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Proc Natl Acad Sci U S A. Oct 4, 2005; 102(40): 14249–14254.
Published online Sep 23, 2005. doi:  10.1073/pnas.0503838102
PMCID: PMC1242290
From the Cover
Biochemistry

Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains

Abstract

Small RNA (sRNA) genes are expressed in all organisms, primarily as regulators of translation and message stability. We have developed comparative genomic approaches to identify sRNAs that are expressed by Staphylococcus aureus, the most common cause of hospital-acquired infections. This study represents an in-depth analysis of the RNome of a Gram-positive bacterium. A set of sRNAs candidates were identified in silico within intergenic regions, and their expression levels were monitored by using microarrays and confirmed by Northern blot hybridizations. Two sRNAs were also detected directly from purification and RNA sequence determination. In total, at least 12 sRNAs are expressed from the S. aureus genome, five from the core genome and seven from pathogenicity islands that confer virulence and antibiotic resistance. Three sRNAs are present in multiple (two to five) copies. For the sRNAs that are conserved throughout the bacterial phylogeny, their secondary structures were inferred by phylogenetic comparative methods. In vitro binding assays indicate that one sRNA encoded within a pathogenicity island is a trans-encoded antisense RNA regulating the expression of target genes at the posttranscriptional level. Some of these RNAs show large variations of expression among pathogenic strains, suggesting that they are involved in the regulation of staphylococcal virulence.

Keywords: nosocomial infections, prokaryotes, small regulatory noncoding RNAs

Small regulatory noncoding RNAs are encoded in both eukaryotic and prokaryotic genomes. Most of these RNA transcripts regulate gene expression by modifying mRNA stability and translation. These RNAs act by pairing with other RNAs, forming part of RNA-protein complexes, or adopting the structures of other nucleic acids (1). Some of these RNAs can also control virulence gene expression in bacterial pathogens in response to host signals (2).

Staphylococcus aureus is the most common cause of hospital-acquired infection and becomes increasingly resistant to antibiotics. Its genomic plasticity facilitates the evolution of virulent and drug-resistant strains, presenting a major and ever-changing clinical challenge. S. aureus lives as a commensal of the human nose and skin in 30-70% of the population. Once it contaminates a skin breach or mucous membranes, it can cause minor skin infections (common) to life-threatening conditions (rare) such as endocarditis, pneumonia, or toxin-mediated diseases (toxic shock syndrome and scalded skin syndrome). Seven complete genome sequences of S. aureus are now available (see supporting information, which is published on the PNAS web site, for the description of the five genomes selected for this study). The strains selected here exhibit genotypic variability, plasticity (3), and alternate ways of expressing virulence, leading to various clinical symptoms. We took advantage of this abundance of sequences to devise a procedure based on comparative genomics and microarrays to identify previously uncharacterized RNAs expressed in S. aureus. Two small RNAs (sRNAs) had previously been shown to be expressed in S. aureus cells: antisense RNA I regulates the replication of S. aureus multiresistance plasmid pSK41 (4), and RNAIII, encoded in the bacterial chromosome, controls virulence genes encoding exoproteins and cell wall-associated proteins (5). Based on sequence comparison, tmRNA, RNase P RNA, 4.5S and 6S RNAs were predicted to be expressed in this bacterium.

Here, we report that at least 12 sRNA transcripts are expressed in S. aureus. Seven are found in S. aureus pathogenicity islands (SaPIs) that contain a subset of genes involved in bacterial virulence that can transfer horizontally between strains; the others are located in the remaining part of the genome (core). Some of these sRNAs are present in multiple copies and localized next to genes implicated in virulence and antibiotic resistance. Some of these sRNAs are differentially expressed among the four tested pathogenic strains. Some sRNAs are conserved throughout bacterial phylogeny, and their secondary structures were inferred from sequence comparison, as has been done for other sRNAs (6). One sRNA is an antisense regulator, and three mRNA targets have been identified. The specific recognition and binding of one of the target mRNAs with its corresponding sRNA was demonstrated in vitro.

