- Email: [email protected]
The sae locus of Staphylococcus aureus encodes a two-component regulatory system
FEMS Microbiology Letters 177 (1999) 15^22
The sae locus of Staphylococcus aureus encodes a two-component regulatory system Ana T. Giraudo a
a
, Aldo Calzolari a , Angel A. Cataldi Rosa Nagel a;c; *
b
, Cristina Bogni
a
,
Departamento de Microbiolog|¨a e Inmunolog|¨a, Facultad de Ciencias Exactas, F|¨sico, Qu|¨mica y Naturales, Universidad Nacional de R|¨o Cuarto, Ruta 36 Km 601, 5800 R|¨o Cuarto, Co¨rdoba, Argentina b Instituto de Biotecnolog|¨a, CICV-INTA, 1708 Moro¨n, Buenos Aires, Argentina c CEFYBO, Serrano 669, 1414 Capital Federal, Argentina Received 10 May 1999; received in revised form 1 June 1999; accepted 3 June 1999
Abstract sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae: :Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Staphylococcus aureus; Exoprotein synthesis ; Sae cloning ; Global regulatory locus; Two-component regulatory system
1. Introduction Staphylococcus aureus is a pathogen of man and animals. It synthesizes many extracellular and cell wall-associated proteins, most of which play a major role in pathogenicity. The expression of these products is subject to coordinated regulation by several loci. The ¢rst and best characterized regulator is agr,
* Corresponding author. Tel.: +54 (11) 4855-7204; Fax: +54 (11) 4856-2751; E-mail: [email protected]
which involves ¢ve genes (agrA, agrB, agrC, agrD and hld). The agr system acts as a positive regulator of secretory proteins like K-, L- and N-hemolysins, proteases, DNase, staphylokinase and toxic shock syndrome toxin-1, and represses the transcription of the genes for protein A, coagulase and other cell wall-associated proteins [1]. The agr locus consists of two divergent transcripts: RNAIII, transcribed from the P3 promoter, which acts in the regulatory control of the target genes and encodes N-hemolysin, and RNAII, transcribed from P2, which encodes the gene products AgrA^D [2,3]. AgrA corresponds to the response regulator and AgrC to the histidine
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protein kinase of two-component signal transduction systems. AgrD would act as an autoinducing peptide interacting with AgrC to transmit a signal via AgrA to activate RNAII and RNAIII expression from P2 and P3 promoters [3,4]. Another characterized regulatory locus is sar. Insertional transposon mutagenesis in the sarA gene lowers the production of K- and L-hemolysins and enhances the expression of protein A and other cellbound proteins. The SarA protein controls the transcription of RNAIII by binding to the DNA region located between agr promoters P2 and P3 [5,6]. We have isolated and characterized a third regulatory locus, designated sae. Inactivation of sae leads to decreased production of K- and L-hemolysins, DNase and coagulase [7]. Northern blot analyses has shown that sae activates exoprotein production at the transcriptional level [8]. Mutations in agr, sar and sae have been shown to decrease virulence in several animal models [9^11]. This work was aimed at the cloning, physical mapping and sequencing of the sae locus. The results of this study revealed that sae encodes a two-component regulatory system, involving an activator gene and a gene encoding a sensor histidine protein kinase, designated saeR and saeS, respectively.
2. Materials and methods 2.1. Bacterial strains, plasmids and media The S. aureus strains used in this work were ISP479, Newman and their isogenic sae: :Tn551 derivatives RC106 and RC300, respectively [7,8]; RN4220, a restriction-de¢cient mutant, was used as a primary host for shuttle plasmid constructs isolated from Escherichia coli cells. E. coli DH5K was used as the recipient strain for cloning experiments involving vectors pUC18 or pBluescript II KS [12]. Plasmid pMK4 [13] was used as shuttle vector and pLTV1 as source for Tn917 [14]. Brain heart infusion (BHI, Difco) and LB broth were used for the growth of S. aureus and E. coli, respectively. Antibiotics were used at the following concentrations: ampicillin (Ap) at 50 Wg ml31 ; erythromycin (Em) at 10 Wg ml31 and chloramphenicol (Cm) at 5 Wg ml31 .
