Opposing roles of σB and σB-controlled SpoVG in the global regulation of esxA in Staphylococcus aureus
© Schulthess et al; licensee BioMed Central Ltd 2011
Received: 28 August 2011
Accepted: 24 January 2012
Published: 24 January 2012
The production of virulence factors in Staphylococcus aureus is tightly controlled by a complex web of interacting regulators. EsxA is one of the virulence factors that are excreted by the specialized, type VII-like Ess secretion system of S. aureus. The esxA gene is part of the σB-dependent SpoVG subregulon. However, the mode of action of SpoVG and its impact on other global regulators acting on esxA transcription is as yet unknown.
We demonstrate that the transcription of esxA is controlled by a regulatory cascade involving downstream σB-dependent regulatory elements, including the staphylococcal accessory regulator SarA, the ArlRS two-component system and SpoVG. The esxA gene, preceding the ess gene cluster, was shown to form a monocistronic transcript that is driven by a σA promoter, whereas a putative σB promoter identified upstream of the σA promoter was shown to be inactive. Transcription of esxA was strongly upregulated upon either sarA or sigB inactivation, but decreased in agr, arlR and spoVG single mutants, suggesting that agr, ArlR and SpoVG are able to increase esxA transcription and relieve the repressing effect of the σB-controlled SarA on esxA.
SpoVG is a σB-dependent element that fine-tunes the expression of esxA by counteracting the σB-induced repressing activity of the transcriptional regulator SarA and activates esxA transcription.
The production of virulence factors in Staphylococcus aureus is coordinated by a network of two-component systems, global regulators and transcription factors, allowing optimal adaptation of the pathogen to a changing environment and stress conditions encountered during the various stages of infection. A central regulatory element of virulence factor production in S. aureus is the accessory gene regulator agr, a two-component quorum sensor regulating gene expression in a growth-dependent manner. The main effector molecule of the agr operon is the regulatory RNAIII , which is responsible essentially for the upregulation of secreted proteins in the post-exponential phase. RNAIII transcription is enhanced by the staphylococcal accessory regulator SarA  and reduced by the alternative sigma factor σB in strain Newman [3, 4]. SarA is a winged helix transcription factor influencing many virulence genes [5, 6]. The transcription of sarA in turn is directly activated by the alternative sigma factor σB , which controls over 250 genes including virulence factors and secondary regulators in a direct or indirect way . Indirectly σB-controlled genes lack a σB consensus promoter sequence, and are thought to be controlled by secondary, σB-dependent regulatory elements. The yabJ-spoVG operon, with SpoVG as effector molecule, is besides SarA one of the directly σB -dependent secondary regulators . SpoVG contributes to methicillin and glycopeptide resistance, stimulates capsule synthesis, and was recently shown to regulate a small σB-subregulon comprising mainly excreted virulence factors including the highly upregulated virulence factor EsxA [8–10].
Secretion of virulence factors is facilitated by several translocation systems in S. aureus , the major Sec pathway, the accessory Sec2 system , the twin-arginine translocation pathway , and the type VII-like specialized ESX secretion pathway (Ess) . The Ess system comprises a cluster of at least nine genes: esxAB, essABC, esaABC and esaD [14, 15] and secretes proteins with a size of approximately 100 amino acids containing a helical structure and a conserved Trp-Xaa-Gly (WXG) motif . Three proteins were so far shown to be exported by the staphylococcal Ess system, two WXG100 family proteins, EsxA and EsxB, and the non-WXG100 substrate EsaC [14, 17]. All three proteins act as pathogenicity factors in a murine model of staphylococcal blood-borne dissemination and abscess formation [14, 17]. The actual role of EsxA, EsxB and EsaC remains unclear. Structural analysis of EsxA suggests a role as transport module or chaperone to assist export of proteins by the Ess secretion pathway rather than being an effector protein itself . The esxA gene seems to be under complex control. Besides being upregulated by SpoVG , esxA was found to be upregulated by ArlR . The two-component system ArlRS [19, 20] itself is activated in an indirect way by σB in strain Newman [3, 9], adding a further level of complexity in the regulation of esxA.
This study analyses the transcriptional control of esxA by σB and the σB-dependent regulatory elements SarA, ArlR, RNAIII and SpoVG.
