ACCEPTED. reduced expression of sars. Jan Oscarsson*, Anna Kanth, Karin Tegmark-Wisell and Staffan Arvidson

Transcription

1 JB Accepts, published online ahead of print on September 00 J. Bacteriol. doi:0./jb.00-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 0 0 SarA is basically a repressor of hla ( -hemolysin) transcription in Staphylococcus aureus: its apparent role as an activator of hla in the prototype strain depends on reduced expression of sars. Jan Oscarsson*, Anna Kanth, Karin Tegmark-Wisell and Staffan Arvidson Department of Microbiology, Tumor and Cell Biology (MTC), Box 0, Karolinska Institutet, S- Stockholm, Sweden. Running title: regulation of hla by sara in S. aureus *Corresponding author: Jan Oscarsson, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, S- Stockholm, Sweden. Phone (), Fax (), Mail: jan.oscarsson@ki.se.present address: Clinical Bacteriology Laboratory, Huddinge University Hospital, S- Stockholm, Sweden Key Words: Staphylococcus aureus, hla ( -hemolysin), regulation, sara, sars, Downloaded from on October, 0 by guest

2 0 0 Abstract In most Staphylococcus aureus strains inactivation of sara increases hla transcription, indicating that sara is a repressor. However, in S. aureus NCTC and its derivatives, used for most studies of hla regulation, inactivation of sara resulted in decreased hla transcription. The disparate phenotype of strain seems associated with its rsbu mutation leading to B deficiency. This has now been verified by the demonstration that sara repressed hla transcription in an rsbu + derivative of strain - (SH000). That sara could act as a repressor of hla in an - background was confirmed by the observation that inactivation of sara in an agr sars rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sara as an activator of hla in - was not a result of the rsbu mutation alone as inactivation of sara in another rsbu mutant, strain V, lead to increased hla transcription. Northern blot analysis revealed much higher levels of sars mrna in strain V than in -, which was likely due to the mutation in the sars activator, tcar, in -, which was not found in strain V. On the other hand, the relative increase in sars transcription upon inactivation of sara was -fold higher in - relative to strain V. Because of this, inactivation of sara in - means a net increase in repressor activity, whereas in strain V inactivation of sara means a net decrease in repressor activity, and therefore enhanced hla transcription. Introduction Staphylococcous aureus is a common human pathogen, which also colonizes the nares and skin of about / of all healthy people. The type of infection caused by this organism ranges from superficial cutaneous infections to life-threatening bacteremias. The pathogenesis of S. aureus is Downloaded from on October, 0 by guest

3 0 0 very complex and virulence depends on the production of an array of extracellular toxins and enzymes, and adhesins, which are regulated by a number of global regulators, e.g. agr (accessory gene regulator) and the sara (staphylococcal accessory regulator) family of regulators (reviewed in (,, 0)). In addition, the alternative sigma factor, sigma B ( B ), seems to be involved in virulence gene expression in addition to its role in regulating stress responses and intermediary metabolism (,,, ). Notably, among the genes controlled by sigma B are the virulence regulators sara and sars (, 0, ). The activity of B is regulated by the anti-sigma factor, RsbW, which binds B thereby inhibiting its association with the RNA-polymerase (, ). The ability of RsbW to bind B depends on the phosphorylation status of RsbV, which is determined by the activity of the phosphatase RsbU (,, 0). Most S. aureus strains produce -hemolysin ( -toxin; hla), which is a kda pore-forming protein that can lyse a wide range of human cells, including lymphocytes and keratinocytes (,, ). In addition, -hemolysin may induce apoptosis in T lymphocytes (), and has also been shown to be required for biofilm formation (0). Similar to most secreted toxins and enzymes in S. aureus, expression of -hemolysin (hla) is positively regulated by agr (,, ). The effector molecule of the agr-dependent regulation is RNAIII, which is transcribed from the agr P promoter at the end of the exponential phase of growth in response to autoactivation of the agr P promoter. Autoactivation is achieved via accumulation of a secreted pheromone, AIP, encoded by the agr operon (0,, ). RNAIII appears to increase -hemolysin expression by three different routes, via a direct interaction with the hla transcript that stimulates translation (), and via rot and sars-dependent pathways respectively. The rot gene locus was first recognized as a suppressor mutation, which partially restored -hemolysin (hla) expression in an Downloaded from on October, 0 by guest

4 0 0 agr null mutant (). Gene array methodology has revealed that rot acts as a global regulator, affecting the transcription of many virulence genes, e.g. hlb, spa, geh, and sspa (). The central role of rot in S. aureus virulence gene regulation was recently demonstrated in a rabbit endocarditis model of infection (). Based on the observation that inactivation of rot had no effect on hla expression in an agr + background, and that transcription of rot was not affected by the agr mutation, it has been suggested that RNAIII acts by sequestering Rot, thereby preventing it from binding to target gene promoters (, ). In a recent study some evidence was presented that RNAIII may even promote degradation of Rot via a clpxp-dependent route (). As rot stimulates transcription of sars (, ), which is a strong repressor of hla (), the suppressive effect of rot on hla transcription is probably indirect, mediated by sars. Whether rot is also a direct repressor of hla transcription is not known. Transcription of hla is also positively regulated by saesr, which encodes a two-component signal transduction system (). Transcription of the sae locus was clearly reduced in both agr and sara mutants during post-exponential phase of growth, indicating that sae is downstream of both RNAIII and sara in the regulatory pathway (). However, the level of hla transcription was approximately ten times higher in the sara relative to the agr mutant (), indicating that sara and sae are also epistatic. In the original S. aureus sara mutant isolated by Cheung and coworkers () production of hemolysin was increased in comparison to the parental strain. The same results were obtained when sara was inactivated in a number of other clinical S. aureus strains (, ). However, when the sara mutation was transferred to the prototype S. aureus strain NTCC it resulted in reduced -hemolysin production, indicating that sara acted as an activator of hla transcription (,, ). Most further studies of hla regulation in S. aureus have been carried out with strains Downloaded from on October, 0 by guest