2.2. DNA manipulations Standard methods were used for plasmid and phage lambda DNA puri¢cation, agarose gel electrophoresis, restriction analysis and ligation [12]. Restriction endonucleases and T4 DNA ligase were purchased from Promega and New England BioLabs. To prepare the probes, plasmids were digested with appropriate restriction enzymes, resolved by agarose gel electrophoresis and puri¢ed by electroelution as described by Sambrook et al. [12]. S. aureus RN4220 was transformed with recombinant shuttle plasmid pRC91 by electroporation using a Bio-Rad Gene Pulser as described by Kraemer and Iandolo [15]. Transduction in S. aureus strains was performed by using phage 80K as described previously [7]. 2.3. Southern blot hybridization This was done as described in Sambrook et al. [12]. Hybond-N membranes (Amersham) were used for DNA transfers. DNA probes were labeled with digoxigenin-11-dUTP (DIG DNA Labelling Kit; Boehringer Mannheim) by random primer extension. Labeled DNA hybrids were detected by using antidigoxigenin FP(ab)2 antibody fragments conjugated to alkaline phosphatase and the chemiluminescent substrate CSPD according to the instructions of the manufacturer (Boehringer Mannheim). 2.4. Physical mapping Chromosomal DNA for clamped homogeneous electric ¢eld (CHEF) electrophoretic analysis was prepared as described by Patel et al. [16]. CHEF electrophoresis was carried out in 1% chromosomal-grade agarose gels with 0.5UTBE and Bio-Rad CHEFIII-DR apparatus. Run parameters were 6 V cm31 with a ramp time of 5^45 s for 26 h at 10³C. DNA fragments were transferred to nylon membrane and hybridized with the sae probe as described above. 2.5. DNA sequencing and sequence analysis DNA sequencing was carried out by the dideoxy chain termination method [17] using an automated
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sequencer (model 373A, ABI Prism). The DNA and protein sequences were analyzed by using the Biology WorkBench programs at the NCSA of the University of Illinois. Homology searches were conducted with the BLAST program [18]. Multiple amino acid sequences alignments were performed with the CLUSTAL W software package. 2.6. Exoprotein analysis Cells were grown in BHI broth in a rotatory shaker at 37³C for 15 h. Cultures were centrifuged for 15 min at 10 000 rpm at 4³C. The supernatants were kept on ice. K- and L-hemolysins, coagulase, and DNase were quanti¢ed by serial dilutions of these supernatants, using 1% washed rabbit and sheep erythrocytes, rabbit plasma (Difco), and toluidineDNA-agar, respectively [7].
3. Results and discussion 3.1. Molecular cloning of the sae region In order to clone a DNA fragment containing part of Tn551 plus adjacent DNA encoding sae, chromosomal DNA from the mutant strain RC106 sae: :Tn551 was digested with several restriction enzymes. The resulting fragments were probed using a digoxigenin-labeled 5-kb EcoRI fragment of pLTV1 carrying Tn917, which has high homology with Tn551 [19]. The HindIII DNA digests gave three positive fragments of 9.5, 5.5 and 1.9 kb (not shown). The 1.9-kb fragment corresponded to the internal HindIII fragment of Tn551 [19]; the other two fragments consisted of part of Tn551 plus adjacent chromosomal DNA. The 5.5-kb HindIII frag-
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ment was cloned into pUC18. Positive clones were screened among E. coli transformants by colony hybridization using the same probe. One positive clone was analyzed and found to harbor a plasmid, designated pRC4, carrying 4.5 kb of chromosomal DNA plus about 1 kb of Tn551 (Fig. 1A). A 1.8-kb XbaIClaI fragment of this plasmid, corresponding to the DNA region £anking the transposon, was cloned into pBluescript II KS vector, and designated pRC5. A 0.5-kb XbaI-T¢I fragment of pRC5 was puri¢ed and labeled to probe a V GEM-12 genomic library prepared in Foster's laboratory from Newman strain. Restriction and hybridization analysis of the DNA of ¢ve positive clones revealed that they carried di¡erent overlapping regions (not shown). A 3.4-kb EcoRI-ClaI fragment of one of these clones was subcloned into pBluescript II KS vector. Southern blot analysis showed that this plasmid, designated pRC9, contains a region homologous to pRC5 plus about 2 kb of chromosomal DNA £anking the other end of the transposon (Fig. 1A). 