Materials and methods
Bacterial strains, plasmids and culture conditions
Strains and plasmids used in this study
Strain or plasmid
Relevant genotype; phenotype
Reference or source
Clinical isolate, ATCC 25904, natural saeS constitutive mutant
Newman ΔesxA, markerless deletion
Newman Δ(yabJ-spoVG)::erm(B); Emr
Newman Δ(rsbUVW-sigB)::erm(B); Emr
Newman sigB1(Am); Tcr
Newman ΔarlR::cat; Cmr
Newman Δagr::ermB; Emr
Newman Δagr::tet(M); Tcr
Newman ΔsarA::ermB; Emr
Newman ΔsarA::tet(L); Tcr
Newman mec, MRSA derivative, Mcr
BB1002 ΔesxA, markerless deletion
Newman GISA derivative, in vitro selected mutant; Ter
NM143 ΔesxA, markerless deletion
E. coli-S. aureus shuttle vector for markerless deletions using the counter selection system
Expression plasmid containing the PBAD promoter and the araC gene; Cmr
pAC7 with a 0.75 kb fragment containing the gene sigB from S. aureus Col; Cmr
E. coli-S. aureus shuttle plasmid with multicloning site from pBluescript II SK (Stratagene) and the rrnT14 terminator sequence from pLL2443; Tcr
pBus1 containing a bacA promoter-yabJ ORF fusion construct; Tcr
pBus1 containing a bacA promoter-spoVG ORF fusion construct; Tcr
pBus1 containing a bacA promoter-yabJ-spoVG operon fusion construct; Tcr
Firefly luciferase casette vector; Apr
pBus1 containing an esxAp-luc+ fusion fragment; Tcr
pesxAp-luc + with deletion of the σA promoter
pesxAp-luc + with deletion of the σB promoter
Promoter probe plasmid; Apr
pSB40N with a 0.6 kb fragment covering the asp23 promoter region fused to the reporter gene lacZα; Apr
pSB40N with a 0.5 kb fragment covering the esxA promoter region fused to the reporter gene lacZα; Apr
pSB40N with a 0.4 kb fragment covering the yabJ promoter region fused to the reporter gene lacZα; Apr
pSB40N with a 0.37 kb fragment covering the capA promoter region fused to the reporter gene lacZα; Apr
Molecular biological methods
General molecular biology techniques were performed according to standard protocols [32, 33]. Sequencing was done using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit and an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed with the Lasergene software package (DNASTAR, Inc., Madison, WI, USA).
Construction of ΔesxAmutants
Oligonucleotide primers used in this study
Sequence (5'-3') a
Construction of BS309 and BS310
The Newman sarA mutant BS309 and the Newman agr mutant BS310 were constructed by transducing the ermB-tagged sarA mutation of NM520 , and the ermB-tagged agr mutation of NM521  respectively, into Newman and selecting for erythromycin resistance. Correct inactivation of the genes was confirmed by sequencing and Southern blot analysis.
For the construction of promoter-lacZ reporter fusions, DNA fragments covering the yabJ or esxA promoter of strain Newman were amplified using primer pairs yab-prom-bam-f/yab-prom-xho-r and Pnwmn0219F/Pnwmn0219R-xho (Table 2), respectively. The PCR products were digested with BamHI and XhoI and ligated into promoter probe plasmid pSB40N  upstream of the lacZα reporter gene to obtain pyabJp and pesxAp.
For the construction of pesxAp-luc + , the esxA promoter region of strain Newman was amplified by PCR using primer pair Pnwmn0219F-hind/Pnwmn0219R (Table 1). The resulting PCR product was HindIII/NcoI-digested and cloned into pSP-luc + upstream of the luciferase reporter gene luc + . The esxA promoter-luc + fusion of the resulting plasmid was amplified using the primers Pnwnm0219F-hind/pSP-Luc XhoI, digested with HindIII and XhoI and cloned into the E. coli-S. aureus shuttle plasmid pBus1 to obtain plasmid pesxA-luc + .
Plasmids pesxApΔσA-luc + and pesxApΔσB-luc + were made by deleting the σA and σB promoter sequences, respectively, from pesxAp-luc + . The corresponding DNA fragments were amplified with primer pairs oBS49/oBS53 and oBS51/oBS54 (Table 2) from pesxAp-luc + and religated.
