INTRODUCTION

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In Staphylococcus aureus, transcription of many virulence genes, encoding extracellular toxins, enzymes, and cell surface proteins, is regulated by a 510-nucleotide (nt)-long RNA, called RNAIII (15, 27). Production of toxins and enzymes is generally positively controlled, while that of cell surface proteins is negatively controlled (14, 18). Synthesis of RNAIII is induced when the concentration of an autocrine octapeptide in the environment has reached a certain level (16, 17). Generally, this happens during the late exponential phase of growth in laboratory cultures, which means that cell surface proteins are produced during the early exponential phase, while secreted toxins and enzymes are produced mainly during the postexponential phase of growth (4, 21, 31). Four genes, agrB, agrD, agrC, and agrA, arranged in an operon (agr) are involved in the synthesis of the inducing octapeptide and the signal transduction that leads to activation of the RNAIII gene, which is closely linked to the agr operon and transcribed in the opposite direction (16).

A precursor of the inducing peptide is encoded by agrD and requires agrB to be properly processed and secreted (16, 17). The agrC and agrA genes code for the components of a classical two-component signal transduction system, where AgrC is the sensor and AgrA is the response regulator, which is required for transcription of the RNAIII molecule and the agr operon itself (26, 27).

Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin (14). Delta-lysin is a 26-amino-acid polypeptide which can form pores in membranes and lyse erythrocytes (8, 12, 19). Delta-lysin is not required for the regulation of target genes by RNAIII (2, 15, 27), though translation of the delta-lysin gene may influence the regulatory function of RNA (3).

An agr locus, organized in the same way as that of S. aureus, has been demonstrated in coagulase-negative Staphylococcus lugdunensis (32). However, RNAIII from S. lugdunensis does not code for delta-lysin, and its role in gene regulation is not known (32).

The production of a deltalike hemolytic activity has been demonstrated in many coagulase-negative staphylococci (CoNS) (6, 7, 9). Amino acid sequencing of the hemolysin from Staphylococcus epidermidis revealed only two amino acids that were different from those in S. aureus delta-lysin (23). This has recently been confirmed by nucleotide sequencing of the S. epidermidis RNAIII gene (28). Based on this high degree of similarity, we asked the question whether delta-lysins from S. epidermidis and other CoNS are encoded by RNAIII-like mRNAs which also have a regulatory function. In this study, RNAIII homologs were identified in S. epidermidis, S. warneri, and S. simulans and were shown to regulate virulence gene expression in S. aureus. In all molecules, the first 50 and last 150 nt were highly conserved, suggesting that these regions are important for the regulatory function.

	MATERIALS AND METHODS

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Bacterial strains. S. aureus WA400 is a mutant of 8325-4 in which the RNAIII gene has been deleted and replaced by the cat86 gene (15). Strain WA400 has an intact agr operon, but due to the lack of functional RNAIII, it has an exoprotein pattern similar to that of an agrA mutant. RN4220 (20) is a restriction-deficient mutant of strain 8325-4. Escherichia coli DH5 (11) was used as the host for plasmid constructions.

Culture conditions. S. aureus strains were precultured overnight in tryptic soy broth (TSB; Difco), and 20 ml of preculture was collected by centrifugation and used to inoculate 100 ml of brain heart infusion (BHI; Difco) in 1-liter baffled flasks. Incubation was at 37°C on a rotary shaker. Bacterial growth was monitored by measuring the optical density at 600 nm, and cultures were harvested by centrifugation at the indicated time points. The appropriate antibiotics were added to the precultures: chloramphenicol (5 µg ml¹) and/or tetracycline (5 µg ml¹).

PCR and sequencing. Chromosomal DNAs were prepared (22) from S. epidermidis, S. cohnii, S. haemolyticus, S. saprophyticus, S. simulans, S. warneri, and S. xylosus and used as the template in PCR with S. aureus RNAIII primers, nt 1095 to 1116 (primer 11) and nt 1554 to 1578 (primer 12) according to the nucleotide sequence numbering of Kornblum et al. (18).

Construction of plasmids. Plasmids used are listed in Table 1. All plasmid constructs were first propagated in E. coli DH5 and then transferred to the restriction-deficient S. aureus RN4220 (20) before they were introduced into S. aureus WA400. Competent E. coli cells were prepared and transformed by the method of Sambrook et al. (29). S. aureus strains were transformed by electroporation by the method of Schenk and Laddaga (30). E. coli transformants were selected on LB (Difco) plates containing 50 µg of ampicillin ml¹ and S. aureus transformants on NYE agar plates (29) containing 5 µg of tetracycline ml¹. Plasmid DNA was extracted by using the Qiagen plasmid mini kit (Qiagen Inc., Valencia, Calif.). Lysostaphin (100 µg ml¹) (Applied Microbiology Inc.) was used to lyse S. aureus cells.

