Molecular Interactions between Two Global Regulators, sar and agr, in Staphylococcus aureus

The expression of many virulence determinants inStaphylococcus aureus is controlled by regulatory loci such as agr and sar. We have previously shown that the SarA protein is required for optimal transcription of RNAII and RNAIII in the agr locus. To define the specific molecular interaction, we overexpressed SarA as a glutathioneS-transferase (GST) fusion protein by cloning the 372-base pair (bp) sarA gene into the vector. The purified GST-SarA as well as cleaved SarA were able to bind specifically to the P2, P3, and the combined P2-P3 promoter fragments of agr in gel shift assays. Using monoclonal antibodies to SarA, we found that SarA is a part of the retarded protein-DNA complex as evidenced by the formation of a supershifted band. The SarA binding site on the agr promoter, mapped by DNase I footprinting assay, covered a 29-bp region between the P2 and P3 promoters devoid of any direct repeats. A synthetic 45-bp fragment encompassing the 29-bp sequence also bound the SarA protein in band shift assays. Serial in-frame deletion analysis of sarA revealed that, with the exception of 15 residues in the N terminus, almost all of SarA (residues 16–124) is essential for agr binding activity. Northern analysis confirmed that only the sar mutant clone containing a truncated sarA gene with a 15-residue deletion in the N terminus (SarA16–124) could activate agrtranscription to a level approaching that of the full-length counterpart (SarA1–124). Taken together, these data indicated that SarA is a DNA-binding protein with binding specificity to the P2 and P3 interpromoter region of agr, thereby activating RNAII and RNAIII transcription. The expression of many virulence determinants inStaphylococcus aureus is controlled by regulatory loci such as agr and sar. We have previously shown that the SarA protein is required for optimal transcription of RNAII and RNAIII in the agr locus. To define the specific molecular interaction, we overexpressed SarA as a glutathioneS-transferase (GST) fusion protein by cloning the 372-base pair (bp) sarA gene into the vector. The purified GST-SarA as well as cleaved SarA were able to bind specifically to the P2, P3, and the combined P2-P3 promoter fragments of agr in gel shift assays. Using monoclonal antibodies to SarA, we found that SarA is a part of the retarded protein-DNA complex as evidenced by the formation of a supershifted band. The SarA binding site on the agr promoter, mapped by DNase I footprinting assay, covered a 29-bp region between the P2 and P3 promoters devoid of any direct repeats. A synthetic 45-bp fragment encompassing the 29-bp sequence also bound the SarA protein in band shift assays. Serial in-frame deletion analysis of sarA revealed that, with the exception of 15 residues in the N terminus, almost all of SarA (residues 16–124) is essential for agr binding activity. Northern analysis confirmed that only the sar mutant clone containing a truncated sarA gene with a 15-residue deletion in the N terminus (SarA16–124) could activate agrtranscription to a level approaching that of the full-length counterpart (SarA1–124). Taken together, these data indicated that SarA is a DNA-binding protein with binding specificity to the P2 and P3 interpromoter region of agr, thereby activating RNAII and RNAIII transcription. Staphylococcus aureus is a major human pathogen with many clinical manifestations (1Crossley K.B. Archer G.L. The Staphylococci in Human Disease. Churchill Livingston, New York1997Google Scholar). Infections due to this organism can range from minor wound infections to severe sepsis. The capacity of S. aureus to cause various infections is probably related to the fact that this organism has an amazing ability to respond to changing environments. The adaptive response is highly coordinated and is generally modulated by regulatory elements via signal transduction pathways. The regulatory elements, in turn, control the transcription of a wide variety of unlinked genes, many of which are involved in pathogenesis (2Waldvogel F.A. Mandell G.L. Douglas R.G.J. Bennett J.E. Principles and Practice of Infectious Diseases. John Wiley 89: 6462-6466Crossref PubMed Scopus (316) Google Scholar, 5Giraudo A.T. Cheung A.L. Nagel R. Arch. Microbiol. 