5 0 0 derived from. In this strain, the sara-dependent stimulation of hla transcription seems to be mediated by sars, which is negatively regulated by sara and acts as a repressor of hla (). However, it has been reported that sara also enhances transcription of hla via agr. This effect seems to be direct, as sara has been shown to bind to the agr promoter region (,,, ), but might also be mediated by sart and saru (, ). Although the reason for the disparate phenotype of strain is not known, it seems associated with the B -deficiency of this strain and its derivatives (), because of a mutation in the rsbu gene (). In addition, strain - has a mutation in the tcar gene locus that could indirectly affect hla expression as tcar stimulates transcription of sars (a repressor of hla) (). Restoration of the B activity in strain derivatives dramatically decreased hla expression relative to the parental strains (, ). This was likely sara-independent, as the overall levels of sara transcription in strains - and SH000 (-, rsbu + ) were very similar (). However, other studies, using rsbu + derivatives of -, revealed increased sara expression during post- exponential phase of growth, from the B dependent promoter (P) (, ). Interestingly, preliminary experiments in our laboratory revealed that inactivation of sara in SH000 (- rsbu + ) resulted in increased hemolysin production on rabbit blood agar, suggesting that sara acts as a repressor of hla in an rsbu + background contrary to the parental rsbu deficient strain, -. The present study was undertaken to understand why sara acts as an activator of hla transcription in -, while being a repressor in the rsbu + derivative (SH000) and in most clinical strains. Materials and Methods Downloaded from on October, 0 by guest

6 0 0 Bacterial strains, plasmids and cultivation conditions Bacterial strains and plasmids used in this study are listed in Table. S. aureus strains were grown on Nutrient agar (NA)-plates (Difco). For screening of protease and -hemolysin production NA-plates supplemented with casein or rabbit erythrocytes were used (, ). S. aureus strains were precultured in Tryptic Soy Broth (TSB) (Difco) for - hours. When required 0 µg ml - tetracycline, 0 µg ml - kanamycin, µg ml - erythromycin or µg ml - lincomycin was added to the culture media. Cells from precultures were centrifuged and inoculated in 00 ml of Brain Heart Infusion (BHI) (Difco) in -liter baffled flasks (culture/flask volume ratio :0) to give an optical density at 00nm (OD 00 ) of 0. and incubated on a rotary shaker (0 r.p.m) at C. For induction of xyla promoter constructs xylose was added to the culture media to a final concentration of 0.%. Escherichia coli strains were cultivated on Luria-Bertani (LB) (Difco) agar plates or in LB medium, when required supplemented with 00 µg ml - ampicillin. Construction of strains and plasmids All plasmids were constructed and cloned in E. coli DH, as described (0), and subsequently transferred to the restriction deficient S. aureus strain RN0 by electroporation () before transfer to appropriate S. aureus strains using phage (). For construction of the sars allelic replacement mutant, WA0, PCR fragments (0 and 0 bp) flanking the sars gene, were synthesized using the primer pairs, sarh-forward ( - TATATAGCATGCCAAATACGCCCGACAACACTC- ) and sarh-reversed ( - TATATACCGCGGCATATCGACTTCAGGCTTGAC- ), and spaf ( - CTGTTAGAGCTCTCAATAATTTAAAAAAGC- ) and sarh-back ( - Downloaded from on October, 0 by guest

7 0 0 TATATAGAGCTCGTTATCAGCTTTCGGTGCTTG- ), respectively. Forward primers contained SphI and SacI restriction sites and reverse primers contained SacII and SacI restriction sites (underlined sequences). The PCR fragments were inserted in two steps at either side of the ermb cassette in the plasmid pkt. In the resulting construct (paw) a tetracycline resistance cassette from plasmid pt was inserted between the ApaI and AatII sites to generate paw. This plasmid was then transferred to S. aureus RN0 by electroporation, and integration in the sars locus was confirmed by PCR analysis. The sars::ermb allele was transferred to S. aureus - via -mediated transduction in order to obtain the double crossover that replaced the sars gene by the ermb cassette. Erythromycin/lincomycin resistant transductants were screened for loss of tetracycline resistance and the sars::ermb allele in one selected clone, WA0, was verified by PCR analysis. The hla::ermb allele of strain DU00 was moved to KT00, generating the derivative WA. The rot::tet allele from strain PM rot::tet was transferred to WA0, generating WA. The sara::km allele from PC was transferred to SH000, V, WA, WA0, WA, and PM rot::tet, generating the derivatives KT00, WA, WA00, WA0, WA, and WA0. The sars::ermb allele from WA0 was transferred to SH000, PC, and KT00, resulting in the derivatives WA0, WA0, and WA0. The construct pkt0, containing the sars gene under the control of the xyla promoter, was transferred to WA0 resulting in the strain WA. The construct pkt0, containing the sara gene under the control of the xyla promoter, was transferred to WA0, generating WA. The construct pik, containing sigb under the control of the xyla promoter, was transferred to strain WA0, generating WA0. Sequence analysis of the tcar operon Downloaded from on October, 0 by guest