Experimental Procedures

Genomic Identification of sRNAs. The genome of S. aureus Mu50 (European Molecular Biology Laboratory accession no. BA000017 rev. 67) was analyzed with the intergenic sequence inspector (7). The genome was partitioned to generate a data set of 1,268 intergenic sequences (IGRs) >120 nt in length. Sequence homology searches were performed with blast against 31 sequenced genomes of firmicutes (as of June 2003, available at www.ebi.ac.uk/genomes/bacteria.html). The IGRs with expected values >0.1 were removed from the data set (972 IGRs remain). Conserved IGRs containing 5 rRNA operons, 6 tRNAs, 41 S. aureus repetitive elements, 8 riboswitches (2 ribof lavine nucleotides, 4 S-adenosylmethionine, and 2 thiamine pyrophosphate) were removed from the data set. The “AT-rich” IGRs containing G+C islands longer than 30 nucleotides with G+C > 35% were selected by using genview, a component of intergenic sequence inspector (sRNA candidates represent only a subset of the IGRs), the rationale for this criterion of selection being that RNA genes present in AT-rich IGRs can be detected based on their G+C content. The final data set contains 191 selected IGRs, including 4.5S RNA (ffs), RNase P RNA (rnpB), tmRNA (ssrA), and RNAIII.

cDNA Labeling and Microarray Hybridization. A microarray was designed by using oligonucleotides derived from the 191 IGRs (the design procedure is available as supporting information). The IGRs were PCR amplified to yield dsDNAs, which were spotted in triplicate. Ten micrograms of total RNA used for each labeling reaction was denatured with random primers (Invitrogen) and reverse transcribed with dCTP-Cy3 or dCTP-Cy5 (Amersham Pharmacia) and 200 units of Superscript II (Invitrogen) at 42°C. After labeling, RNAs are removed by NaOH/HCl treatment and precipitated with isopropanol. For hybridization, half of the samples were denatured 2 min at 100°C, mixed with DigEasyHyb (Roche Diagnostics) and applied to each slide. After 16 h at 55°C, the slides were washed for 5 min successively in 0.01% SDS/2× SSC, 0.2× SSC, and 0.1× SSC. Slides were scanned with Packard Biosciences Scanarray. Spot intensities were analyzed with GenePix Pro-4 (Axon Instruments). Microarray analysis and normalization data are available as supporting information.

Bacterial Growth, RNA Extraction, Labeling, and Northern Blots. For each sample, one colony was suspended in 20 ml of LB and incubated at 37°C overnight. Each culture was started by diluting the precultures to an OD600 = 0.06 and was then incubated at 37°C [120 rpm/in Infors, unitron incubator (Bottmingen, Switzerland)] and stopped at mid-exponential phase (E, OD600 = 1.4), early stationary phase (S1, OD600 = 3) and late stationary phase (S2, OD600 = 5). RNA extraction was performed as described in ref. 8. DNA was removed by treatment with 10 units of FPLCpure DNase I (Amersham Pharmacia) at 37°C for 1 h in 40 mM Tris·HCl/6 mM MgCl2, pH 7.5. RNAs were purified by phenol/ether extraction. 5′ RNA labeling was performed as in ref. 9. The DNA sequences used to detect sRNAs are available as supporting information. Total RNA (15 μg) was denatured and separated on denaturing 8% PAGE with a radiolabeled RNA ladder (Novagen) and electrotransferred onto a Zeta probe GT membrane (Bio-Rad) in 0.5× 90 mM Tris/64.6 mM boric acid/2.5 mM EDTA, pH 8.3. Specific 32P probes were hybridized with ExpressHyb solution (Clontech) for 90 min, washed in 2× SSC for 20 min, 0.2× SSC/0.1% SDS for 20 min, and exposed and scanned with a PhosphorImager (Molecular Dynamics).