3.2. Complementation analysis sae: :Tn551 mutants have a greatly reduced production of several exoproteins [7]. In order to determine whether pRC9 carried the sae region, the 3.4kb EcoRI-SalI chromosomal fragment of pRC9 was subcloned into pMK4 shuttle vector. This plasmid, designated pRC91, was introduced into RN4220 by electroporation, and re-transferred from one Cmr electrotransformant with transducing phage 80K to two isogenic pairs of strains, Newman and its sae: :Tn551 derivative mutant RC300, and ISP479 and its sae: :Tn551 derivative RC106. Culture supernatants of a Cmr transductant of each strain were
Table 1 Exoprotein production by sae: :Tn551 mutants harboring pRC91 Production (U ml31 )
Strains Newman sae
RC300 sae: :Tn551
Newman (pRC91)
RC300 (pRC91)
ISP479 sae
RC106 sae: :Tn551
ISP479 (pRC91)
RC106 (pRC91)
K-hemolysin L-hemolysin Coagulase DNase (U104 )
770 3* 1 600 32
20 3 4 3.2
800 3 800 64
700 3 800 64
600 24 000 16 40
45 300 62 10
770 27 000 64 40
1 300 50 000 256 40
Data are the mean of two experiments. 3 : non-production ; *Newman strain is a L-hemolysin non-producer.
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Fig. 1. A: Physical map of S. aureus DNA fragments containing saeS and saeR genes cloned into pBluescript II KS vector. pRC4 and pRC5 contain DNA from RC106, and pRC9 from Newman strain. A schematic drawing indicates the location and orientation of saeRsaeS operon and of part of an incomplete ORF with homology to cbsB of B. subtilis (GenBank accession number L77099). S indicates the insertion site of Tn551. Restriction sites : A, AccI; C, ClaI; E, EcoRI; H, HindIII; S, SalI; T, T¢I; V, EcoRV; X, XbaI. B: Nucleotide sequence of the sae locus and the predicted protein products. Potential Shine-Dalgarno sequences are underlined and indicated by SD. Asterisks mark the translational termination codons. Two convergent arrows at the non-coding region downstream of saeS indicate a putative transcription termination region. Two hydrophobic sequences that may correspond to transmembrane regions located at the Nterminal region of SaeS protein are underlined with dots. The sequence data reported here have been submitted to the GenBank database and have been assigned the accession number AF29010.
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assayed for coagulase, K-hemolysin and DNase. The presence of the region carried by multicopy plasmid pRC91 restored to both sae: :Tn551 mutants the coagulase, K-hemolysin and DNase levels of the parental strains (Table 1). We have no explanation for the high levels of coagulase, and K- and L-hemolysins of strain RC106 (pRC91) compared to ISP479 (pRC91). 3.3. Physical mapping of the sae locus In order to map the sae locus, SmaI digests of the ISP479 chromosome were subjected to pulse-¢eld gradient gel electrophoresis. The digoxigenin-labeled EcoRI-ClaI fragment of pRC9 containing the sae locus was found to hybridize with the band corresponding to the SmaI-D fragment of the S. aureus chromosome (data not shown). 3.4. Sequence analysis of the sae locus Restriction fragments from the 3.4-kb EcoRI-ClaI fragment of pRC9, which was shown to complement the sae: :Tn551 exoprotein de¢ciency, were subcloned and sequenced (Fig. 1B). Sequence data of the subclones of pRC9 revealed that Tn551 was inserted into an open reading frame (ORF) encoding a product that exhibits high homology to the response regulator of bacterial two-component regulatory systems. This gene was designated saeR. A second ORF located downstream of saeR, designated saeS, is transcribed in the same direction and encodes a product with homology to sensor histidine protein kinases (Fig. 1). The GC contents of saeR (33.6%) and saeS (31.2%) are similar to the overall content of the S. aureus chromosome (33%). The saeR coding region,