8 0 0 The tcar gene locus of strain V was amplified by PCR and sequenced in both directions using the oligonucleotide primers tcar ( - CAATGATTGCTGAGGACAGCTGATAAAAATAGTAATTAG- ) and tcar ( - CAATTCTGTTCCTCTTCAGCTGTTATATGTATATCAC- ), which were based on the tcar sequence of strain COL (GeneBank accession no. AY00). DNA sequence analysis was performed using the ABI PRISM BigDye terminator Cycle Sequencing kit v. (Applied Biosystems), and the Applied Biosystem DNA sequencer. Our nucleotide sequence data for the tcar locus in strain V have been deposited in the EMBL database as entry AM0. Northern blot analysis Total S. aureus RNA was prepared using the FAST RNA-blue kit (Bio 0) according to instructions of the manufacturer. Concentration of RNA was determined by measuring the absorbance at 0nm. Samples containing 0µg of total RNA were analyzed by Northern blotting as described previously (). For Northern hybridization, internal fragments of hla (nucleotides [nt] to ; GeneBank accession number X0), RNAIII (nt 0-; X), sara (nt to 0; U), sars (nt 0-; locus SACOL00: CP0000), and s rrna (nt -0; X) were amplified by PCR, radio labeled with ( - P)-dCTP (Amersham) using a random prime labeling kit (Roche Molecular Biochemicals) and used as probes. Radioactivity was detected by a radioisotope imaging system (Phosphorimager SI; Molecular Dynamics), and quantified using the ImageQuant software (Molecular Dynamics). Each experiment was repeated three times. Analysis of -hemolysin in culture supernatants Downloaded from on October, 0 by guest

9 0 0 For Western blot analysis of -hemolysin, S. aureus culture supernatants were harvested at postexponential phase (h) and an amount of supernatant corresponding to a bacterial density of 0. OD 00 units (equal to ml OD 00 = 0.) was separated in % SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) based membranes. Membranes were incubated with monoclonal mouse anti- -hemolysin antibodies (), and bound antibodies were subsequently detected using horseradish peroxidase (HRP)-conjugated sheep anti-mouse antibodies (Amersham). RESULTS Whether sara acts as a repressor or activator of hla in strain - depends on the rsbu activity Inactivation of sara in several clinical S. aureus strains resulted in increased -hemolysin production, indicating that sara is a repressor of hla (, ). However, in strain - (RN0) sara appeared to act as an activator of hla transcription, as inactivation of sara resulted in decreased hla expression (,, ). It has been suggested that the disparate phenotype of - could be due to the rsbu mutation of this strain, resulting in B - deficiency (). To analyze this further, we inactivated the sara gene locus of SH000 (- rsbu + ) by allelic replacement, generating strain KT00. As indicated by slightly increased hemolysis on rabbit blood agar (Fig. A) expression of hla seemed to be upregulated in KT00 relative to SH000. Immunoblotting and inactivation of the hla locus in KT00 confirmed that the increased zone of hemolysis was indeed due to production of -hemolysin (Fig. B and C). The enhanced -hemolysin production of KT00 (SH000 sara) was accompanied by elevated (- to -fold) hla mrna Downloaded from on October, 0 by guest

10 0 0 levels in KT00 relative to SH000, during post-exponential phase of growth (Fig. ), indicating that sara acts as a repressor of hla transcription in SH000. Taken together, our findings suggest that the apparent role of sara as an activator of hla transcription in strain - depends on the rsbu activity. The immunoblot analysis (Fig. C) also revealed partial degradation of -hemolysin in KT00, likely a result of derepressed protease expression in the absence of SarA, consistent with previous observations (, 0). The kda protein band in supernatants from SH000 (Fig. C) represents the extracellular fraction of protein A, as indicated by its reactivity with non-specific IgG, and by its pronounced upregulation in an agr mutant (data not shown). The lack of protein A in KT00 was probably due to increased production of proteases, which is consistent with previous results showing that protein A is degraded by extracellular proteases (). The reduced protein A production in strain WA0 (SH000 sars) is in agreement with sars being an activator of spa (). Whether sara acts as a repressor or activator of hla depends on the relative activity of sars To test whether the effect of rsbu on hla regulation by sara in - is a general phenomenon, we analyzed another natural B deficient S. aureus strain, V, which has an IS element inserted in the rsbu gene (). In this strain, inactivation of sara resulted in a - to -fold increase in hla transcription during post-exponential phase of growth (h and h) (Fig. A), indicating that it is not B activity per se that determines whether sara acts as a repressor or activator of hla transcription in a given strain, but other factors are also involved. As a result of the B deficiency strains - and V had low levels of the B -dependent sara P transcript during postexponential phase of growth (). As sara-dependent regulation of hla transcription is partly mediated by sars, which is a repressor of hla (), the level of sars mrna was determined in Downloaded from on October, 0 by guest 0

11 0 0 -, SH000 and V, and in their corresponding sara mutants (PC, KT00 and WA). In agreement with previous findings (), sars mrna levels were almost not detectable in - (Fig. and Fig. B). On the other hand, we observed a - to -fold increase in sars mrna levels in SH000 relative to - (Fig, ). This is consistent with the observation that transcription of sars is enhanced by B (, ). However, we did not detect the B -dependent sars transcript (P) in SH000, but only increased levels of the A -dependent sars transcript (P, same as the sars transcript that was upregulated in the - sara mutant, PC ()) (Fig. ). This indicates that the upregulation of sars in SH000 is not a direct effect of the B activity. As shown in Fig. B, the level of sars transcription was much higher in strain V than in -. A possible explanation would be that the tcar gene locus, which encodes an activator of sars transcription, is inactive in - (). DNA sequence analysis (see Materials and Methods) confirmed that the tcar allele in strain V was intact and identical to that of strain COL (GenBank accession no. AY00). Consistent with previous results (), inactivation of sara in - (strain PC) resulted in highly elevated (more than 0-fold) sars transcription during post-exponential phase of growth (Fig. and Fig. B). Interestingly, inactivation of sara in SH000 or in V only resulted in an approximately twofold increase in sars transcription (Fig. and Fig. B). Therefore, whether inactivation of sara will increase or decrease hla transcription seems to depend on the relative levels of sara and sars expression in the parental strain, and to what extent sars is upregulated in a cognate sara mutant. The reason why sara appears as an activator of hla transcription in - would thus be that the levels of both sara and sars are low (essentially no repression of hla transcription), and that inactivation of sara results in a very dramatic increase in sars transcription and therefore suppression of hla. To confirm that sara is basically acting as a repressor of hla transcription even in -, a Downloaded from on October, 0 by guest