RNA Sequencing and Binding Assays. Total RNA (200 μg) from S. aureus N315 was separated on 5% PAGE. The unknown RNAs to be sequenced were extracted from the gel, eluted in 10 mM Tris·HCl/250 mM NaCl/1 mM EDTA, pH 7.5, precipitated, labeled, and gel purified. RNA sequencing was performed as in ref. 9, aligned with the S. aureus N315 genome by using fasta. SprA and a 159-nt fragment corresponding to the 3′ UTR of ORF2216, a putative mRNA target of sprA, were PCR amplified from genomic DNA of S. aureus Mu50 with oligonucleotides containing a T7 promoter and transcribed in vitro. The RNAs were 3′ labeled and gel purified. Binding assays between 3′-labeled sprA and increasing amounts of a purified mRNA fragment of ORF2216 (the 19 last codons followed by 102 nt of the predicted 3′ UTR) were performed as described in ref. 10, with the following modifications: the purified RNAs were denatured independently for 3 min at 90°C and allowed to refold separately at room temperature for 30 min in 5 mM MgCl2/20 mM NH4Cl/10 mM Hepes KOH, pH 6.9. The RNAs were pooled with 8 μl of 10 mM spermidin/3 mM MgCl2/200 mM NH4Cl/10 mM Hepes KOH, pH 6.9, supplemented with 1 μl of RNasin (Promega) and incubated at 37°C for 30 min. Binding specificity was tested by adding either a 50- to 200-fold molar excess of unlabeled sprA or a 100- to 1,000-fold molar excess of bulk yeast tRNA to a 1:10 molar ratio of labeled sprA versus the unlabeled mRNA fragment derived from the ORF SA2216. The samples were separated by 5% nondenaturing acrylamide gel electrophoresis, and the results were analyzed on a PhosphorImager (Molecular Dynamics).

Supporting Information. See supporting information for the location of the sRNA genes sprA and 6S RNA, the set of tested IGRs, microarray design, the normalized expression levels of 191 IGRs in S. aureus, the comparative analysis of strains N315, MRSA252, 502A and COL, and the sequence of oligonucleotides used for the Northern blots.

Results

Purification and Sequence Determination of Highly Expressed sRNAs in S. aureus. Evidence of the existence of sRNAs other than 5S rRNA and tRNAs came with the development of PAGE to analyze total RNA (11). Besides tRNAs, 5S, 16S, and 23S rRNAs, additional bands can be detected by ethidium bromide staining (Fig. 1A). A ≈270-nt RNA has a length and expression profile compatible with the predicted sequence of the RNA component (4.5S) of the signal recognition particle. Two weaker bands at ≈350 nt are compatible with sizes predicted from the sequences of tmRNA (available upon request) and of the RNA component of the ribonuclease P (12). Finally, three additional unidentified RNAs can be visualized (Fig. 1A). One has a length of ≈230 nt; its expression level is high (comparable with that of predicted 4.5S RNA) and further increases during cell growth. Two additional ≈150- and ≈500-nt-long RNAs are expressed at lower levels. These three RNAs were gel purified, eluted, radiolabeled, and sequenced by using ribonucleases T1 and U2 (Fig. 1B). The three partial RNA sequences were aligned against the genomic sequence of S. aureus N315 by using fasta3 (13). The ≈500-nt RNA sequence matches a 500-nt sequence of the 23S rRNA, whereas the ≈150-nt and the ≈230-nt RNA sequences each match a single genomic sequence (supporting information). Their expression profiles and sizes were confirmed by microarrays (see below) and Northern blots analysis (Fig. 1C). Recently, the gene encoding 6S RNA, a regulator of bacterial RNA polymerase (14), has been identified in S. aureus based on blast searches (15), and it matches the 230-nt RNA sequence. Gene names were assigned to the candidates that were confirmed to be expressed as RNA transcripts in S. aureus. The nomenclature is based on their location on the bacterial chromosome: <<spr>> for <<small pathogenicity island RNA>>, followed by a capital letter.

Fig. 1.
Experimental identification of highly expressed sRNAs in S. aureus. (A) Total RNA extracted at exponential (E) or early stationary (S1) phase analyzed by 8% PAGE stained by ethidium bromide (L, RNA ladder). (B) Three unknown RNAs or RNA fragments (1- ...

Identification of Candidate sRNA Genes by Homology and Whole Genome Expression Analysis. It remained possible that the genome of S. aureus might express additional sRNAs at low levels, which would be undetectable by electrophoretic fractionation of total RNA. To look for these RNAs, a computational approach combined with whole genome expression analysis was applied to the genome of S. aureus N315. Of 2,293 IGRs, 191 candidate IGRs were selected by the Intergenic Sequence Inspector (7) (see Experimental Procedures for selection of the candidates). Microarrays were constructed to allow detection of transcripts from these 191 selected IGRs. Total RNA, isolated from S. aureus N315 cells grown to exponential early and late stationary phases, was used to generate fluorolabeled cDNAs that were hybridized to the microarrays. Expression patterns from the selected IGRs were used as additional criteria to select among candidate regions, leading to a set of 25 IGR candidates, listed in supporting information, that were analyzed further by Northern hybridization by using strand-specific probes.