of 687 bp, begins with an ATG codon, ends with a TAA stop codon and is preceded by a potential Shine-Dalgarno ribosome binding site (AGAGG) at the appropriate position. A putative promoter region is located upstream of the Shine-Dalgarno sequence (Fig. 1B). The saeS gene, of 1062 bp, starts with a GTG codon, is preceded by a putative Shine-Dalgarno sequence located in the saeR ORF and ends with a TAA stop codon. The fact that there are only two nucleotides at the end of the saeR gene and upstream of the coding region of saeS suggests that these two genes are cotranscribed. An incomplete ORF transcribed in the opposite direction was identi¢ed downstream of saeS (Fig. 1B). This sequence, which shows a high homology with the csbB gene of Bacillus subtilis, would have no relation to the sae genes. An inverted repeat (vG = 39 kcal or 337 kJ at 37³C) located in the non-coding region lying downstream of saeS and of the cbs homolog ORF might act as terminator of transcription (Fig. 1B). The saeR and saeS ORFs encode proteins of 228 and 353 amino acids, with predicted molecular masses of 26 959 and 39 504 Da, and isoelectric points of 5.2 and 6.8, respectively. The N-terminal region of the protein encoded by saeR, which is the most conserved among response regulators [20], contains the predicted aspartate phosphorylation site (D at position 51), two aspartate residues (D at positions 8 and 9) and the conserved lysine (K at position 101) (Figs. 1B and 2A). Examination of the hydropathy pro¢le of the N-terminal portion of the SaeS protein reveals two putative transmembrane sequences, spanning amino acids 10^27 and 40^58 (Fig. 1B), that are characteristic of the N-terminal region of sensor proteins [20]. The C-terminal region shows the conserved functional subdomains [20] with the predicted histidine autophosphorylation site (H
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Fig. 2. Alignments of the deduced amino acid sequences of (A) SaeR and (B) SaeS proteins with other bacterial two-component regulatory systems. Sequences of SaeR correspond to N-terminal conserved regions. Sequences of the histidine protein kinase correspond to Cterminal H, N, G1, F and G2 motifs. The residues that are strictly conserved are indicated in boldface, residues that are identical in most members of each group of protein are shaded, and the putative phosphorylation sites are indicated with asterisks. Numbers indicate amino acid positions. Abbreviations of organism names : Sa, S. aureus ; Tm, Thermotoga maritima ; Bs, B. subtilis; Ll, Lactococcus lactis; Ef, Enterococcus faecium; Mt, Mycobacterium tuberculosis; Ec, Escherichia coli. The protein sequences are from GenBank and the accession numbers are indicated in parentheses: DrrA and HpkA (U67196) ; ResD (P35163) ; PhoP (M16775) ; ArcA (AJ001103) ; VanR (Q06239) ; MtrA (Z95121); PhoR (P23545); YkoH (AJ002571) ; VanS (Q06240) ; BaeS (P30847) ; ResE (P35164).
at position 131), the highly conserved asparagine residue (NA motif at position 251), the two glycine-rich G1 and G2 motifs (DXGXG and GXG at positions 281 and 313) and the conserved phenylalanine (F at position 295) (Figs. 1B and 2B). The sequences of putative SaeR and SaeS proteins were analyzed for homology to other sensor histidine protein kinases and regulatory proteins of two-component systems. The SaeR sequence shows the highest homology to DrrA of Thermotoga maritima and ResD and PhoP of B. subtilis with 39% identity and 60% similarity over the entire length of the protein (Fig. 2A). All these proteins show the C-terminal e¡ector domain characteristic of the OmpR-PhoB subfamily of response regulators. The C-terminal portion of the SaeS protein, which is the conserved region of the histidine protein kinases, shows the highest homology to PhoR and YkoH of B. subtilis and VanS of Enterococcus faecium with about 27% identity and 47% amino acid similarity, within an overlap of 250 and 300 amino acids (Fig. 2B).