12 0 0 plasmid carrying sara behind a xylose-inducible promoter (pkt0) was transferred to a sars mutant derived from strain -, resulting in WA. As shown in Fig., induction of sara expression with 0.0% xylose, which did not affect bacterial growth (data not shown), and only very slightly reduced RNAIII production at h and h, completely repressed hla transcription. This is in agreement with the observation that SarA bound to the hla promoter in vitro (, ) and repressed hla transcription in an in vitro assay (), supporting a model in which sara can also suppress hla transcription in a direct way. High hla transcription can be obtained in an agr mutant if sara, sars and rot are inactivated Our present results indicate that hla transcription is suppressed by three regulators, sara, sars and rot. As Rot, which is an activator of sars transcription (), is supposed to be neutralized by RNAIII (), the relative impact of all three repressors was analyzed in an agr mutant. For this, we constructed agr sars sara and agr sara rot triple mutants (WA and WA0), and an agr sars rot sara quadruple mutant (WA0). As shown in Fig. A, hla mrna levels were equally low, relative to the parental strain (RN0), in WA (agr sars sara), WA0 (agr sara rot), and in an agr sars rot triple mutant (WA0). Interestingly, the quadruple mutant (WA0) had clearly increased (up to -fold) hla mrna levels relative to the triple mutants, during post-exponential phase of growth (h and h) (Fig. A). These results indicate that sara, sars and rot are equally potent and sufficient to suppress hla transcription. As rot appeared to suppress hla transcription independent of sars and sara this regulatory effect might be direct. It can also be concluded that RNAIII per se was not required for high transcription of hla in the absence of repression by sara, sars and rot, as the level of hla transcription was very similar in Downloaded from on October, 0 by guest

13 0 0 the quadruple mutant (WA0) and the parental strain, RN0. However, it must be pointed out that the quadruple mutant produced much less -hemolysin than the parental strain (Fig. B and data not shown), confirming that RNAIII is still essential for translation of the hla mrna (). The B -mediated reduction in hla transcription in strain SH000 is independent of sara, sars and rot Considering that both sara and sars can act as repressors of hla transcription, the reason for the low hla mrna levels in strain SH000 would be the increased B -dependent expression of these regulators during post-exponential phase of growth. To further investigate this, the relative impact of sara and sars on hla transcription in SH000 was analyzed by comparing the hla mrna levels in sara and sars single and sara sars double mutants, derived from SH000 (Fig., and data not shown). As hla transcription remained very low in all mutants compared to -, we concluded that sara and sars were not the sole factors responsible for the low hla transcription in SH000. In agreement with this, induction of B expression in an - sara sars double mutant, containing a xylose-inducible sigb gene (strain WA0), resulted in severe suppression of hla transcription (Fig. ), confirming that sara and sars were not required for B - dependent repression of hla. Notably, RNAIII production was impaired (- to -fold) upon induction of B in WA0 (Fig. ), suggesting that the reduction in hla transcription was the result of low RNAIII levels. A similar reduction of RNAIII levels was observed in SH000 relative to its parental strain ()(Fig. ). As RNAIII was not required for transcription of hla in derivatives of - lacking sara, sars and rot (Fig. A), the impact of the reduced RNAIII levels in SH000 on hla transcription was analyzed by construction of a sara sars rot triple Downloaded from on October, 0 by guest

14 0 0 mutant of SH000 (WA00). As hla transcription was hardly upregulated in WA00 relative to SH000 (Fig. ), we concluded that the B -mediated reduction in hla transcription in SH000 was independent of sara, sars and rot, or reduced RNAIII levels. Notably, although inactivation of sars in SH000 derivatives had no marked effect on hla mrna levels (Fig. and data not shown), these strains (WA0 and WA0) were completely non-hemolytic on rabbit blood agar (Fig. A) and lacked detectable -hemolysin (Fig. C and data not shown). This is consistent with the reduced (up to -fold) RNAIII levels in these strains (Fig. and data not shown), as RNAIII is required for translation of the hla mrna (). As complementation of WA0 with pkt0, encoding sars, restored RNAIII expression to parental (SH000) levels (Fig. ), we concluded that sars enhanced RNAIII production in SH000, in contrast to in -. Discussion The present study was undertaken to find an explanation to why sara appears to be an activator of hla transcription in the prototype strain NTCC and its derivatives, while being a repressor of hla in most other S. aureus strains (, ). Our results clearly show that the role of sara as an activator of hla transcription in - was associated with the rsbu mutation in this strain, which leads to B deficiency, as sara acted as a repressor of hla in an rsbu + derivative of - (SH000) (Fig. and Fig. ). However, this was not a direct effect of the B deficiency, as inactivation of sara in another B deficient strain, V, resulted in increased hla transcription (Fig. A). The possibility that strains V and - respond differently to sara inactivation because of their different rsbu mutations can not be excluded. Our results indicate that whether inactivation of sara leads to increased or decreased hla transcription depends on the relative Downloaded from on October, 0 by guest