Small RNA Transcripts Detected by Northern Hybridization. The 25 IGR candidates were analyzed by Northern hybridization with RNA extracted from S. aureus N315 cells grown to exponential (E), early (S1), and late (S2) stationary phases. Northern analysis was carried out by using strand-specific oligonucleotide probes to determine gene orientation (W, clockwise; C, counterclockwise). IGR candidates gave distinct bands consistent with the expression of sRNAs (Figs. (Figs.11 and and2).2). The results from the strand-specific probes generally agreed with predictions, one exception being sprE. In one case, an sRNA is expressed from both the W and C strands within the IGR (sprF and sprG, Fig. 2). For this IGR, two or more RNA species were detected with a single probe. There is no evidence of the existence of conserved ORFs within the genes expressing these sRNAs. tmRNA, RNase P RNA, and 4.5S RNA are expressed from their predicted genomic locations (Fig. 2B). The size of all these sRNAs is in agreement with sequence comparison predictions. RNAIII is not expressed in the N315 strain, as has been described in ref. 16. Ten candidates did not display any detectable transcript from either the W or the C DNA strands, although the microarray analysis detects expression from the 10 IGRs containing these sRNAs. The sequence corresponding to these predicted sRNAs represents only a part of the IGRs spotted on the microarray. For these 10 cases, the positive signals on the microarray are probably due to regulatory regions of adjacent genes. Overall, based on microarrays and northern hybridizations, at least 12 sRNA genes are expressed in S. aureus (Table 1). Five are expressed from the core genome and seven from pathogenicity islands.

Fig. 2.
Experimental verification of the predicted sRNAs in S. aureus. Northern hybridization was performed by using strand-specific probes. (A) The labeled probes target either an sRNA candidate expressed from the Watson (W) or the Crick (C) strands. Total RNA ...
Table 1.
The sRNAs expressed in S. aureus

Distribution and Expression of sRNA Genes in S. aureus Pathogenic Strains. The expression levels of sRNAs were monitored in all four strains of S. aureus. Methicillin-resistant MRSA252 is the most genetically diverse strain sequenced to date, with ≈6% of its genome being novel compared with the other published genomes. The MRSA COL strain is at the far end from strain MRSA252 on a neighbor-joining tree based on multilocus sequence typing. Strain 502A is among the least pathogenic strains and lacks multiple antibiotic drug resistance. Finally, N315 was studied because its genome was sequenced when the work was initiated.

Fig. 3A details blast search results regarding the presence or absence of the sRNA genes sprA-G, tmRNA, rnpB, RNAIII, ffs (4.5S RNA), and ssrS (6S RNA) within the genomes of the four strains. sRNAs sprA, sprE, sprF, and sprG are present in multiple (two to five) copies. Note that the additional copies of sprA and sprFG are in the core genome, not in the pathogenicity islands. Fig. 3B shows the genomic location of all of the sRNA genes identified in strain N315. Fig. 3C shows the genes flanking some of the novel sRNAs, with emphasis to those located next to genes involved in bacterial virulence. For example, sprB flanks a gene encoding a β-lactamase, sprC is situated next to lukD and lukE expressing the Panton Valentine leukotoxine responsible for outbreaks of severe skin infections (18), and sprE is next to a staphylokinase that promotes bacterial spread in tissues.

Fig. 3.
Copy number and genomic location of the sRNA genes in S. aureus. (A) The copy number of the sRNA genes within the genomic sequences of S. aureus N315, MRSA252, and COL; in brackets are the locations of the sRNA genes. tmRNA, 4.5S RNA, and RNase P RNA ...