3.5. sae and other S. aureus regulatory loci In this work we show that sae is a two-component regulatory locus. Two-component regulatory systems have been shown to play a crucial role in the signal transduction implicated in sensing di¡erent environmental conditions. Many of these two-component systems control the expression of virulence determinants. Two other two-component regulatory systems have been identi¢ed in S. aureus. One of them corresponds to lytS and lytR genes which control the rate of autolysis by a¡ecting the expression of murein hydrolases [21]. The other involves the agrA and agrC genes which belong to the agr locus. A low homology was detected between the proteins of these two regulatory two-component systems and the corresponding ones of the sae system. AgrC-AgrA and SaeR-SaeS are two di¡erent sensor-e¡ector systems that modulate exoprotein synthesis. In the agr system the response regulator pro-
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tein AgrA would be controlling exoprotein synthesis indirectly, by activating RNAIII transcription from agr P3 promoter, probably by an as yet unknown interaction with SarA [6]. AgrA would be activated through the interaction of AgrC with an autoinducing peptide encoded by agrD [4]. Northern blot analysis has shown that sae is necessary for transcription of hla, hlb and coa genes and that its inactivation does not exert any e¡ect on agr or sarA transcription [8]. From these observations we could infer that SaeR exerts its e¡ect by direct interaction with the target genes which are also subject to regulation by agr. However, a direct interaction between sae and agr cannot at present be excluded. Whatever the mechanism, sae and agr would be acting, directly or indirectly, on the expression of several common target genes (hla, hlb, nuc and coa). This action would result in additive, synergistic or epistatic e¡ects, as has in fact been observed for several exoproteins in studies with a double sae agr null mutant [11]. Additive e¡ects were observed with the sae agr null double mutant in relation to diminished virulence, which could result from the reduction in the production of several exoproteins and cell-bound proteins involved in pathogenicity [11]. Further work will be necessary to understand the regulation of sae expression during the life cycle and under di¡erent environmental conditions, as well as its interactions with other regulatory systems.
Acknowledgements Thanks are due to Dr. T. Foster for the generous gift of the V genomic library of S. aureus. A.G., A.A.C. and R.N. are Career Investigators of the Consejo Nacional de Investigaciones Cient|¨¢cas y Te¨cnicas (CONICET). This work was supported by grants from CONICET, CONICOR, and UNRC (Argentina). References [1] Kornblum, J., Kreiswirth, B.N., Projan, S.J., Ross, H. and Novick, R.P. (1990) agr: a polycistronic locus regulating exoprotein synthesis in Staphylococcus aureus. In: Molecular Biology of the Staphylococci (Novick, R.P., Ed.), pp. 373^402. VCH Publishers, New York.
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[2] Janzon, L. and Arvidson, S. (1990) The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. EMBO J. 9, 1391^1399. [3] Novick, R.P., Projan, S.J., Kornblum, J., Ross, H.F., Ji, G., Kreiswirth, B., Vandenesch, F. and Moghazeh, S. (1995) The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus. Mol. Gen. Genet. 248, 446^458. [4] Lina, G., Jarraud, S., Ji, G., Greenland, T., Pedraza, A., Etienne, J., Novick, R.P. and Vandenesch, F. (1998) Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus. Mol. Microbiol. 28, 655^662. [5] Bayer, M.G., Heinrichs, J.H. and Cheung, A.L. (1996) The molecular architecture of the sar in Staphylococcus aureus. J. Bacteriol. 178, 4563^4570. [6] Chien, Y. and Cheung, A.L. (1998) Molecular interactions between two global regulators, sar and agr, in Staphylococcus aureus. J. Biol. Chem. 273, 2645^2652. [7] Giraudo, A., Raspanti, C., Calzolari, A. and Nagel, R. (1994) Characterization of a Tn551-mutant of Staphylococcus aureus defective in the production of several exoproteins. Can. J. Microbiol. 40, 677^681. [8] Giraudo, A., Cheung, A.L. and Nagel, R. (1997) The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level. Arch. Microbiol. 168, 53^58. [9] Abdelnour, A., Arvidson, S., Bremell, T., Ryde¨n, C. and Tarkowski, A. (1993) The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infect. Immun. 61, 3879^3885. [10] Cheung, A.L., Yeaman, M.R., Sullam, P.M., Witt, M.D. and Bayer, A.S. (1994) Role of the sar locus of Staphylococcus aureus in induction of endocarditis in rabbit. Infect. Immun. 62, 1719^1725. [11] Giraudo, A., Rampone, H., Calzolari, A. and Nagel, R. (1996) Phenotypic characterization and virulence of a sae3 agr3 mutant of Staphylococcus aureus. Can. J. Microbiol. 42, 120^123. [12] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning : A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [13] Sullivan, M.A., Yasbin, R.E. and Young, F.E. (1984) New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene 20, 21^26. [14] Camilli, A., Portnoy, D.A. and Youngman, P. (1990) Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA £anking transposon insertions. J. Bacteriol. 172, 3738^3744. [15] Kraemer, G.R. and Iandolo, J.J. (1990) High-frequency transformation of S. aureus by electroporation. Curr. Microbiol. 21, 373^376. [16] Patel, A.H., Foster, T.J. and Pattee, P.A. (1989) Physical and genetic mapping of the protein A gene in the chromosome of Staphylococcus aureus. J. Gen. Microbiol. 135, 1799^1807. [17] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463^5467.