15 0 0 activity of sara and sars, respectively, i.e., their basal levels of expression, and how much sars transcription is elevated when sara is inactivated. In the parental strain -, the basal postexponential levels of sara and sars mrnas are low due to the impaired B activity ( B enhances sara and sars transcription (, )) and the mutation in tcar (tcar stimulates sars transcription ()). In strain V, which was found to have an intact tcar locus, the level of sars mrna was much higher than in strain - (Fig. B), while the post-exponential level of sara expression was essentially the same in these strains (). We assume that the level of sara in these strains is sufficient to partly repress hla transcription, whereas the suppressive effect of sars is only significant in strain V, which has a lower level of hla mrna than - (our unpublished data). However, inactivation of sara resulted in about -fold higher relative increase in sars transcription in - than in V (Fig. and Fig. B). A likely explanation why this lead to reduced hla transcription in strain -, and increased hla transcription in strain V, would be that the loss of repression of hla due to inactivation of sara in - was counteracted by the massive increase in sars, whereas the level of repression of hla in response to the increase in sars transcription in the V sara mutant (WA) was less than the derepression of hla due to loss of sara activity. Our present findings strongly suggest that sara is basically a repressor of hla transcription in S. aureus strains, including - and its derivatives. Repression of hla transcription by sara seems to be independent of agr (RNAIII), sars and rot. As indicated in Fig., transcription of hla is negatively controlled by three regulators, sara, sars and rot, all of which had to be inactivated or neutralized to achieve full hla transcription. It has been reported that hla transcription is also negatively controlled by sart (). However, it is not clear whether this Downloaded from on October, 0 by guest

16 0 0 effect is direct or mediated by sars, which is stimulated by sart (). On the other hand, as sart is strongly repressed by both sara and agr (), the possible direct repression of hla by sart seems not to be of major importance, as very similar hla mrna levels were obtained in the wildtype (sart repressed by both sara and agr) and in an agr sara sars rot quadruple mutant (Fig. A). The positive effect of RNAIII on hla transcription seems mainly be due to its capability of downregulating sars transcription via neutralization of Rot and repression of sart, which are both required for sars transcription (,,, ). This is consistent with the observation that RNAIII was not required for hla transcription in the absence of sara, sars and rot (Fig. A). Interestingly, inactivation of these regulators in strain SH000 (-, rsbu + ) did not result in elevated hla transcription (Fig. ), indicating that other B -dependent factor(s) (indicated by X with an asterisk in Fig. ) are responsible for the low hla transcription in this strain. The observed requirement of an intact sars allele for RNAIII production in SH000 (Fig. and Fig. ) might be explained by sars acting as a negative regulator of a B -dependent repressor of agr (RNAIII) expression. This and additional B -dependent factors involved in the regulation of hla transcription might be found among the hypothetical proteins identified in a micro array based analysis of the B regulon (). Although the much studied and genetically well characterized strain - and its derivatives seem not representative of most clinical S. aureus isolates because of the mutations in rsbu and tcar, it is still very useful in exploration of the regulatory networks governing virulence gene expression if compared to other strains. ACKNOWLEDGEMENTS We are grateful to Agneta Wahlquist, Lena Norenius and Caroline Harlos for valuable technical assistance. We wish to thank Drs. Simon Foster and Tim Foster for sending us the strains Downloaded from on October, 0 by guest

17 0 0 0 SH000 and DU00, respectively. This work was financed by funds from the Swedish Research Council (VR), the Swedish Foundation for Strategic Research (SSF), and by the Swedish Society for Medical Research (SSMF). REFERENCES. Arvidson, S. 00. Virulence gene regulation and their role in pathogenesis of disease, p. -. In D. Ala'Aldeen and K. Hiramatsu (ed.), Staphylococcus aureus, molecular and clinical aspects. Horwood Publishing, Chichester.. Arvidson, S., and K. Tegmark. 00. Regulation of virulence determinants in Staphylococcus aureus. Int. J. Med. Microbiol. :-0.. Benson, A. K., and W. G. Haldenwang.. Bacillus subtilis B is regulated by a binding protein (RsbW) that blocks its association with core RNA polymerase. Proc. Natl. Acad. Sci. USA 0:0-.. Bischoff, M., P. Dunman, J. Kormanec, D. Macapagal, E. Murphy, W. Mounts, B. Berger-Bächi, and S. Projan. 00. Microarray-based analysis of the Staphylococcus aureus sigmab regulon. J. Bacteriol. :0-0.. Björklind, A., and S. Arvidson. 0. Mutants of Staphylococcus aureus affected in the regulation of exoprotein synthesis. FEMS Microbiol. Lett. :0-0.. Björklind, A., and S. Arvidson.. Occurrence of an extracellular serineproteinase among Staphylococcus aureus strains. Acta. Path. Microbiol. Scand. :-0.. Blevins, J., A. Gillaspy, T. Rechtin, B. Hurlburt, and M. Smeltzer.. The Staphylococcal accessory regulator (sar) represses transcription of the Staphylococcus aureus collagen adhesin gene (cna) in an agr-independent manner. Mol. Microbiol. :-.. Blevins, J. S., K. E. Beenken, M. O. Elasri, B. K. Hurlburt, and M. S. Smeltzer. 00. Strain-dependent differences in the regulatory roles of sara and agr in Staphylococcus aureus. Infect. Immun. 0:0-0.. Bronner, S., H. Monteil, and G. Prevost. 00. Regulation of virulence determinants in Staphylococcus aureus: complexity and applications. FEMS Microbiol. Rev. : Caiazza, N. C., and G. A. O'Toole. 00. Alpha-toxin is required for biofilm formation by Staphylococcus aureus. J Bacteriol :-.. Chan, P. F., and S. J. Foster.. Role of SarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus. J. Bacteriol. 0:-.. Chan, P. F., S. J. Foster, I. Ingham, and M. O. Clements.. The Staphylococcus aureus alternative sigma factor B controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J. Bacteriol. 0:0-0. Downloaded from on October, 0 by guest