1997; 168: 53-58Crossref PubMed Scopus (165) Google Scholar). the agr locus consists of two divergent transcripts, RNAII and RNAIII, driven by two distinct promoters, P2 and P3, respectively (3Kornblum J. Kreiswirth B. Projan S.J. Ross H. Novick R.P. Novick R.P. Molecular Biology of the Staphylococci. VCH Publishers, New York1990: 373-402Google Scholar). RNAIII is the effector of the agr response that involves up-regulation of genes involved in exoprotein synthesis and down-regulation of genes encoding surface proteins (6Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3977Crossref PubMed Scopus (834) Google Scholar, 7Morfeldt E. Taylor D. von Gabain A. Arvidson S. EMBO J. 1995; 14: 4569-4577Crossref PubMed Scopus (350) Google Scholar). Another regulatory locus, designated sar, was uncovered in our laboratory (4Cheung A.L. Koomey J.M. Butler C.A. Projan S.J. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6462-6466Crossref PubMed Scopus (316) Google Scholar). Unlike agr, the sar locus activates the synthesis of both extracellular (e.g.hemolysins) and cell wall-associated proteins (e.g.fibronectin-binding protein (8Cheung A.L. Eberhardt K.J. Chung E. Yeaman M.R. Sullam P.M. Ramos M. Bayer A.S. J. Clin. Invest. 1994; 94: 1815-1822Crossref PubMed Scopus (246) Google Scholar, 23Cheung, A. L., and Wolz, C. (1997) Abstracts of the 37th ICAAC, B60, Toronto, Canada.Google Scholar)). The sar locus, encoded within a 1.2-kb 1The abbreviations used are: kb, kilobase(s); GST, glutathione S-transferase; bp, base pair(s); PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-β-d-galactopyranoside; MOPS, 4-morpholinepropanesulfonic acid; nt, nucleotide(s); ORF, open reading frame. DNA fragment, encompasses three overlapping transcripts (9Bayer M.G. Heinrichs J.H. Cheung A.L. J. Bacteriol. 1996; 178: 4563-4570Crossref PubMed Scopus (138) Google Scholar). These transcripts, designated sarA (0.58 kb), sarB (0.8 kb), and sarC (1.2 kb), have common 3′ ends but three distinct promoters. A major 372-bp ORF (sarA) together with extensive (≈800 bp) upstream sequence is present within the largest transcript, sarB. Phenotypic analysis revealed that the sar locus is necessary for hemolysin production, probably mediated by the interaction of sar gene products with the P2 and, to a lesser extent, P3 promoter of agr (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google Scholar, 11Morfeldt E. Tegmark K. Arvidson S. Mol. Microbiol. 1996; 21: 1227-1237Crossref PubMed Scopus (143) Google Scholar). The ensuing transcription of RNAII and RNAIII would lead to activation of exoprotein gene transcription (7Morfeldt E. Taylor D. von Gabain A. Arvidson S. EMBO J. 1995; 14: 4569-4577Crossref PubMed Scopus (350) Google Scholar). In contrast to the RNAIII-mediated control of exoprotein synthesis inagr, we recently reported that the SarA protein likely modulates α-hemolysin production in S. aureus (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google Scholar). Transcriptional and deletion analyses indicated that the intact SarA protein is required for full agr expression (i.e.RNAII and RNAIII) in S. aureus (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google Scholar). In this report, we examined the binding of GST-SarA fusion protein as well as purified SarA to the agr promoters. Our results demonstrated that both of these proteins bind to the P2 and P3 promoters of agr in a dose- dependent fashion. The binding affinity of GST-SarA appears to be higher with the P2 than with the P3 promoter. DNase I footprinting analysis of SarA binding to the agrpromoter region revealed that the SarA binding site resides in a 29-bp sequence between the P2 and P3 promoter region (−73 to −101 upstream of the P2 transcription start). Interestingly, the binding site excludes the 7-bp consensus repeat (AGTTAAG) within the P2-P3 interpromoter region previously reported by Morfeldt et al. (11Morfeldt E. Tegmark K. Arvidson S. Mol. Microbiol. 1996; 21: 1227-1237Crossref PubMed Scopus (143) Google Scholar). A labeled 45-bp DNA oligonucleotide probe encompassing the 29-bp binding site also bound the GST-SarA fusion protein in a gel retardation assay. In-frame deletion analysis of the SarA protein revealed that, with the exception of the 15 residues in the N terminus, almost all of the SarA protein (residues 16–124) is essential for binding activity to the agr promoter (P2-P3). Northern analysis also confirmed that only the sar mutant clones containing either the full-length the truncated (SarA16–124) could activate RNAII and RNAIII transcription of the agr locus. Taken together, our data indicated that SarA is a DNA-binding protein with binding specificity to the interpromoter region between P2 and P3 of agr, thereby to activation of P2 and P3 transcription. The and used in this are in was used as the for S. and were used for the of S. aureus (4Cheung A.L. Koomey J.M. Butler C.A. Projan S.J. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6462-6466Crossref PubMed Scopus (316) Google Scholar, R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3977Crossref PubMed Scopus (834) Google was used for were used the and and aureus R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3977Crossref PubMed Scopus (834) Google mutant of that DNA R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3977Crossref PubMed Scopus (834) Google that on and has a genetic to that of A.L. Eberhardt K. Heinrichs J.H. 1997; PubMed Google mutant of J.H. Bayer M.G. Cheung A.L. J. Bacteriol. 1996; 178: PubMed Google with with with A.L. Projan S.J. J. Bacteriol. 1994; PubMed Google with with highly for the cloning cloning for direct cloning of fragments J.H. Bayer M.G. Cheung A.L. J. Bacteriol. 1996; 178: PubMed Google gene fusion J.H. Bayer M.G. Cheung A.L. J. Bacteriol. 1996; 178: PubMed Google encompassing and sarB with a GST-SarA fusion containing an from the N designated with a GST-SarA fusion containing a from the N designated with a GST-SarA fusion containing a from the N designated with a GST-SarA fusion containing a from the N designated with a GST-SarA fusion containing a from the N designated with a GST-SarA fusion containing the full-length with a GST-SarA fusion containing an from the designated containing a sar fragment that and sarB. encompassing and sarB with an in-frame deletion of 15 from the N terminus of encompassing and sarB with an in-frame deletion of from the N terminus of in a The intact 372-bp sarA gene A.L. Projan S.J. J. Bacteriol. 1994; PubMed Google was by a and containing and to sarA gene fragments for the truncated SarA with in-frame in the N and were by products were with and and into The containing the gene are in The GST-SarA fusion clones were by and DNA expression of various GST-SarA was by to a of the were by for in and on was by for 15 and the was to the The affinity was the of the the fusion protein was cleaved with to the full-length SarA SarA and fusion proteins were by monoclonal on and the was by gel SarA protein GST-SarA fusion proteins were used for the gel shift and footprinting assays. SarA protein was by the 15 residues the N terminus with protein protein was used to to monoclonal antibodies as Fischetti V.A. J. PubMed Scopus Google Scholar). from were by with SarA antibodies and were purified from as Fischetti V.A. J. PubMed Scopus Google Scholar). To in-frame within the sarA was with the A fragment encompassing the sar locus was into to as a for oligonucleotide (9Bayer M.G. Heinrichs J.H. Cheung A.L. J. Bacteriol. 1996; 178: 4563-4570Crossref PubMed Scopus (138) Google and (9Bayer M.G. Heinrichs J.H. Cheung A.L. J. Bacteriol. 1996; 178: 4563-4570Crossref PubMed Scopus (138) Google were for the the DNA was into The in-frame deletion in was confirmed by DNA DNA fragments containing the were gel purified and into of S. with containing the within the sar locus was as previously S. Microbiol. 1992; 94: Google Scholar). was used to of containing the with sar The was used to the sar mutant as (4Cheung A.L. Koomey J.M. Butler C.A. Projan S.J. Fischetti V.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6462-6466Crossref PubMed Scopus (316) Google Scholar). The of was confirmed by were and as H. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). monoclonal antibodies were to with the for by of with a of were as PubMed Scopus Google Scholar). protein were used for molecular of S. aureus were in and to and The were and with a in with and a as A.L. Eberhardt K. Fischetti V.A. 1994; PubMed Scopus Google Scholar). of was a gel in MOPS, of was with the 179: 3963-3971Crossref PubMed Google Scholar, J.H. Bayer M.G. Cheung A.L. J. Bacteriol. 1996; 178: PubMed Google that SarA with the agr locus by binding to the P2 promoter. To the interaction between SarA and agr promoter, purified GST-SarA and SarA from E. were used in band shift P2 and P3 promoters. shown in A and the purified GST-SarA fusion protein bound to the agr P2 and P3 promoters in a with retardation for P2 and P3 promoter by and of the fusion the combined P2-P3 promoter fragment binding activity of GST-SarA to be band shift with of The for the P2 promoter was than P3 that SarA with higher affinity to the P2 than to the P3 agr promoter. the combined P2-P3 promoter has the affinity for the SarA protein The of P2-P3 fragment with the formation of the binding complex between the labeled probe and the GST-SarA fusion protein of a promoter region from DNA We also the binding of the full-length SarA by to the agr P2-P3 promoter purified SarA was able to bind to the P2-P3 promoter, with the binding to that of the fusion protein and The data indicated that SarA from S. aureus is of binding to the agr promoter. To that SarA is a part of the