Northern blotting and primer extension analyses. Total S. aureus RNA was prepared by extraction of lysostaphin-treated cells, with hot phenol as described previously (13). Concentration of RNA was determined spectrophotometrically as absorbance at 260 nm. Electrophoresis of RNA (10 µg of total RNA per lane), transfer to a Biodyne B nylon membrane (Pall Ultrafine Filtration Corp.), and hybridization were carried out as described previously (24). Internal fragments of the genes coding for alpha-toxin (nt 487 to 1930 [10]), serine protease (nt 435 to 1364 [5]), protein A (nt 815 to 1072 [22]), S. aureus RNAIII (nt 1095 to 1578 [18]), S. epidermidis RNAIII (nt 78 to 620 [this study]), S. simulans RNAIII (nt 80 to 640 [this study]), and S. warneri RNAIII (nt 80 to 748 [this study]) were amplified by PCR, radiolabeled with [-³²P]dCTP (Amersham) using a random prime labelling kit (Boehringer Mannheim Biochemicals), and used as probes. For a common probe for RNAIII, primer 28 (nt 1133 to 1156 [18]) was labeled with [-³²P]ATP (Amersham), using polynucleotide kinase.

Sequence analysis. The multiple alignment of the RNAIII gene sequences was done with the PileUp program, and the secondary structure predictions were done with the Squiggles program (energy minimization method of Zuker) (both programs from the Genetics Computer Group Inc.).

Nucleotide sequence accession numbers. Sequence data have been submitted to the EMBL Nucleotide Sequence Database under the accession numbers AJ223774 (S. epidermidis hld gene), AJ223775 (S. simulans hld gene), and AJ223776 (S. warneri hld gene).

	RESULTS

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Identification and sequencing of the RNAIII gene in CoNS. Seven different staphylococcal species (S. cohnii, S. epidermidis, S. haemolyticus, S. saprophyticus, S. simulans, S. warneri, and S. xylosus) were tested for the presence of sequences related to the S. aureus RNAIII gene by PCR. Internal RNAIII primers based on the S. aureus sequence were used. A major PCR product, ranging from 500 to 700 nt, was obtained only from S. epidermidis, S. simulans, and S. warneri (Fig. 1). The presumed internal RNAIII gene fragments generated from S. epidermidis, S. simulans, and S. warneri were cloned and sequenced. A high degree of identity to the S. aureus RNAIII gene was found in all cases, which confirmed that the amplified PCR products indeed were internal RNAIII gene fragments. In order to obtain flanking sequences, additional PCR and inverted PCR were carried out (see Materials and Methods). Based on the derived sequences, new primers were designed to amplify and clone the entire RNAIII determinants, which were then sequenced in both directions (Fig. 2).

View larger version (100K): [in this window] [in a new window]   FIG. 1.   Agarose gel analysis of PCR products, amplified with internal S. aureus RNAIII primers, from chromosomal DNAs of CoNS. Lanes: 1, size markers (in base pairs); 2, S. aureus; 3, S. cohnii; 4, S. epidermidis; 5, S. haemolyticus; 6, saprophyticus; 7, S. simulans; 8, S. warneri; 9, S. xylosus; 10, negative control.

View larger version (60K): [in this window] [in a new window]   FIG. 2.   Alignment of the RNAIII gene sequences from S. aureus (S.a) and three different CoNS species, S. epidermidis (S.e), S. warneri (S.w), and S. simulans (S.s). The alignment was done with the PileUp program from the Genetics Computer Group Inc., and the sequences were manipulated by introducing gaps (indicated by dashes) to fit transcription start points, the delta-lysin open reading frames, and secondary structure predictions. Positional sequence identity for at least three of the sequences (boxes) and conserved direct repeats (shaded boxes), which are important for regulation of RNAIII in S. aureus, are indicated. The transcriptional start site (+1) and the putative 10 and 35 promoter elements, direct and indirect repeats (arrows), Shine-Dalgarno sequence (S.D.), and the N-terminal methionine of the predicted delta-lysin open reading frames and stop codons (bold type) are indicated.

Transcription of the RNAIII gene in S. epidermidis, S. simulans, and S. warnerii. Northern blot analysis revealed that the RNAIII gene was expressed in all three strains (Fig. 3). The mobility of the transcripts corresponded to the lengths deduced from the DNA sequencing data and primer extension analysis. Upstream of the transcription start points (indicated in Fig. 2), putative 10 and 35 promoter elements were found, similar to those in S. aureus. The 3' end of the molecules was identified by the striking sequence similarity to the terminator region of RNAIII in S. aureus. Based on these results, the estimated lengths of RNAIIIs are 560 nt for S. epidermidis, 573 nt for S. simulans, and 684 nt for S. warneri.