All plasmids constructs were confirmed by sequence analyses.
Northern blot analysis
Overnight cultures were diluted 1:100 into LB, grown for 2 h, and then used to inoculate 100 ml of pre-warmed LB to an optical density of 600 nm [OD600 nm] of 0.05. Cell samples were taken at the time points indicated, centrifuged at 12,000 × g and 4°C for 2 min, the pellets were snap-frozen in liquid nitrogen. Total RNA was isolated according to Cheung et al. . RNA samples (8 μg) were separated in a 1.5% agarose gel containing 20 mM guanidine thiocyanate in 1 × Tris-borate-EDTA buffer . RNA transfer and detection were performed as previously described [41, 42]. Digoxigenin (DIG) labelled probes were amplified using the PCR DIG Probe synthesis kit (Roche, Basel, Switzerland). The primer pairs used for amplification of the esxA, spoVG, asp23, arlR, sarA and RNAIII probes are listed in Table 2.
RNA was extracted from LR15 cultures that were grown to OD600 nm 2.0, as described by Cheung et al. . Primer extension reactions were performed using 20 μg of total RNA and 3 pmol of the 5'-biotin-labelled primers pe_esxA_1 and pe_esxA_2 (Table 2) using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturers instructions. Sequencing reactions were performed using the Thermo Sequenase Cycle Sequencing Kit (USB Corporation, Cleveland, OH, USA) and template DNA amplified with primers Pnmmn0219F and esxA_term-r from Newman genomic DNA. The Biotin Chromogenic Detection Kit (Fermentas, Burlington, Ontario, Canada) was used for biotin detection.
Testing of the interaction of S. aureus promoters with E. coli RNA polymerase containing S. aureus σB was done essentially as described earlier . The promoter-reporter plasmids pasp23p (asp23 promoter); pyabJp (yabJ promoter); pesxap (esxA promoter); and pSTM07 (capA promoter); or the empty plasmid pSB40N, were transformed into E. coli DH5α containing either pAC7-sigB or pAC7. The color production of the clones was analyzed on LBACX-ARA plates (LB agar containing 5 mg ml-1 lactose; 100 μg ml-1 ampicillin; 40 μg ml-1 chloramphenicol; 20 μg ml-1 X-Gal (5-bromo-4-chloro3-indolyl-D-galactopyranoside) and 2 μg ml-1 arabinose) .
Luciferase activity was measured as described earlier  using the luciferase assay substrate and a Turner Designs TD-20/20 luminometer (Promega).
The proteolytic activity of S. aureus strains was determined on skim milk (Becton Dickinson, 75 g l-1) agar plates as clear zones surrounding colonies.
To compare the hemolytic activity, S. aureus strains were grown on sheep blood agar and the clear halos around the colonies were analyzed.
Plates containing an antibiotic gradient were prepared and inoculated by swabbing a 0.5 McFarland cell suspension in physiological NaCl solution along the gradient as described before . Growth was read after 24 h and 48 h of incubation at 35°C. Teicoplanin and oxacillin minimal inhibitory concentrations (MICs) were determined using Etests according to the manufacturer's instructions (AB-Biodisk, Solna, Sweden).
Results and discussion
Transcriptional analysis of esxA
The 294 bp esxA gene (nwmn_0219, GenBank accession no. NC_009641), coding for a small secreted protein involved in staphylococcal virulence, is the first of at least nine genes of the ess gene cluster encoding the type VII-like ESX-1 secretion pathway (Ess) in S. aureus (Figure 1A) [14, 15]. Although esxA seems to belong transcriptionally to the ess gene cluster , transcriptional profiling produced one single esxA-specific transcript with a size of about 0.45 kb appearing in early growth phase after 1 h and increasing slightly within time (Figure 1B). No esxA-specific signals were detected in the corresponding ΔesxA mutant BS304, confirming the esxA deletion. The deletion of esxA had no polar effects on the expression of the downstream ess genes, nor on the divergently transcribed gene directly upstream of esxA, predicted to be involved in staphyloxanthin synthesis [37, 44, 45] (data not shown). Our results suggest that esxA is located on a monocistronic transcript and is not co-transcribed with the remaining genes of the ess gene cluster.
esxApromoter and terminator sequence analysis
Primer extension analysis located the transcriptional start point (TSP) of esxA 74 bp upstream of the start codon of esxA (Figure 1A-C). It was preceded by the predicted -10 and -35 σA promoter elements, and further up by the σB promoter.