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[18] Altschul, S.F., Madden, T.L., Scha«¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^ 3402. [19] Shaw, J.H. and Clewell, D.B. (1985) Complete nucleotide sequence of macrolide-lincosamide-streptogramin B-resistance transposon Tn917 in Streptococcus faecalis. J. Bacteriol. 164, 782^796.
[20] Stock, J.B., Surette, M.G., Levit, M. and Park, P. (1995) Twocomponent signal transduction systems : structure-funcion relationships and mechanisms of catalysis. In: Two-Component Signal Transduction (Hoch, J.A. and Silhavy, T.J., Eds.), pp. 25^51. ASM Press, Washington, DC. [21] Brunskill, E.W. and Bayles, K.W. (1996) Identi¢cation and molecular characterization of a putative regulatory locus that a¡ects autolysis in Staphylococcus aureus. J. Bacteriol. 178, 611^618.
FEMSLE 8875 15-7-99
The sae locus of Staphylococcus aureus encodes a two-component regulatory system Ana T. Giraudo a
a
, Aldo Calzolari a , Angel A. Cataldi Rosa Nagel a;c; *
b
, Cristina Bogni
a
,
Departamento de Microbiolog|¨a e Inmunolog|¨a, Facultad de Ciencias Exactas, F|¨sico, Qu|¨mica y Naturales, Universidad Nacional de R|¨o Cuarto, Ruta 36 Km 601, 5800 R|¨o Cuarto, Co¨rdoba, Argentina b Instituto de Biotecnolog|¨a, CICV-INTA, 1708 Moro¨n, Buenos Aires, Argentina c CEFYBO, Serrano 669, 1414 Capital Federal, Argentina Received 10 May 1999; received in revised form 1 June 1999; accepted 3 June 1999
Abstract sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae: :Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Staphylococcus aureus; Exoprotein synthesis ; Sae cloning ; Global regulatory locus; Two-component regulatory system
1. Introduction Staphylococcus aureus is a pathogen of man and animals. It synthesizes many extracellular and cell wall-associated proteins, most of which play a major role in pathogenicity. The expression of these products is subject to coordinated regulation by several loci. The ¢rst and best characterized regulator is agr,
* Corresponding author. Tel.: +54 (11) 4855-7204; Fax: +54 (11) 4856-2751; E-mail: [email protected]
which involves ¢ve genes (agrA, agrB, agrC, agrD and hld). The agr system acts as a positive regulator of secretory proteins like K-, L- and N-hemolysins, proteases, DNase, staphylokinase and toxic shock syndrome toxin-1, and represses the transcription of the genes for protein A, coagulase and other cell wall-associated proteins [1]. The agr locus consists of two divergent transcripts: RNAIII, transcribed from the P3 promoter, which acts in the regulatory control of the target genes and encodes N-hemolysin, and RNAII, transcribed from P2, which encodes the gene products AgrA^D [2,3]. AgrA corresponds to the response regulator and AgrC to the histidine
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protein kinase of two-component signal transduction systems. AgrD would act as an autoinducing peptide interacting with AgrC to transmit a signal via AgrA to activate RNAII and RNAIII expression from P2 and P3 promoters [3,4]. Another characterized regulatory locus is sar. Insertional transposon mutagenesis in the sarA gene lowers the production of K- and L-hemolysins and enhances the expression of protein A and other cellbound proteins. The SarA protein controls the transcription of RNAIII by binding to the DNA region located between agr promoters P2 and P3 [5,6]. We have isolated and characterized a third regulatory locus, designated sae. Inactivation of sae leads to decreased production of K- and L-hemolysins, DNase and coagulase [7]. Northern blot analyses has shown that sae activates exoprotein production at the transcriptional level [8]. Mutations in agr, sar and sae have been shown to decrease virulence in several animal models [9^11]. This work was aimed at the cloning, physical mapping and sequencing of the sae locus. The results of this study revealed that sae encodes a two-component regulatory system, involving an activator gene and a gene encoding a sensor histidine protein kinase, designated saeR and saeS, respectively.
