RNAIII in the respective mutant clones (integrated area units of 9,120 and 8,144 for ALC854 and ALC853, respectively) compared with the control vector (ALC475).

Phenotypic characterization.

In previous studies, we have shown that the sarA gene of S. aureus is necessary for activating hemolysin production (alpha, beta, and delta). To determine if the sarA homolog from S. epidermidis has similar functional capacities in an S. aureus sar mutant, we assessed the production of hemolysins of ALC853 and ALC854 on blood agar plates. As shown in Table 2, the secretion of alpha- and beta-hemolysins in the sar mutant control (ALC475) was lower than in the parent strain but was enhanced in clones complemented with sar fragments from S. epidermidis (ALC853 and ALC854). This result concurred with that of the quantitative assays in which supernatants from clones carrying sar fragments of S. epidermidis had intermediate hemolytic titers (1:4 to 1:8), while the parental control RN6390 lysed the RBC at a 1:32 dilution (Table 2). As expected, neither the sar mutant vector control nor S. epidermidis 6937 exhibited any hemolytic effect.

TABLE 2.

Phenotypic characterization of sar-like clones

Strain	Presence ofa:		Hemolytic titerb (alpha-hemolysin)
	Alpha-hemolysin	Beta-hemolysin
S. aureus RN6390	+	±	32
S. aureus ALC475	−	−	1
S. epidermidis 6937	−	−	No lysis
S. aureus ALC853	+	+	4–8
S. aureus ALC854	+	+	4–8

^aAssayed by measuring the zones of hemolysis on cross-streaked rabbit RBC agar plates. 

^bReciprocal of the highest dilution of complete hemolysis. This result concurred with that of an immunoblot probed with rabbit anti-alpha-hemolysin antibody (data not shown). 

Gel mobility of the P2 promoter region (Fig. 6).

FIG. 6.

Gel shift assays of cell extracts of sar mutant clones (with S. epidermidis sar fragments) on the mobility of a radiolabeled agr promoter fragment of S. aureus. Control, agr fragment without cell extract; I, specific competition with 100 ng of cold agr fragment; N, nonspecific competition with 100 ng of a 200-bp PCR fragment of the S. aureus protein A gene. The amount of unlabeled fragment represents ≈20-fold excess.

We recently showed that the sar gene product(s) of S. aureus likely interact with the P2 promoter of the RNAII operon in agr (15). To determine if the sarA gene product of S. epidermidis reveals similar DNA binding activity, cell extracts of sar mutant clones ALC853 and ALC854 were prepared and used in gel shift assays with a γ-³²P-labeled 171-bp P2 promoter fragment of S. aureus [nt 1603 to 1773 according to a published sequence (15)]. The sar mutant strain carrying the 1.5-kb fragment encoding the entire sar locus of S. epidermidis (ALC853) was able to retard the mobility of the labeled P2 promoter, whereas the sar mutant complemented only with the homologous sarA fragment (ALC854) at an equivalent cell extract protein concentration had no effect. This discrepancy in gel retardation activity between sarA and the entire sar locus has also been observed with analogous S. aureus sar fragments. As predicted, the cell extract of the sar mutant control did not demonstrate any gel shift activity with the agr promoter fragment (ALC475).

DISCUSSION

Agr-like sequences, including that of an RNAIII homolog, have been described in coagulase-negative species such as S. lugdunensis (25) and S. epidermidis (26). Based on the interactions between sar and agr in S. aureus, we assessed and confirmed the existence of a sar homolog in S. epidermidis, a major nosocomial pathogen (13). Sequence analysis revealed that the SarA protein of S. epidermidis is highly homologous to its S. aureus counterpart. Despite protein sequence similarity between two sarA genes from S. epidermidis and S. aureus, significant divergence at the nucleotide level is apparent in the region flanking the sarA coding region. This finding explains why we failed to amplify the heterologous sar locus of S. epidermidis with primer pairs derived from flanking sar sequence of S. aureus.

Based on transcriptional (Fig. 3) and primer extension studies (Fig. 4), it is likely that the sar locus in S. epidermidis is composed of three overlapping transcripts originating from three distinct promoters. Sequence analysis revealed that the smallest transcript was 0.64 kb in size. The putative −10 (GGGTAT) and −35 (TAGATAT) core promoter boxes (P1), spaced ∼14 bp apart, possess a striking homology to the stress response ςB-dependent promoters (consensus −37 RGGX TT-N14-GGGTAT) (14). A shuttle plasmid carrying a fragment including the proximal P1 promoter and the homologous sarA coding region indicated that the P1 promoter within sar in S. epidermidis is maximally transcribed during the postexponential phase (data not shown), contrasting with the P1 proximal promoter of sar in S. aureus, which is most active during the exponential phase of growth (1). The central promoter (P2), lying 216 bp upstream of the sarA homolog translation start, with the putative −10 (TATAAT) and −35 (TTTACT) core promoter boxes spaced 17 bp apart, corresponds to the canonical hexamer of the ςA-dependent promoter of Bacillus subtilis. The most distal promoter (P3) contains core promoter boxes that did not display unambiguous consensus sequence. The TERMINATOR program allowed us to identify a single putative transcription termination signal (nt 1381 to 1406) with a termination site at position 1433 (P = 3.81) by applying a normalized dinucleotide distribution matrix (P > 3.5) alone (3). We have verified the predicted termination site by conducting probe walking near the C terminus of the sarA-like gene. As anticipated, a probe outside the predicted termination site did not hybridize with any sar-like transcript, whereas a probe internal to it led to restoration of all three sar-related transcripts (data not shown).