Effect of σB and σB-controlled SpoVG on esxAexpression
To determine if either yabJ or spoVG inactivation was responsible for the reduction of esxA transcription, we complemented Newman, SM148 and IK184 in trans with a series of plasmids expressing constitutively either yabJ (pyabJ), spoVG (pspoVG), or yabJ-spoVG (pyabJspoVG), circumventing the requirement of σB to transcribe the yabJ-spoVG operon. Northern blot analysis revealed that the constructs containing spoVG or yabJ-spoVG, but not the one carrying yabJ, did restore the esxA transcription to wild type level in SM148 (Figure 3C). In IK184, showing stronger esxA transcription signals than the wild type, the esxA transcription was even further enhanced by the complementation with pspoVG or pyabJspoVG, confirming that SpoVG, but not YabJ, had a positive effect on esxA expression in presence and absence of σB. However, the fact that esxA transcription was strongly induced in IK184 lacking not only σB, but consequently also the σB-dependent SpoVG, suggested that esxA transcription may be activated by SpoVG but repressed by other σB-dependent factors.
Influence of major regulators SarA, RNAIII and ArlR on esxA
Influence of EsxA on regulatory elements and itself
EsxA itself had no influence on the signal intensity or activity of any of the above regulatory genes, neither on asp23, as an indicator of σB activity [37, 44, 50], nor on spoVG, arlR, sarA or RNAIII, when comparing their expression in strain Newman and in the ΔesxA mutant BS304 during the growth cycle (Additional file 1). We could also rule out any autoregulatory effects of EsxA on its own transcription, since luciferase activity patterns of pesxAp-luc + were congruent over the entire growth cycle in Newman and BS304 (data not shown).
Influence of SarA, RNAIII, σB, ArlR and SpoVG on each other
Further, minor changes in transcription were observed in the ΔsarA mutant where RNAIII was downregulated and arlR transcripts were slightly upregulated, and in the ΔarlR mutant where sarA transcription was increased (Additional file 2: Figure S2A). However, these dependencies could not explain the changes in esxA transcription in the corresponding mutants.
Phenotypic characteristics of the ΔesxAmutant
Oxacillin and teicoplanin MICs
MIC (μg ml-1)
Our data suggest that the repression of esxA by σB is due the σB-induced transcription of sarA, leading to a strong and dominating SarA-mediated repression of esxA. The activation of esxA transcription, on the other hand, is stimulated by the agr quorum sensing system, the response regulator ArlR, and the effector protein SpoVG; whereby arlR is controlled indirectly, and spoVG directly by σB. Thereby the activating effect of ArlR seems to be more profound than the effect of SpoVG and agr. Moreover, virulence gene regulation in S. aureus is very complex and additional factors might contribute to the regulation of esxA transcription.
The mode of function of SpoVG, named after the stage V sporulation protein G in Bacillus subtilis , and SpoVG homologues in other bacterial species is yet unknown, nor have any SpoVG interacting partners been reported. SpoVG does not affect σB activity as seen from the expression of asp23, which is a measure of σB activity in S. aureus. SpoVG does also not interfere with the transcription of sarA, arlRS nor agr in strain Newman.
By which mechanisms SpoVG counteracts the postulated SarA-mediated repression of esxA remains open. The affinity of SarA binding to DNA can be enhanced by phosphorylation , but a postulated interaction of SpoVG with SarA or other proteins has yet to be investigated. Interestingly, the same stimulating effect by ArlRS and SpoVG is seen in S. aureus capsule synthesis . We therefore can not rule out that SpoVG and ArlR may interact or have some common target. SpoVG by itself seems also to enhance transcription of esxA when artificially overexpressed in a sigB mutant. The absence of predicted DNA binding motifs in SpoVG may not fully exclude its interaction with nucleic acids or with factors involved in transcription. In conclusion, we have presented here SpoVG, an interesting new player in the regulatory cascade modulating S. aureus virulence factors.
This study was carried out with financial support from the Forschungskredit of the University of Zurich to BS, and from the Swiss National Science Foundation grant 31-117707 to BBB.
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