2. Materials and methods 2.1. Bacterial strains, plasmids and media The S. aureus strains used in this work were ISP479, Newman and their isogenic sae: :Tn551 derivatives RC106 and RC300, respectively [7,8]; RN4220, a restriction-de¢cient mutant, was used as a primary host for shuttle plasmid constructs isolated from Escherichia coli cells. E. coli DH5K was used as the recipient strain for cloning experiments involving vectors pUC18 or pBluescript II KS [12]. Plasmid pMK4 [13] was used as shuttle vector and pLTV1 as source for Tn917 [14]. Brain heart infusion (BHI, Difco) and LB broth were used for the growth of S. aureus and E. coli, respectively. Antibiotics were used at the following concentrations: ampicillin (Ap) at 50 Wg ml31 ; erythromycin (Em) at 10 Wg ml31 and chloramphenicol (Cm) at 5 Wg ml31 .
2.2. DNA manipulations Standard methods were used for plasmid and phage lambda DNA puri¢cation, agarose gel electrophoresis, restriction analysis and ligation [12]. Restriction endonucleases and T4 DNA ligase were purchased from Promega and New England BioLabs. To prepare the probes, plasmids were digested with appropriate restriction enzymes, resolved by agarose gel electrophoresis and puri¢ed by electroelution as described by Sambrook et al. [12]. S. aureus RN4220 was transformed with recombinant shuttle plasmid pRC91 by electroporation using a Bio-Rad Gene Pulser as described by Kraemer and Iandolo [15]. Transduction in S. aureus strains was performed by using phage 80K as described previously [7]. 2.3. Southern blot hybridization This was done as described in Sambrook et al. [12]. Hybond-N membranes (Amersham) were used for DNA transfers. DNA probes were labeled with digoxigenin-11-dUTP (DIG DNA Labelling Kit; Boehringer Mannheim) by random primer extension. Labeled DNA hybrids were detected by using antidigoxigenin FP(ab)2 antibody fragments conjugated to alkaline phosphatase and the chemiluminescent substrate CSPD according to the instructions of the manufacturer (Boehringer Mannheim). 2.4. Physical mapping Chromosomal DNA for clamped homogeneous electric ¢eld (CHEF) electrophoretic analysis was prepared as described by Patel et al. [16]. CHEF electrophoresis was carried out in 1% chromosomal-grade agarose gels with 0.5UTBE and Bio-Rad CHEFIII-DR apparatus. Run parameters were 6 V cm31 with a ramp time of 5^45 s for 26 h at 10³C. DNA fragments were transferred to nylon membrane and hybridized with the sae probe as described above. 2.5. DNA sequencing and sequence analysis DNA sequencing was carried out by the dideoxy chain termination method [17] using an automated
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sequencer (model 373A, ABI Prism). The DNA and protein sequences were analyzed by using the Biology WorkBench programs at the NCSA of the University of Illinois. Homology searches were conducted with the BLAST program [18]. Multiple amino acid sequences alignments were performed with the CLUSTAL W software package. 2.6. Exoprotein analysis Cells were grown in BHI broth in a rotatory shaker at 37³C for 15 h. Cultures were centrifuged for 15 min at 10 000 rpm at 4³C. The supernatants were kept on ice. K- and L-hemolysins, coagulase, and DNase were quanti¢ed by serial dilutions of these supernatants, using 1% washed rabbit and sheep erythrocytes, rabbit plasma (Difco), and toluidineDNA-agar, respectively [7].