Using microarrays (Fig. 4) and Northern blots (Fig. 5), a comparative expression analysis of the 12 sRNAs from strains MRSA252, COL, N315, and 502A was performed with total RNA extracted at exponential (E), early (S1), and late (S2) stationary phases. Each sRNA sequence, when detected, is conserved in all four strains and was detected with a single oligonucleotide; one exception was sprA, for which only the expression of the copy located in the pathogenicity island was monitored. Of the four strains, N315 expresses the highest levels of all of the sRNAs, with the exception of RNAIII. Conversely, the COL strain expresses very few of the sRNAs and at low levels. 6S RNA, also encoded in the core genome of the four strains, has higher expression levels at stationary phase, with the appearance of a shorter transcript. As predicted from their absence from the genomes, neither sprC in MRSA252 strain nor sprD in strain COL are expressed in vivo (Fig. 5 B and C). Several sRNAs that are encoded in all strains, however, display huge variations of expression between strains, especially those located in pathogenicity islands (sprA-C, Fig. 5). As confirmed by Northern blots, RNAIII is encoded within the genomes of the four strains but displays large variations of expression profiles (Fig. 5B). RNAIII is expressed at high levels in strain 502A but is not expressed in the agr-negative strain N315 (16). SprA is highly expressed only in strain N315. SprA, sprF, and sprG are expressed by strain 502A, whereas sprD and sprE are expressed by strain MRSA252. Therefore, even though all of the sRNA genes are present in their genomes, each bacterial strain has a specific expression pattern under the tested growth conditions.

Fig. 4.
Comparative expression profiles of S. aureus sRNAs across microarrays. (A) Three-dimensional diagram of the normalized intensity of expression of sRNAs sprA-sprFG among four strains of S. aureus at exponential (E), early (S1), and late (S2) stationary ...
Fig. 5.
Expression profiles of the sRNAs among four S. aureus pathogenic strains. Northern hybridization was performed by using strand-specific probes. (A) RNAIII is expressed only in MRSA252 (weakly) and 502A (strongly), and 6S RNA is expressed in all four strains. ...

Structural Study of Selected sRNAs from S. aureus. Phylogenetic comparative methods have proven effective for inferring the secondary and tertiary structures of RNAs (19). Based on sequence comparison with other species, the secondary structures of tmRNA, 4.5S RNA, and RNase P RNA from S. aureus have been predicted, and that of RNAIII was deduced from structural probes (17). Among the other sRNA genes identified in the genome of S. aureus, we gathered sufficient divergent gene sequences of 6S RNA and sprA to deduce their structures by phylogenetic comparison among 184 bacterial genomes and 30 plasmid sequences of S. aureus.

An analogue of Escherichia coli 6S RNA was identified initially in Bacillus subtilis (20), and its secondary structure was recently inferred by structural probes combined with comparative sequence analysis (14). In this report, 6S RNA was also identified in 37 bacterial genomes from 7 families and 2 genera (see supporting information for details). We propose an RNA structure for the 6S RNA (Fig. 6A) that is, overall, similar to earlier reports for a variety of bacterial species (14, 15), with specific comments. In the proposed structure for 6S RNA, 53% of the nucleotides participate in pairing. In 6S RNA, H4 is followed by a U-rich stretch (detected in nine sequences), probably acting as a transcription terminator. At late stationary phase, the ≈230-nt-long 6S RNA coexists with a ≈180-nt-long RNA (Figs. (Figs.1D1D and and5A).5A). The length of the longer RNA corresponds to the sequence including H4 (Fig. 6A), whereas the size of the shorter one is compatible with an RNA starting and ending at H1a, probably processed at its 5′ and 3′ ends.

Fig. 6.
Secondary structures of S. aureus 6S RNA (A) and sprA (B) based on comparative sequence analysis (supporting information). Phylogenetic support for H1ab and H4a in sprA is weak. The bars are the GC and AU pairs, and the dotted lines are the GU pairs. ...

Forty-five genes sequences of sprA have been detected and aligned (supporting information) within 9 genomes of staphylococcaceae (26 sequences) and 14 accessory genetic elements (19 sequences from plasmids). An alignment of sequences with sufficient sequence variations could identify seven conserved base paired regions H1abc, H2, H3, and H4ab. The primary nucleotide sequence of H1a, H1b, and H4a is conserved, and pairings are not supported by covariations. In sprA, only 43.8% of the nucleotides are paired (Fig. 6B), due to a 57- to 59-nt-long region with no proposed structure between H1a and H2. Predicted pairings between H2 and H3 forms an RNA pseudoknot. H2 and H3 are entangled and connected by three nucleotide stretches. Four to 6 intervening nucleotides intercalate between the two stems. Stem H3 abuts on H4a in all of the sequences but one (S. aureus pMW2), suggesting coaxial stacking.

