trans-Complementation of a Staphylococcus aureus agr Mutant by Staphylococcus lugdunensis agr RNAIII

Abstract

RNAIII from Staphylococcus lugdunensis (RNAIII-sl) in a Staphylococcus aureus agr mutant partially restored the Agr phenotype. A chimeric construct consisting of the 5′ end of RNAIII-sl and the 3′ end of RNAIII from S. aureus restored the Agr phenotype to a greater extent, suggesting the presence of independent regulatory domains.

In Staphylococcus aureus, the expression of several exoproteins and surface proteins involves the well-characterized global regulator agr, which combines a density-sensing cassette (AgrD and -B) and a two-component sensory transduction system (AgrA and -C), all encoded within the P2 operon (5, 11). The actual effector of the agr system of S. aureus is a 517-nucleotide (nt) transcript, RNAIII-sa of the P3 operon, which incidentally encodes the 26-amino-acid delta-hemolysin (4). RNAIII-sa acts primarily at the level of transcription, stimulating expression of postexponentially expressed extracellular toxins and enzymes, such as alpha- and beta-hemolysins, enterotoxins, toxic shock syndrome toxin, lipase, nuclease, and serine protease, and repressing expression of exponential-phase surface proteins, such as protein A and coagulase (3, 6, 12, 16, 17). RNAIII homologs have been identified in several coagulase-negative staphylococci (CoNS), including S. lugdunensis (19), S. epidermidis, S. warneri, and S. simulans (18). RNAIIIs from the last three species are very similar to that of S. aureus, notably in the first 50 and last 150 nt and in the presence of open reading frames encoding a delta-hemolysin (18). In contrast, RNAIII from S. lugdunensis (RNAIII-sl) differs considerably from RNAIII-sa, having no delta-hemolysin gene and low overall homology despite some conservation at the 5′ and 3′ ends (Fig. 1). A recent study has shown that RNAIIIs from several CoNS can influence the expression of S. aureus agr-regulated genes. RNAIII from S. epidermidis, S. warneri, or S. simulans, when introduced into an RNAIII-deficient mutant of S. aureus (WA400), completely repressed transcription of protein A but stimulated transcription of alpha-hemolysin (hla) and serine protease (sasp) genes less efficiently. A fusion between the 5′ half of RNAIII-sa and the 3′ half of S. epidermidis RNAIII increased transcription of hla and sasp, although it increased sasp transcription less efficiently than wild-type RNAIII-sa. The converse fusion also increased transcription of hla and sasp, though with lesser efficiency than RNAIII-sa. This suggests than both the 5′ and 3′ halves of RNAIII are important for regulatory functions and that the impaired stimulatory activity of S. epidermidis RNAIII was mainly due to sequence differences in the 5′ half of the molecule (18). The present study describes the ability of S. lugdunensis RNAIII-sl, which contains no delta hemolysin gene and has low homology with RNAIII-sa, to regulate the expression of S. aureus target genes by introducing RNAIII-sl into S. aureus WA400.

FIG. 1.

Alignment of the P3 operon of S. aureus (sa) with that of S. lugdunensis (sl), showing consensus sequences of promoters P2 and P3 (−10, −35), the open reading frame (hld), and direct repeats (arrows). The region of partial complementarity between hla mRNA and the 5′ end of RNAIII-sa is underlined. Sequences correspond to nt 1756 to 1036 of S. aureus (GenBank accession no. X52543) and to nt 1315 to 693 of S. lugdunensis (GenBank accession no. L13334). The indicated SpeI restriction site was used for construction of the chimeric RNAIII molecule. RBS, ribosome binding site; IR, inverted repeat.

Transcription of RNAIII-sl.

In order to clone full-sized S. lugdunensis rnaIII (rnaIII_SL) DNA, including its promoter, on the staphylococcal vector pE194, the size and transcription start site of RNAIII-sl were first determined. Whole-cell extracts of S. lugdunensis RN8160 were prepared according to the method of Kornblum et al. (7) and were analyzed by Northern blotting (10) with an RNAIII-sl-specific probe synthesized by PCR using the S. lugdunensis agr (agr_SL) sequence (GenBank accession no. L13334) and labeled with digoxigenin-labeled UTP (Boehringer Mannheim). RNAIII-sl was estimated to be 450 nt long (Fig. 2), and reverse transcription from a primer corresponding to nt 1061 to 1087 of the published sequence of agr_SL showed that the transcript began with A1130 (Fig. 1), as for RNAIII-sa (4). This suggests strong conservation of the P3 promoter sequences (Fig. 1). The entire rnaIII_SL DNA, including the promoter, from nt 17 to 1394, was then cloned by PCR into the staphylococcal vector pE194, forming pLUG150. This was electroporated into S. aureus RN6390 (agr⁺), RN6911 (agr null), or WA400 (lacking agr RNAIII) (Table 1). Northern blot analysis using the RNAIII-sl probe showed that RNAIII-sl was not expressed in RN6911/pLUG150 but was expressed in RN6390/pLUG150 and WA400/pLUG150 (Fig. 2, lanes 2, 4, and 6). Probing of the same blot by using an RNAIII-sa-specific probe synthesized by PCR from S. aureus agr (GenBank accession no. X52543) showed that RN6390/pLUG150 expresses the two RNAIIIs, distinguishable by a size difference, simultaneously and in virtually identical amounts (Fig. 2, lane 2). The level of RNAIII-sl production in RN6390 does not appear different from that in S. lugdunensis RN8160 (Fig. 2, lane 7). These results indicate that the RNAIII-sl promoter is agr dependent in the S. aureus background and requires a functional P2 operon, as observed for other CoNS RNAIII promoters (18). This reinforces the possible role of a highly conserved repeat, present in all staphylococci examined (18) (Fig. 1), upstream of the −35 element as important for RNAIII transcription (14). These repeats might be targets recognized by SarA, a transcriptional regulator important for full agr function in S. aureus (1). Preliminary experiments indicate that sequences with homology to sarA are present in S. lugdunensis (1a).