18 Cheung, A. L., M. G. Bayer, and J. H. Heinrichs.. sar genetic determinants necessary for transcription of RNAII and RNAIII in agr locus of Staphylococcus aureus. J. Bacteriol. :-.. Cheung, A. L., J. M. Koomey, C. A. Butler, S. J. Projan, and V. A. Fischetti.. Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. Proc. Natl. Acad. Sci. USA :-.. Cheung, A. L., K. Schmidt, B. Bateman, and A. C. Manna. 00. SarS, a SarA homolog repressible by agr, is an activator of protein A synthesis in Staphylococcus aureus. Infect. Immun. :-.. Cheung, A. L., and P. Ying.. Regulation of alpha- and beta-hemolysins by the sar locus of Staphylococcus aureus. J. Bacteriol. :0-.. Chien, Y., and A. L. Cheung.. Molecular interactions between two global regulators, sar and agr, in Staphylococcus aureus. J. Biol. Chem. :-.. Chien, Y., A. Manna, and A. Cheung.. SarA level is a determinant of agr activation in Staphylococcus aureus. Mol. Microbiol. 0:-00.. Chien, Y. T., A. C. Manna, S. J. Projan, and A. L. Cheung.. SarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation. J. Biol. Chem. :-. 0. Deora, R., T. Tseng, and T. K. Misra.. Alternative transcription factor B of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar. J. Bacteriol. :-.. Dinges, M. M., P. M. Orwin, and P. M. Schlievert Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. :-.. Dufour, A., and W. G. Haldenwang.. Interactions between a Bacillus subtilis antisigma factor (RsbW) and its antagonist (RsbV). J. Bacteriol. :-0.. Frees, D., K. Sorensen, and H. Ingmer. 00. Global virulence regulation in Staphylococcus aureus: pinpointing the roles of ClpP and ClpX in the sar/agr regulatory network. Infect. Immun. : Giachino, P., S. Engelmann, and M. Bischoff. 00. B Activity Depends on RsbU in Staphylococcus aureus. J. Bacteriol. :-.. Giraudo, A. T., A. L. Cheung, and R. Nagel.. The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level. Arch. Microbiol. :-.. Hanahan, D.. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. :-0.. Heinrichs, J. H., M. G. Bayer, and A. L. Cheung.. Characterization of the sar locus and its interaction with agr in Staphylococcus aureus. J. Bacteriol. :-.. Horsburgh, M. J., J. L. Aish, I. J. White, L. Shaw, J. K. Lithgow, and S. J. Foster. 00. B modulates virulence determinant expression and stress resistance: charecterization of a functional rsbu strain derived from Staphylococcus aureus. J. Bacteriol. :-.. Janzon, L., S. Löfdahl, and S. Arvidson.. Evidence for a coordinate transcriptional control of alpha-toxin and protein A in Staphylococcus aureus. FEMS Microbiol. Lett. :-. Downloaded from on October, 0 by guest

19 Ji, G., R. C. Beavis, and R. P. Novick.. Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. USA :0-0.. Jonas, D., I. Walev, T. Berger, M. Liebetrau, M. Palmer, and S. Bhakdi.. Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation. Infect. Immun. :0-.. Karlsson, A., and S. Arvidson. 00. Variation in extracellular protease production among clinical isolates of Staphylococcus aureus due to different levels of expression of the protease repressor sara. Infect. Immun. 0:-.. Karlsson, A., P. Saravia-Otten, K. Tegmark, E. Morfeldt, and S. Arvidson. 00. Decreased amounts of cellwall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sara mutants due to up-regulation of extracellular proteases. Infect. Immun. :-.. Karlsson-Kanth, A., K. Tegmark-Wisell, S. Arvidson, and J. Oscarsson. 00. Natural human isolates of Staphylococcus aureus selected for high production of proteases and alpha-hemolysin are sigma B-deficient. Int. J. Med. Microbiol. :-.. Khan, S. A., G. K. Adler, and R. P. Novick.. Functional origin of replication of pt plasmid DNA is contained within a -base-pair segment. Proc. Natl. Acad. Sci. USA :0-.. Kreiswirth, B., S. Löfdahl, M. J. Betley, M. O'Reilly, M. P. Schleivert, M. S. Bergdoll, and R. P. Novick.. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 0:0-.. Kullik, I., and P. Giachino.. The alternative sigma factor B in Staphylococcus aureus: regulation of the sigb operon in response to growth and heat shock. Arch. Microbiol. :-.. Kullik, I., P. Giachino, and T. Fuchs.. Deletion of the alternative sigma factor B in Staphylococcus aureus reveals its function as a global regulator of virulence genes. J. Bacteriol. 0:-0.. Lina, G., S. Jarraud, G. Ji, T. Greenland, A. Pedraza, J. Etienne, R. P. Novick, and F. Vandenesch.. Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus. Mol. Microbiol. :-. 0. Lindsay, J., and S. Foster.. Interactive regulatory pathways control virulence determinant production and stability in response to environmental conditions in Staphylococcus aureus. Mol. Gen. Genet. :-.. Manna, A. C., and A. L. Cheung. 00. saru, a sara homolog, is repressed by SarT and regulates virulence genes in Staphylococcus aureus. Infect. Immun. :-.. Mayville, P., G. Ji, R. Beavis, H. Yang, M. Goger, R. P. Novick, and T. W. Muir.. Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc. Natl. Acad. Sci. USA :-.. McCallum, N., M. Bischoff, H. Maki, A. Wada, and B. Berger-Bächi. 00. TcaR, a putative MarR-like regulator of sars expression. J. Bacteriol. :-.. McNamara, P. J., and A. S. Bayer. 00. A rot mutation restores parental virulence to an agr-null Staphylococcus aureus strain in a rabbit model of endocarditis. Infect. Immun. :0-0. Downloaded from on October, 0 by guest