Functional Study of sprA. Bacterial sRNAs can function as antisense RNAs (21). Complementary sites between the 3′ end of sprA and the 3′ ends of three mRNA 3′ UTRs from S. aureus were detected (Fig. 7A; 29 nt, of 34-35, are predicted to interact through Watson-Crick GC or AU pairs), by using fasta3 (13). One of the predicted mRNA targets encodes a ≈3,521-nt-long ABC transporter operon, including two ORFs, SA2216 and SA2217 (S. aureus N315, genome nomenclature). The other two mRNA targets of sprA encode a putative α-acetolactate decarboxylase (SA2007) and a hypothetical protein (SA1944). Twenty-nine base pairs (14 GC and 15 AU pairs with SA2216, Fig. 7B) are predicted to form between nucleotides from stem-loop H4b in sprA and nucleotides embedded into the upper portion of predicted stem loops at the 3′ UTR of each mRNA target (Fig. 7A).

Fig. 7.
SprA has antisense functions and targets selected mRNA 3′ UTRs. (A) Three mRNA 3′ UTRs have sequence complementarities with sequences at the 3′ end of sprA (H4b). The ORFs are numbered according to ref. 22. The termination codons ...

In vitro duplex formation between sprA (a purified 202-nt-long transcript) and a T7-transcribed mRNA fragment containing the 3′-terminal 19 codons of SA2216 ORF from the ABC transporter operon, extended by 102 nt from its predicted 3′ UTR (a 159-nt-long transcript), was investigated by gel retardation assays. The two RNAs were refolded separately and independently, cooled down at room temperature, and labeled sprA (3 pmol) was incubated with increasing concentrations of unlabeled mRNA at 37°C for 30 min. The reactions were analyzed by gel electrophoresis by using nondenaturing polyacrylamide gels (Fig. 7C). At a 1:1 molar ratio, nearly all sprA is in complex with its mRNA target. Therefore, the interaction does not require the participation of protein Hfq in vitro. The binding between sprA and the mRNA fragment is specific, because a 100- to 1,000-fold molar excess of total tRNA from yeast does not displace sprA from a preformed sprA-mRNA complex, whereas a 50-fold excess of cold sprA competed labeled sprA out of the complex (Fig. 7C). The observed binding is saturable with half-maximal binding at a free ORF concentration of ≈85 nM (Kd).

Discussion

Very few sRNAs have been identified in Gram-positive species (4, 5, 23, 24). Our report shows that the RNome of the Gram-positive bacterium, S. aureus, contains at least 12 RNA species. This work serves as a blueprint for the prediction, detection, and characterization of a group of novel sRNA genes in Gram-positive bacteria. Our multifaceted search strategy to detect sRNA genes in the genome of S. aureus was validated by the identification of eight previously uncharacterized sRNAs and four predicted to be expressed based on sequence comparison (Table 1). Northern analysis determined that 15 of 25 candidate intergenic regions express RNA transcripts, some present in multiple copies and some expressing more than one RNA. The microarray analysis shows that in the four strains studied, all of the IGRs in the first megabase pairings whose expression was monitored are generally not transcribed (supporting information). The previously uncharacterized transcripts range from 90 to 400 nt in length, their expression levels and profiles varying during cell growth.

The expression of the RNome is subjected to large variations among S. aureus strains. Indeed, sprA-G and RNAIII show significant variations of expression levels among clinical isolates. Among the four strains studied, there are huge variations in RNAIII expression levels. Although it is encoded in the genome, strain N315 does not express RNAIII, whereas sprA, sprB, and sprC are expressed to high levels. Conversely, RNAIII expression in strains 252 and 502A is high, whereas the expression levels of sprA and sprC, and, to a lesser extent, sprB, are switched off.

Of the 12 sRNAs detected, 6S RNA is conserved among eubacteria (14, 15) and is highly expressed in S. aureus, allowing its detection and isolation by direct purification. In E. coli, 6S RNA inhibits sigma factor σ70 and activates σS in response to nutriment starvation (15). In S. aureus, three sigma factors are known, the housekeeping σA (a σ70 homolog), σB, and σH. σB is involved in general stress response of many Gram-positive bacteria and plays important roles in antibiotic resistance and virulence factors in S. aureus (25). Although σS and σB have functional similarities, these proteins are not homologous in sequence and regulation. These differences suggest that the role of 6S RNA in S. aureus and E. coli may be different, especially concerning virulence.