FIG. 2.

Northern blot analysis of agr-RNAIII transcripts from S. aureus and S. lugdunensis. RNAs from post-exponential-phase cultures were simultaneously probed with RNAIII-sa- and RNAIII-sl-specific probes. Lane 1, S. aureus RN6390 (agr⁺); lane 2, S. aureus RN6390/pLUG150 (RNAIII-sl); lane 3, S. aureus WA400 (agr mutant); lane 4, S. aureus WA400/pLUG150 (RNAIII-sl); lane 5, S. aureus RN6911 (agr null); lane 6, S. aureus RN6911/pLUG150 (RNAIII-sl); lane 7, S. lugdunensis RN8160 (agr⁺). Positions of migration of 16S rRNA (1,541 bases) and 23S rRNA (2,904 bases) are indicated.

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid	Relevant characteristics	Reference or source
Strains
S. aureus
8325-4	NCTC8325 cured of three prophages	15
RN4220	Restriction mutant of 8325-4	8
RN6390	Derivative of 8325-4; agr positive	17
RN6911	RN6390 Δagr 1057-4546::tetM	16
WA400	8325-4 ΔRNAIII region::cat86	3
WA400/pEX072	WA400/pEX072	3
S. lugdunensis RN8160	Standard agrSL positive	19
Plasmids
pEX072	Shuttle plasmid (pRN8054::pSP64)::2.1-kb BglII-PstI fragment of rnaIIISA region	3
pE194	3.728-kb S. aureus plasmid; inducible MLSa resistance (erm)	2
pLUG150	pE194::rnaIIISL	This work
pLUG226	pE194::rnaIIISL-SA	This work

^aMacrolide-lincosamide-streptogramin B. 

Regulatory effect of RNAIII-sl on S. aureus exoprotein genes.

The expression of six exoproteins and surface proteins in strain WA400/pLUG150 (RNAIII-sl) and strain WA400/pEX072 (a plasmid containing RNAIII-sa DNA, including the promoter and upstream sequences [3]) was compared. Briefly, culture supernatants were collected in the early stationary phase and analyzed as follows. Alpha-hemolysin was detected by observation of zones of hemolysis on rabbit erythrocyte agar plates (bioMérieux) and by immunoblotting (9) using a rabbit antibody (1:2,500 dilution; provided by A. Cheung) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Bio-Rad). Protein A was detected by using HRP-linked immunoglobulin. Expression of beta-hemolysin was analyzed on sheep erythrocyte agar plates (bioMérieux) (20). Lipase esterase activity was evaluated by colorimetric assay on an API ZYM strip (bioMérieux). Nuclease production was analyzed on toluidine blue DNA agar plates (Sanofi Diagnostic Pasteur). Serine protease was assayed spectrophotometrically on casein from cow’s milk coupled with activated resorufin (Boehringer Mannheim). Exoprotein gene expression was analyzed by Northern blotting with specific gene probes synthesized by PCR using primers selected from GenBank sequences for the following genes: alpha-hemolysin (hla) (accession no. X01645), beta-hemolysin (hlb) (X13404), nuclease (nuc) (JO1785), lipase (glycerol ester hydrolase; geh) (M12715), serine protease (sasp) (Y00356), and protein A (spa) (A04518). As noted previously (3), pEX072 fully restored the Agr⁺ phenotype, with some overexpression of RNAIII-sa and the corresponding exoprotein mRNAs (Fig. 3 and Table 2). In contrast, WA400/pLUG150 resulted in a level of RNAIII-sl very similar to the amount of RNAIII-sa in agr⁺ strains of S. aureus (Fig. 3 and Table 2). The pLUG150-complemented strain expressed most of the exoproteins normally produced by agr⁺ strains of S. aureus; only nuclease was undetectable. The quantity of hla mRNA was essentially identical to that produced by RN6390, but the level of alpha-hemolysin secreted was considerably reduced (Table 2 and Fig. 3). This agrees with a previous observation that alpha-hemolysin is regulated by RNAIII at both the transcriptional and the translational level (13). Translational activation, which involves a partial complementarity between the 5′ end of RNAIII-sa (Fig. 1) and a stretch of approximately 80 bp within the ribosome binding site of the hla transcript (13), is not observed with RNAIII-sl, which differs widely in this region. Both mRNA and secreted proteins showed slightly reduced beta-hemolysin levels and severely reduced serine protease and lipase levels. Down-regulation of the expression of protein A was achieved normally in WA400/pEX072 but not in WA400/pLUG150. To elucidate the participation of the 5′ and 3′ regions of RNAIII in the regulation of gene expression, we constructed a hybrid consisting of the 5′ end of RNAIII-sl grafted to the terminal 120 nt from RNAIII-sa. It was constructed by replacing a 445-bp SpeI-EcoRV fragment from pLUG150, encompassing the last 130 nt of RNAIII-sl, by a 422-bp PCR fragment isolated from S. aureus RN6390 by using primers sa757 (nt 757 to 771 of agr_SA) and sa1179 (nt 1179 to 1161), forming pLUG226. The chimeric RNAIII (RNAIII-sl-sa) was expressed in WA400 at levels comparable to those of RNAIII-sl in the same background (Fig. 3, lanes 4 and 5). The amounts of mRNAs and exoproteins expressed in WA400/pLUG226 were generally marginally higher (e.g., for beta-hemolysin, lipase, and serine protease) than those expressed in WA400/pLUG150, but there was no restoration of production of nuclease. The hybrid RNAIII-sl-sa was, however, as efficient at suppressing protein A mRNA as was RNAIII-sa (Fig. 3, lane 5). The difference in complementation observed with these constructs (pLUG150 and pLUG226) cannot be attributed to variation in the level of RNAIII production, which was identical in the two cases (Fig. 3, lanes 4 and 5).