View larger version (102K): [in this window] [in a new window]   FIG. 3.   Northern blot analysis of RNAIII molecules from S. aureus, S. epidermidis, S. simulans, and S. warneri. Ten micrograms of total RNA from postexponential cells of each strain was loaded onto a 1.2% denaturing agarose gel. A mixture of DNA probes specific for RNAIII from S. aureus (lane 1), S. epidermidis (lane 2), S. simulans (lane 3), and S. warneri (lane 4) was used.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Predicted amino acid sequences of delta-lysin in S. epidermidis, S. simulans, and S. warneri compared with the amino acid sequence of S. aureus delta-lysin (14). Two delta-lysin genes were predicted in S. warneri (S. warneri-I and S. warneri-II). Charged residues are indicated by bold type.

Regulatory effect of RNAIII from different CoNS. To test the ability of the RNAIII molecules from the CoNS to regulate transcription of virulence genes in S. aureus, plasmids containing the CoNS RNAIII genes were introduced into the RNAIII-deficient S. aureus strain, WA400. In order to ensure that the same level of RNAIII was expressed, the different RNAIII genes were put under the control of S. aureus RNAIII promoter. A similar plasmid with the S. aureus RNAIII gene was used as a control. The expression of RNAIII, alpha-toxin (hla), serine protease (ssp), and protein A (spa) mRNAs was analyzed by Northern blotting. To be able to compare the levels of RNAIII, a 24-nt primer recognizing a 100% conserved region was used as a probe. As can be seen in Fig. 5, comparable amounts of the different CoNS RNAIII molecules were produced by S. aureus WA400. All three CoNS RNAIII molecules stimulated the expression of the protease and alpha-toxin genes. However, the levels of transcripts were generally reduced compared to those seen in strain WA400 expressing S. aureus RNAIII. Transcription of the protein A gene was completely repressed in all cases. Taken together, these results show that RNAIII molecules from S. epidermidis, S. simulans, and S. warneri have the ability to regulate transcription of the alpha-toxin, serine protease, and protein A genes in S. aureus in the same manner as S. aureus RNAIII.

View larger version (62K): [in this window] [in a new window]   FIG. 5.   Northern blot analysis of RNAIII, alpha-toxin, serine protease, and protein A transcripts in strains WA400, WA400(pEX0128), WA400(pEX0124), WA400(pEX0125), and WA400(pEX0122) at different time points during growth. The pEX part of plasmid designations is not shown in the figure. Strain WA400 without plasmids () is the control. The same filter was hybridized with each of the specific probes.

Complementation with S. aureus-S. epidermidis hybrids. Though S. epidermidis RNAIII could stimulate the transcription of both serine protease and alpha-toxin genes, it was less efficient than wild-type S. aureus RNAIII. To test which part of RNAIII is most important for the regulatory function, hybrid molecules were created by using a conserved StyI site (Fig. 2), dividing the RNAIII gene into two parts of roughly the same length. A fusion between the 5' half of S. aureus RNAIII and the 3' half of S. epidermidis (pEX0130) increased the transcription of hla and ssp about fourfold compared to S. epidermidis wild-type RNAIII (pEX0124). However, the fusion molecule was slightly less efficient than wild-type S. aureus RNAIII in stimulating ssp expression (Fig. 6). The fusion between the 3' half of S. epidermidis RNAIII and the 5' half of S. aureus (pEX0129) also increased transcription of hla and ssp compared to pEX124 (S. epidermidis), though it was less efficient than pEX0130. The same differences were seen when zones of proteolysis on casein agar plates were compared (Fig. 7).

View larger version (62K): [in this window] [in a new window]   FIG. 6.   Northern blot analysis of RNAIII, alpha-toxin, serine protease, and protein A transcripts in strains WA400(pEX0128), WA400(pEX0129), WA400(pEX0130), WA400(pEX0124), and WA400 at different time points during growth. The pEX part of plasmid designations is not shown in the figure. Strain WA400 without plasmids () is the control. The same filter was hybridized with each of the specific probes.

View larger version (36K): [in this window] [in a new window]   FIG. 7.   Zones of proteolysis in different strains. The RNAIII-deficient S. aureus strain WA400 (zone 1) and the same strain expressing S. epidermidis RNAIII (pEX0124) (zone 2), S. aureus RNAIII (pEX0128) (zone 3), and S. aureus-S. epidermidis RNAIII hybrids pEX0129 (zone 4) and pEX0130 (zone 5) (see Table 1) were used.