There are two previously described examples of sRNA regulation by antisense mechanisms in S. aureus. The 5′ end of RNAIII positively controls the translation of hla, encoding α-hemolysin (26), and RNAI controls the replication of plasmid pSK41 from S. aureus (4). This report shows that there is at least a third one, SprA, that does not require the contribution of protein Hfq to bind in vitro one of its three predicted mRNA targets, but Hfq might be required in vivo. SprA is in multiple copies in all of the sequenced S. aureus strains, suggesting an important functional role. Multiple copies of sprA probably allow the fine-tuning of their expression levels to induce a physiological response that is dictated by external signals, as described for the redundant RNAs that regulate the quorum sensing and virulence in V. cholerae (27). In all 45-gene sequences of sprA identified, the gene is always flanked by at least one, sometimes two, insertion sequences that are either complete (probably functional) or have sustained deleterious genomic reshuffling. Three mRNA targets of sprA have been identified and are probably functionally related. They include the 3′ UTR of an mRNA encoding an ABC exporter. Antisense sRNAs such as gcvB (28), sgrS (29), or rydC (9) regulate the expression of transporters representing up to 10% of the functional genes in eubacteria, suggesting that transport regulation by sRNAs is widely used in prokaryotes. Most bacterial antisense-box sRNAs described thus far bind to the 5′ UTR of their target mRNAs (21). Binding of sprA to its mRNA targets, however, is at their 3′ UTRs. In an archaeon, predicted interactions of recently identified sRNAs (30) are also located in 3′ UTRs of target mRNAs. Initially, a loop-loop contact between the antisense and target RNAs, as suggested for sprA, is followed by the propagation of intermolecular helices. These antisense RNAs, or their targets, contain YUNR (Y, pyrimidine; U, uracil; n, any nucleoside; R, purine) U-turn motifs required for efficient bimolecular interaction (31). When aligning the 45 genes sequences of sprA, sequence conservations of the recognition loop H4 have been identified, with the motif YBGCRR (B = any nucleoside except A), that might also mediate efficient pairing with target mRNAs.

What are the functions of the remaining sRNAs? As in other species, some might act as antisense RNA because natural antisense controls are found in bacterial plasmids, transposons, and bacteriophages. In addition, protein Hfq is encoded in the genome of S. aureus, linked to the action of the sRNAs that use pairing interactions to regulate the expression of target mRNAs. It seems likely that these sRNAs will be found to perform regulatory roles in cellular stress responses. The next challenges are to elucidate their functions in S. aureus gene regulation.

Virulence factors are differently distributed and regulated among S. aureus strains (32). S. aureus genes involved in pathogenesis are controlled by a complex regulatory network that includes at least four two-component systems. The intracellular effector of one of these systems, the agr regulon, is RNAIII, up-regulating transcription of extracellular protein genes, down-regulating many surface protein genes, also acting at the translational level (33). We report the identification of sRNA genes in bacterial pathogenicity islands. Several copies of sprA and sprE-G are located in pathogenicity islands of S. aureus (Fig. 3B). Our findings provide insights and raise provocative questions and hypotheses regarding bacterial virulence. Of these previously undescribed sRNAs genes expressed from the genome of S. aureus, seven are expressed from pathogenicity islands and integrated bacteriophages, some in multiple copies. These genetic elements carry genes with virulence or antibiotic resistance functions and accounts for ≈25% of any S. aureus genome, mostly consisting of mobile, or once mobile, genetic elements that were initially transferred horizontally and then vertically, if positively selected. This report demonstrates that small, noncoding RNA molecules are expressed in S. aureus pathogenicity islands, probably an interesting finding that will have important implications in the understanding of S. aureus virulence.

Supplementary Material

Supporting information:

Acknowledgments

We thank Dr. Y. Leloir (Institut National de la Recherche Agronomique, Rennes, France) and the Network on Antimicrobial Resistance in S. aureus network (Herndon, VA) for the S. aureus strains and Drs. F. Hubler and A. Monnier (Unité Mixte de Recherche 6061, Rennes) for their help with the microarrays. C.P. has Ph.D. financial support from the région Bretagne (France).

Notes

Author contributions: C.P. and B.F. designed research; C.P. and B.F. analyzed data; and B.F. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: IGR, intergenic sequence; SaPI, S. aureus pathogenicity islands; sRNA, small RNA.

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