FIG. 3.

Northern blot analysis of exoprotein mRNA expression. RNAs from post-exponential-phase cultures were hybridized with probes corresponding to RNAIII-sa plus RNAIII-sl (RNAIII), the alpha-hemolysin gene (hla), the beta-hemolysin gene (hlb), the lipase gene (geh), the nuclease gene (nuc), the serine protease gene (sasp), and the protein A gene (spa). Lane 1, RN6390 (agr⁺); lane 2, WA400 (agr mutant); lane 3, WA400/pEX072 (RNAIII-sa); lane 4, WA400/pLUG150 (RNAIII-sl); lane 5, WA400/pLUG226 (RNAIII-sl-sa). Positions of migration of 16S rRNA (1,541 nt) and 23S rRNA (2,904 nt) are indicated.

TABLE 2.

Expression of exoproteins in S. aureus agr⁺, agr mutant, and complemented agr mutant strains

Strain	Expressiona of:
	Alpha-hemolysin		Beta-hemolysin		Lipase		Nuclease		Serine protease		Protein A
	Pheb	mRNA	Phec	mRNA	Phed	mRNA	Phee	mRNA	Phef	mRNA	Pheg	mRNA
8325-4	++	++	++	++	++	++	+	+	+++	+++	−	−
WA400	+/−	+/−	+/−	+/−	−	−	−	−	−	−	++	+++
WA400/pEX072	+++	+++	+++	+++	+++	+++	+	++	+++	+++	−	−
WA400/pLUG150	+	++	+	+	+	+/−	−	−	+	+	−	++
WA400/pLUG226	+	++	++	++	+	+	−	−	++	++	−	−

^a+++, strong expression; ++, moderate expression; +, weak expression; +/−, very weak expression; −, no expression; Phe, phenotype. 

^bWide zone of complete hemolysis on rabbit erythrocyte agar plates; confirmed by immunoblotting of culture supernatant with alpha-hemolysin antibody provided by A. Cheung. 

^cZone of incomplete hemolysis (hot-cold hemolysis) on sheep blood agar plates. 

^dDetermined on an API ZYM strip. 

^eDetermined on toluidine blue DNA agar plates. 

^fDetermined on protease substrate (casein from cow’s milk with indicator). 

^gDetermined by immunoblots with HRP-conjugated goat immunoglobulin G. 

Concluding remarks.

These results confirm the expected lack of importance of the delta-hemolysin gene in the regulatory function of RNAIII (3, 6, 16), since RNAIII-sl and the hybrid RNAIII-sl-sa, both of which lack hld, restored the agr defect to an almost normal level for several exoproteins. Confirming results in other CoNS (18), the finding that restoration of exoprotein expression in the different agr constructs using agr_SL, agr_SA, or chimeras differed for the various exoproteins considered suggests that the regulatory functions for several target genes are independent. Moreover, we propose that the 3′ end of the RNAIII molecule is important for repressive activity and may constitute an intrinsic domain.