20 McNamara, P. J., K. C. Milligan-Monroe, S. Khalili, and R. A. Proctor Identification, cloning, and initial characterization of rot, a locus encoding a regulator of virulence factor expression in Staphylococcus aureus. J. Bacteriol. :-0.. Miyazaki, E., J. M. Chen, C. Ko, and W. R. Bishai.. The Staphylococcus aureus rsbw (orf) gene encodes an anti-sigma factor of SigB. J. Bacteriol. :-.. Morfeldt, E., L. Janzon, S. Arvidson, and S. Löfdahl.. Cloning of a chromosomal locus (exp) which regulates the expression of several exoprotein genes in Staphylococcus aureus. Mol. Gen. Genet. :-0.. Morfeldt, E., D. Taylor, A. von Gabain, and S. Arvidson.. Activation of alphatoxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J. :-.. Morfeldt, E., K. Tegmark, and S. Arvidson.. Transcriptional control of the agrdependent virulence gene regulator, RNAIII, in Staphylococcus aureus. Mol. Microbiol. :-. 0. Novick, R. P. 00. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol. Microbiol. :-.. Novick, R. P.. Genetic systems in staphylococci. Methods Enzymol. 0:-.. Novick, R. P.. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology :-.. Novick, R. P., and D. Jiang. 00. The staphylococcal saers system coordinates environmental signals with agr quorum sensing. Microbiology :0-.. Novick, R. P., H. F. Ross, S. J. Projan, J. Kornblum, B. Kreiswirth, and S. Moghazeh.. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J. :-.. O'Reilly, M., J. C. de Azavedo, S. Kennedy, and T. J. Foster.. Inactivation of the alpha-haemolysin gene of Staphylococcus aureus - by site-directed mutagenesis and studies on the expression of its haemolysins. Microb. Pathog. :-.. Oscarsson, J., C. Harlos, and S. Arvidson. 00. Regulatory role of proteins binding to the spa (protein A) and sars (staphylococcal accessory regulator) in Staphylococcus aureus NTCC -. Int. J. Med. Microbiol. :-.. Oscarsson, J., K. Tegmark-Wisell, and S. Arvidson. 00. Coordinated and differential control of aureolysin (aur) and serine protease (sspa) transcription in Staphylococcus aureus by sara, rot and agr (RNAIII). Int. J. Med. Microbiol. :in press.. Recsei, P., B. Kreiswirth, M. O'Reilly, P. Schlievert, A. Gruss, and R. P. Novick.. Regulation of exoprotein gene expression in Staphylococcus aureus by agr. Mol. Gen. Genet. 0:-.. Said-Salim, B., P. M. Dunman, F. M. McAleese, D. Macapagal, E. Murphy, P. J. McNamara, S. Arvidson, T. J. Foster, S. J. Projan, and B. N. Kreiswirth. 00. Global regulation of Staphylococcus aureus genes by Rot. J. Bacteriol. : Sambrook, J. E., E. F. Fritsch, and T. Maniatis.. Molecular cloning: a laboratory manual, nd ed ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.. Schenk, S., and R. A. Laddaga.. Improved methods for electroporation of Staphylococcus aureus. FEMS Microbiol. Lett. :-.. Schmidt, K. A., A. C. Manna, and A. L. Cheung. 00. SarT influences sars expression in Staphylococcus aureus. Infect. Immun. :-. Downloaded from on October, 0 by guest 0

21 0 0. Schmidt, K. A., A. C. Manna, S. Gill, and A. L. Cheung. 00. SarT, a repressor of alpha-hemolysin in Staphylococcus aureus. Infect. Immun. :-.. Sterba, K. M., S. G. Mackintosh, J. S. Blevins, B. K. Hurlburt, and M. S. Smeltzer. 00. Characterization of Staphylococcus aureus SarA binding sites. J. Bacteriol. :0-.. Söderquist, B., P. Colque-Navarro, L. Blomqvist, P. Olcén, H. Holmberg, and R. Möllby.. Staphylococcal -toxin in septicemic patients; detection in serum, antibody response and production in isolated strains. Serodiagn. Immunother. Infect. Dis. :-.. Tegmark, K Regulation of virulence gene expression in Staphylococcus aureus. Karolinska Institutet, Stockholm.. Tegmark, K., A. Karlsson, and S. Arvidson Identification and characterization of SarH, a new global regulator of virulence gene expression in Staphylococcus aureus. Mol. Microbiol. :-0.. Voelker, U., A. Dufour, and W. G. Haldenwang.. The Bacillus subtilis rsbu gene product is necessary for RsbX-dependent regulation of B. J. Bacteriol. :-.. Walev, I., E. Martin, D. Jonas, M. Mohamadzadeh, W. Muller-Klieser, L. Kunz, and S. Bhakdi.. Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions. Infect. Immun. :-. 0. Wise, A., and C. Price.. Four additional genes in the sigb operon of Bacillus subtilis that control activity of the general stress factor B in response to enviromental signals. J. Bacteriol. :-. FIGURE LEGENDS Downloaded from on October, 0 by guest

22 FIG.. A. Hemolysin production by strains - (rsbu) and SH000 (rsbu + ) and their corresponding sara (PC and KT00), sars (WA0 and WA0), and sara sars (WA0 and WA0) mutant derivatives, grown on a rabbit blood agar plate. B. Hemolysin production by KT00 (SH000 sara) and WA (SH000 sara hla), grown on a rabbit blood agar plate. C. Immunoblot analysis of -hemolysin in culture supernatants of PC (- sara) (), - (), SH000 ( and ), KT00 (SH000 sara) () and WA0 (SH000 sars) (). Downloaded from on October, 0 by guest

23 FIG.. Northern blot analysis of sars, sara, RNAIII, s rrna and hla in strain SH000 (rsbu + ) and its derivatives WA0 (sars) and KT00 (sara), and in strain - (rsbu) and its derivative, PC (sara). The main sara transcript seen is the sigma B-dependent sara P transcript, which is markedly enhanced in SH000. The lower panel (hla) shows a reduced exposure. Samples were taken at the specified time points during growth of a representative culture. Downloaded from on October, 0 by guest