	DISCUSSION

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In this study, we have identified RNAIII homologs in three different CoNS, i.e., S. epidermidis, S. warneri, and S. simulans. RNAIII molecules from these three species turned out to be very similar to that of S. aureus. The inability to detect an RNAIII gene in S. cohnii, S. xylosus, S. haemolyticus, and S. saprophyticus by the method used in this study does not unambiguously exclude the possibility that these species lack the gene. However, similar results were obtained by Donvito et al. (6) in a screening for RNAIII and delta-lysin in several CoNS species by PCR and Southern blotting, except that they did not obtain a positive signal in S. simulans. In addition, Vandenesch et al. (32) have identified RNAIII in S. lugdunensis. In contrast to the RNAIII molecules identified in this study, the S. lugdunensis RNAIII did not contain an open reading frame encoding delta-lysin. However, the remaining parts of S. lugdunensis RNAIII are very similar to the RNAIII molecules identified here, suggesting a common function.

RNAIII from all sequenced strains contained open reading frames encoding delta-lysin homologs with a high degree of sequence identity to the S. aureus delta-lysin. In particular, the distribution of the charged amino acid residues was conserved, suggesting that amphipatic -helices can be formed and that they can function as pore-forming toxins, as described for the S. aureus delta-lysin (19). This is in agreement with the finding that delta-lysin-like activity is produced by many CoNS (9).

Northern blot analysis revealed that S. epidermidis, S. simulans, and S. warneri produced large amounts of RNAIII. Though the entire agr locus has not yet been identified, sequencing of the region upstream of the CoNS RNAIII determinants revealed a putative divergent promoter, similar to P2 of S. aureus, followed by the beginning of a tentative agrB gene (unpublished results). The finding that the CoNS RNAIII promoters were regulated in an agr-dependent way in S. aureus also supports the existence of an agr operon in some CoNS species. This was also supported by the observation that RNAIII in S. simulans was expressed only during the late exponential and postexponential phases of growth (unpublished results). Otto et al. (28) have recently sequenced the entire agr locus from an S. epidermidis strain, showing that it has the same architecture in S. aureus. Their sequence agreed completely with our data.

A number of big insertions and/or deletions have occurred mainly in the 5' halves of the CoNS RNAIII molecules compared to that of S. aureus, while the 3' halves seem more conserved. RNAIII molecules from S. aureus, S. epidermidis, and S. warneri seem to be the most closely related, while S. simulans RNAIII is less closely related, except for the first 50 and last 150 nt where all molecules are highly identical. It seems likely that these conserved regions are important for the regulatory function of RNAIII, as indicated by the ability of the CoNS RNAIII molecules to complement the RNAIII defect of S. aureus WA400.

The finding that the CoNS RNAIII repressed transcription of spa as efficiently as S. aureus RNAIII, while stimulation of hla and ssp transcription was impaired, suggests that these regulatory functions are independent. This is also indicated by the finding that some deletions in the S. aureus RNAIII gene affected transcription of spa but not transcription of hla and hlb and vice versa (27). It may also be concluded that the repressing activity should reside in those parts of the molecules with highly conserved sequences. However, conserved secondary structures in regions with less sequence identity may also be important for the regulatory function. On the other hand, since most of the predicted secondary structures seem to be conserved, the impaired stimulatory function of the CoNS RNAIII molecules compared to that of S. aureus RNAIII might be due to differences in the primary nucleotide sequence.

Slightly different effects were seen between the different RNAIII molecules. In particular, the kinetics of hla mRNA production was different with S. simulans RNAIII (pEX0125 in Fig. 5) compared to the other RNAIII species. This may be related to the fact that S. simulans RNAIII is the most distantly related to S. aureus RNAIII. Balaban and Novick (3) have presented evidence that S. aureus RNAIII alters between different conformations with different regulating activities during the growth cycle. Differences in the nucleotide sequence of the RNAIII molecule might influence the kinetics of these conformational changes and hence the regulation of hla transcription. It should be pointed out that maximum amounts of ssp mRNA were still obtained after 4 h with plasmid pEX0125, suggesting that slightly different mechanisms of regulation may operate.

The evidence that the S. epidermidis RNAIII molecule can be improved in its stimulatory function by substituting either the 5' or 3' half of the molecule for the S. aureus equivalent indicates that both parts are involved in the regulation. However, the hybrid RNAIII consisting of the 5' part of S. aureus RNAIII and the 3' part of S. epidermidis RNAIII was slightly more efficient than the inverse hybrid (Fig. 6 and 7), suggesting that the impaired stimulatory activity of S. epidermidis RNAIII was mainly due to sequence differences in the 5' half of the molecule. Whether this means that independent regulatory domains are present or that the 5' and 3' parts of the molecule interact to create a functional structure is not known. Secondary structure predictions have indicated such an interaction (27), but these predictions must be demonstrated by biochemical methods.