Even with a triple-promoter organization similar to that in S. aureus, distinct differences in the promoter region, possibly reflecting evolutionary divergence, can be observed. First, a ςB-like promoter (P1) preceded by an upstream ςA promoter (P2) lies proximal to the homologous sarA coding region in S. epidermidis, while the arrangement in S. aureus is reversed, with a ςA-dependent promoter most proximal to the sarA starting codon. Second, the three promoters are more closely spaced in S. epidermidis than in S. aureus (345 and 745 bp upstream of the sarA coding region for S. epidermidis and S. aureus, respectively). The proximity of these promoters in S. epidermidis reflects a lack of ORFs that are interspersed among the three promoters as seen with S. aureus (e.g., ORF3 and ORF4 potentially encoding peptides of 39 and 18 amino acids, respectively, within the sar promoter region of S. aureus) (15). Third, as opposed to 85% homology within the sarA coding region of S. aureus, the DNA sequence outside the coding region is ≈50% homologous to its S. aureus counterpart, thus implying significant sequence divergence. We surmise that these structural differences may conceivably reflect functional divergence between these two organisms. Alternatively, discrepancy in the promoter region may imply disparity in the mode of sar activation between these two species. Unlike S. aureus, S. epidermidis does not secrete alpha-hemolysin or synthesize protein A. We recently reported that ORF3, located between two proximal sar promoters, in conjunction with SarA protein, may play a role in repressing protein A transcription in S. aureus (4, 6). Likewise, complementation analysis of an S. aureus sar mutant with a single copy of a fragment encoding sarA and ORF3 from S. aureus indicated that ORF3 may be required for restoring alpha-hemolysin production to the parental level (4). In the absence of protein A and alpha-hemolysin expression in S. epidermidis, we speculate that the selective pressure to maintain the sequence encoding ORF3 may be diminished. Consequently, neither the required element ORF3 nor its encoded sequence will be preserved during the evolution of this species. This hypothesis seems plausible considering that S. epidermidis is commensal and becomes invasive only in the presence of artificial devices, while S. aureus is an invasive pathogen with a broad host range specificity (e.g., primates and poultry).

Despite differences in the respective promoter regions, it is clear that the sarA gene itself is highly conserved in S. epidermidis and S. aureus, with both deduced proteins having similar molecular sizes and basic pIs (Fig. 2B). Preliminary hybridization studies with a sarA-specific probe indicated that homologous sarA sequences are likely to be conserved in other related species (e.g., S. hemolyticus) and possibly across the staphylococcal genus. Due to a relative lack of genetic manipulative tools for S. epidermidis, the construction of an S. epidermidis sar mutant to ascertain the resultant phenotype has not been successful to date. Nevertheless, phenotypic studies as reported here likely imply functional conservation as evidenced by the ability of the sarA homolog of S. epidermidis to partially restore alpha- and beta-hemolysin production in an S. aureus sar mutant (Table 2). In recent studies with sarA of S. aureus, we have shown that purified SarA protein, the major regulatory determinant (4), binds to the promoter of an intermediary regulatory element (e.g., agr) (12) to activate target gene transcription (e.g., alpha-hemolysin). More importantly, inactivation of the sarA gene in S. aureus has led to a significant diminution in virulence in several animal models of infection (2, 7, 21). These findings led us to propose that the sarA gene product, being structurally and functionally conserved across staphylococcal species, may be a good target for the development of novel antimicrobial agents against multidrug-resistant staphylococci.

An agr homolog has been described in S. epidermidis (26). With the exception of RNAIII containing a delta-hemolysin-like sequence, the homology with RNAII of S. aureus is less dramatic, with 50, 49, and 36% homology at the nucleotide level to agrC, agrB, and agrD homologs, respectively (26). The discovery of a sar homolog in S. epidermidis makes it reasonable to hypothesize that the S. epidermidis sar gene product may interact with the homologous agr promoter in S. epidermidis as well.

Little has been known about the regulation of virulence genes in S. epidermidis. The discovery of a sar homolog with similarity (sarA) to and differences (promoter region) from its S. aureus counterpart is a significant step in our initial approach toward understanding gene regulation in this pathogen. Without the typical virulence determinants of S. aureus, an obvious question is what role sar plays in the control of virulence genes (e.g., slime or capsular genes) in S. epidermidis. Clearly, the gene targets under the control of sar and agr in S. epidermidis are ill defined. It will be of significant clinical interest to define these target genes and to ascertain if similar sar homologs can be found in other staphylococcal pathogens, such as S. hemolyticus and S. saprophyticus. Ultimately, it will be important to determine if a single agent can be used to block sar gene activation among staphylococcal species.