retarded protein-DNA we used monoclonal antibodies and to these monoclonal antibodies with the containing SarA and labeled P2-P3 we that bound to the retarded complex as evidenced by the formation of a supershifted complex in and a the of SarA any that the supershifted band is due to the binding of to the agrpromoter probe and Our band shift have shown that SarA specifically to the P2 and P3 promoter region of To the binding site we protein-DNA by the DNase I J. Molecular a Scholar). to both and was examined of purified GST-SarA was a region of was on both A and In both the are and to to −101 upstream of the P2 transcription site to upstream of a 29-bp region We also the footprinting with purified SarA from The is the The transcription between two agr promoters and are by a sequence containing several direct and repeats. The upstream of P2 and P3 are in that of the These have been to be the binding site for a common regulatory with SarA a (11Morfeldt E. Tegmark K. Arvidson S. Mol. Microbiol. 1996; 21: 1227-1237Crossref PubMed Scopus (143) Google Scholar). our footprinting analysis indicated that the SarA protein to a 29-bp region between the P2 and P3 promoters. region the repeats. To our footprinting we band shift with a labeled 45-bp oligonucleotide probe the 29-bp SarA binding shown in the 45-bp DNA fragment was retarded by the GST-SarA fusion protein in a In a fragment containing the but the SarA binding site to bind to the SarA protein These data that the direct are the binding site for To the specificity of this we also a of with of labeled and The results that the 45-bp DNA was able to with labeled P2, P3, 45-bp DNA fragments for the binding to the SarA protein To the DNA-binding of SarA to the agr P2 and P3 promoters, we an of fusion proteins full-length to truncated of SarA These were with and purified to as under shift were used to of these truncated fusion proteins with the agr promoter sequence in an to the SarA protein sequence that can as a DNA-binding protein to 15 residues bound the P2-P3 promoter but to residues and any protein-DNA A of as as residues to bind to the P2-P3 promoter probe These results that an extensive region from 15 to the terminus of SarA is required for DNA binding to the agr promoter. To in of truncated SarA protein to the agr promoter region with activation of RNAII and RNAIII aureus we for transcription in sar mutant clones and To as the which in-frame of sarA can be we a previously sar mutant clone a that the sar locus sarA and extensive upstream sequence (≈800 activation of RNAII and RNAIII in this clone has been shown to be optimal (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google Scholar). We two in-frame to and to sar mutant clones and To of the full-length truncated SarA protein in these deletion we monoclonal to the of truncated SarA proteins in cell from these S. aureus clones clone containing the intact sarA gene in a the full-length SarA and truncated SarA protein of and The and sar mutant as the and A level of SarA expression in as with be to the on our that truncated SarA protein with a 15-residue deletion in the N terminus was able to bind the promoter we also that containing this of truncated SarA, was for RNAII and RNAIII expression in a sar mutant with approaching that of the full-length SarA protein in with the results of the gel shift assay, clone with a in the N terminus to activate transcription from the agr promoter Taken together, these data indicated that the sarA sequence in was expression in a sar of virulence determinants in S. aureus is dependent in part on two regulatory loci, sar and that the sar locus is of three overlapping and we have previously the binding of agrpromoter fragment to cell from sar mutant clones containing a of the (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google Scholar). with in these data that the SarA protein likely hemolysin production by RNAII and RNAIII transcription of direct binding of SarA to the agr promoter region is In this report, we gel shift and footprinting data that purified SarA specifically to the agr P2-P3 promoter from response analysis that the SarA protein with higher affinity to the P2 than to the P3 promoter of GST-SarA for retardation of the labeled P2 and P3 A and The affinity in promoter binding of SarA with our that the of was on RNAII than on RNAIII transcription (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google Scholar). a of and for P2, and P3, binding between SarA and the agr promoters. with this was our that a sar mutant clone with a of the sarB SarA and a higher level of RNAII than the sarA counterpart encoding SarA (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google