24 FIG.. A. Northern blot analysis of hla and RNAIII in strain V and WA (V sara). B. Northern blot analysis of sars and s rrna in strain PC (- sara), -, WA (V sara), and V. Samples were taken at the specified time points during growth. FIG.. Northern blot analysis of hla, RNAIII and s rrna in WA (- sars, containing pkt0 with the xylose-inducible sara). Samples were taken at the indicated time points during growth in liquid medium with or without 0.0% xylose (final concentration). Downloaded from on October, 0 by guest

25 0 FIG.. A. Northern blot analysis of hla and s rrna in WA0 (agr sars rot sara), WA0 (agr sars rot), WA0 (agr rot sara), WA (agr sars sara), and RN0 (wt), grown in liquid cultures with samples taken at the indicated time points. B. Hemolysin production by strains from left to right, grown on a rabbit blood agar plate, row : - (wt) and WA (agr sars); row : PM rot::tet (agr rot) and WA0 (agr sars rot sara); row : PM (agr) and WA0 (agr sars rot). Downloaded from on October, 0 by guest

26 FIG.. Northern blot analysis of RNAIII and hla in WA0 (- sara sars, containing pik with the xylose-inducible sigb). Samples were taken at the indicated time points during growth in liquid medium with or without 0.0% xylose (final concentration). FIG.. Northern blot analysis of hla and s rrna in WA00 (sars sara rot triple mutant of SH000) and -. Samples were taken at the indicated time points during growth of a representative culture. Downloaded from on October, 0 by guest 0 FIG.. Effect of complementation of the sars mutant derived from SH000. Northern blot analysis of RNAIII in WA (SH000 sars with pkt0 carrying sars), WA0 (SH000

27 0 sars), and SH000. Samples were taken at the indicated time points during growth of a representative culture. sara sars RNAIII rot sart hla Rot FIG.. Schematic overview of regulatory interactions involving sara, sars, sart, rot, agr (RNAIII) and B in the control of hla transcription. Arrows indicate stimulation and bars repression. An asterisk (*) indicates genes known to be upregulated by B. In exponential phase of growth hla transcription is suppressed by sara, sars and rot (, )(Fig. ). Transcription of sars is stimulated by rot (, ) and sart (), and is suppressed by sara (, ). Transcription of sart is repressed by sara and agr (RNAIII) (). RNAIII expression is enhanced by sara (,, ). In post-exponential phase RNAIII is supposed to neutralize Rot activity (, ), whereas transcription of sara and sars is B -dependent (,, )(Fig. ). In addition, B seems to suppress hla transcription via mechanisms not involving sara, sars and rot (referred to as X) (Fig. ). For further explanation see text. X Downloaded from on October, 0 by guest

28 TABLE. Bacterial strains and plasmids used in this study Strain / plasmid Relevant characteristics Source E.coli DH S. aureus E.coli strain used for propagation of all plasmid constructs () - Prototype S. aureus strain, rsbu () DU00 -, hla::ermb (Em r ) () KS0 Clinical strain () KT00 SH000, sara::km (Km r ) This study NA KS0, sara::km (Km r ) () PC -, sara::km (Km r ) () PM RN0, agr null () PM rot::tet RN0, agr null, rot::tet (Tc r ) P.J. McNamara RN0 Restriction deficient mutant of - () Downloaded from on October, 0 by guest RN0 Laboratory S. aureus strain that is derived from -, () rsbu SH000 - with functional rsbu () V High protease producing strain from which the four ATCC

29 major staphylococcal proteases (sspa, sspb, aur and scp) were originally characterized WA0 -, sars::ermb (Em r ) This study WA0 SH000, sars::ermb (Em r ) This study WA0 -, sara::km, sars::ermb (Km r Em r ) This study WA0 -, sara::km, sars::ermb (pik) (Km r Em r Tc r ) This study WA0 SH000, sara::km, sars::ermb (Km r Em r ) This study WA WA0 (pkt0) (Tc r ) This study WA V, sara::km (Km r ) This study WA SH000, sars::ermb, rot::tet (Em r Tc r ) This study WA00 SH000, sars::ermb, sara::km, rot::tet (Em r Km r Tc r ) This study WA0 RN0, agr null, sars::ermb, rot::tet (Em r Tc r ) () WA0 RN0, agr null, sars::ermb, rot::tet, sara::km (Em r Tc r Km r ) This study WA KT00, hla::ermb (Em r ) This study WA RN0, agr null, sars::ermb (Em r ) () WA RN0, agr null, sars::ermb, sara::km (Em r Km r ) This study Downloaded from on October, 0 by guest WA0 RN0, agr null, rot::tet, sara::km (Tc r Km r ) This study WA -, sars::ermb (pkt0) (Em r Tc r ) This study Plasmids paw pkt with upstream and downstream fragments of the This study r

30 sars locus inserted at either side of ermb (Em r ) paw paw with Tc r cassette from pt (Tc r Em r ) This study pgem-t Easy E. coli cloning vector for PCR products (Amp r ) Promega pkt pgem-t Easy with ermb of Tn (Amp r Em r ) () pik pkt0 pkt0 S. aureus plasmid with sigb under control of xyla promoter (Tc r ) pspt with xylr and the sars under control of xyla promoter (Tc r ) S. aureus plasmid with sara under control of xyla promoter (Tc r ) () () () pspt Shuttle vector (Tc r ) () pt A tetracycline-resistant plasmid from S. aureus (Tc r ) () Amp r = resistance to ampicillin, Km r = resistance to kanamycin/neomycin, Em r = resistance to erythromycin/lincomycin, Tc r = resistance to tetracycline Downloaded from on October, 0 by guest 0