Scholar). In we that SarA, in with encoded by upstream of be required for optimal binding to the agr promoter. In this we have data to that the ORF upstream of sarA be required for full and A. regulatory be involved in this DNase I footprinting analysis has uncovered the SarA binding site to be a 29-bp sequence in the interpromoter region between P2 and P3 can be from this the that the binding site is to the P2 than to the P3 promoter would be with the that the SarA protein probably modulates P2 than P3 transcription. be required to bind with SarA to the P3 promoter Kornblum J. Novick R.P. J. Bacteriol. PubMed Scopus Google Scholar). this sequence any of the 7-bp (AGTTAAG) that have been by Morfeldt et al. (11Morfeldt E. Tegmark K. Arvidson S. Mol. Microbiol. 1996; 21: 1227-1237Crossref PubMed Scopus (143) Google to be the binding site for SarA In of our is the that a 45-bp labeled fragment encompassing this 29-bp sequence but any of the bound to the SarA a fragment containing two of the in to the to binding and a of band shift (11Morfeldt E. Tegmark K. Arvidson S. Mol. Microbiol. 1996; 21: 1227-1237Crossref PubMed Scopus (143) Google the footprinting data as well as the formation of a retarded complex and a binding site purified SarA protein was used for the binding assay. The for the results from the fact that purified protein was used in our affinity purified were used in (11Morfeldt E. Tegmark K. Arvidson S. Mol. Microbiol. 1996; 21: 1227-1237Crossref PubMed Scopus (143) Google Scholar). the by Morfeldt et al. (11Morfeldt E. Tegmark K. Arvidson S. Mol. Microbiol. 1996; 21: 1227-1237Crossref PubMed Scopus (143) Google be with DNA binding In this our has purified distinct DNA-binding protein with binding specificity to the agr promoter revealed of to the SarA this protein almost with the SarA protein in The of a complex also with the formation of a supershifted band the of monoclonal antibodies to the retarded protein-DNA complex A of the supershifted revealed two distinct of The and have distinct supershifted that than the promoter In the a supershifted with of the of these antibodies revealed that the for and resides the is the of the SarA and A. monoclonal with the DNA-binding of the SarA in of the protein-DNA to be to this In the SarA DNA-binding by of the sarA gene we found that only containing the full-length a truncated (SarA16–124) could bind to the agr promoter on gel shift assays. the bp) of the SarA site is that the DNA-binding almost the SarA to that, with the exception of 15 residues in the N terminus, the residues in SarA to the of the DNA-binding of the to binding to the agr promoter. are to define the DNA-binding of is the fact that in an in the terminus, the previously reported SarA of S. aureus (9Bayer M.G. Heinrichs J.H. Cheung A.L. J. Bacteriol. 1996; 178: 4563-4570Crossref PubMed Scopus (138) Google the full-length is to that of S. aureus and The of to bind to agr with our data in which the cell of a sar mutant clone containing the truncated sarA gene either as a J.H. Bayer M.G. Cheung A.L. J. Bacteriol. 1996; 178: PubMed Google as a (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google bind to the agr promoter fragment in gel shift assays. To this we the from and that a to have in the E. clone containing the sarA gene of was confirmed by the sarA with of and In with our in binding data Northern analysis of S. aureus sar mutant clone containing the truncated revealed that this clone could activate RNAII and RNAIII transcription to a level approaching that of the full-length SarA counterpart from the gel shift assay, the clone containing the deletion in sarA activate RNAII and RNAIII transcription as as the intact SarA control and and the gel shift the protein was of RNAII transcription a level as with the sar mutant control that the truncated SarA was of level agr is we have previously found that Northern analysis for transcription is than (10Cheung A.L. Bayer M.G. Heinrichs J.H. J. Bacteriol. 1997; 179: 3963-3971Crossref PubMed Google Scholar, J.H. Bayer M.G. Cheung A.L. J. Bacteriol. 1996; 178: PubMed Google Scholar). to the SarA protein is likely to be a DNA-binding protein to promoters. Our has shown that the SarA binding site on the agr promoter resides in a 29-bp sequence that is of is highly for S. is that this sequence is found a in the We that this be a common for SarA binding promoters. a of to be to this we have and a sarA in Staphylococcus and A. (1997) Abstracts of the of the for Scholar). that an agr in Projan S.J. Kreiswirth B. J. Novick R.P. Microbiol. 1993; PubMed Google Scholar, A. and J. of the of the for New be of to a SarA binding site in S. and related