3. Results and discussion 3.1. Molecular cloning of the sae region In order to clone a DNA fragment containing part of Tn551 plus adjacent DNA encoding sae, chromosomal DNA from the mutant strain RC106 sae: :Tn551 was digested with several restriction enzymes. The resulting fragments were probed using a digoxigenin-labeled 5-kb EcoRI fragment of pLTV1 carrying Tn917, which has high homology with Tn551 [19]. The HindIII DNA digests gave three positive fragments of 9.5, 5.5 and 1.9 kb (not shown). The 1.9-kb fragment corresponded to the internal HindIII fragment of Tn551 [19]; the other two fragments consisted of part of Tn551 plus adjacent chromosomal DNA. The 5.5-kb HindIII frag-
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ment was cloned into pUC18. Positive clones were screened among E. coli transformants by colony hybridization using the same probe. One positive clone was analyzed and found to harbor a plasmid, designated pRC4, carrying 4.5 kb of chromosomal DNA plus about 1 kb of Tn551 (Fig. 1A). A 1.8-kb XbaIClaI fragment of this plasmid, corresponding to the DNA region £anking the transposon, was cloned into pBluescript II KS vector, and designated pRC5. A 0.5-kb XbaI-T¢I fragment of pRC5 was puri¢ed and labeled to probe a V GEM-12 genomic library prepared in Foster's laboratory from Newman strain. Restriction and hybridization analysis of the DNA of ¢ve positive clones revealed that they carried di¡erent overlapping regions (not shown). A 3.4-kb EcoRI-ClaI fragment of one of these clones was subcloned into pBluescript II KS vector. Southern blot analysis showed that this plasmid, designated pRC9, contains a region homologous to pRC5 plus about 2 kb of chromosomal DNA £anking the other end of the transposon (Fig. 1A). 3.2. Complementation analysis sae: :Tn551 mutants have a greatly reduced production of several exoproteins [7]. In order to determine whether pRC9 carried the sae region, the 3.4kb EcoRI-SalI chromosomal fragment of pRC9 was subcloned into pMK4 shuttle vector. This plasmid, designated pRC91, was introduced into RN4220 by electroporation, and re-transferred from one Cmr electrotransformant with transducing phage 80K to two isogenic pairs of strains, Newman and its sae: :Tn551 derivative mutant RC300, and ISP479 and its sae: :Tn551 derivative RC106. Culture supernatants of a Cmr transductant of each strain were
Table 1 Exoprotein production by sae: :Tn551 mutants harboring pRC91 Production (U ml31 )
Strains Newman sae
RC300 sae: :Tn551
Newman (pRC91)
RC300 (pRC91)
ISP479 sae
RC106 sae: :Tn551
ISP479 (pRC91)
RC106 (pRC91)
K-hemolysin L-hemolysin Coagulase DNase (U104 )
770 3* 1 600 32
20 3 4 3.2
800 3 800 64
700 3 800 64
600 24 000 16 40
45 300 62 10
770 27 000 64 40
1 300 50 000 256 40
Data are the mean of two experiments. 3 : non-production ; *Newman strain is a L-hemolysin non-producer.
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Fig. 1. A: Physical map of S. aureus DNA fragments containing saeS and saeR genes cloned into pBluescript II KS vector. pRC4 and pRC5 contain DNA from RC106, and pRC9 from Newman strain. A schematic drawing indicates the location and orientation of saeRsaeS operon and of part of an incomplete ORF with homology to cbsB of B. subtilis (GenBank accession number L77099). S indicates the insertion site of Tn551. Restriction sites : A, AccI; C, ClaI; E, EcoRI; H, HindIII; S, SalI; T, T¢I; V, EcoRV; X, XbaI. B: Nucleotide sequence of the sae locus and the predicted protein products. Potential Shine-Dalgarno sequences are underlined and indicated by SD. Asterisks mark the translational termination codons. Two convergent arrows at the non-coding region downstream of saeS indicate a putative transcription termination region. Two hydrophobic sequences that may correspond to transmembrane regions located at the Nterminal region of SaeS protein are underlined with dots. The sequence data reported here have been submitted to the GenBank database and have been assigned the accession number AF29010.
6
assayed for coagulase, K-hemolysin and DNase. The presence of the region carried by multicopy plasmid pRC91 restored to both sae: :Tn551 mutants the coagulase, K-hemolysin and DNase levels of the parental strains (Table 1). We have no explanation for the high levels of coagulase, and K- and L-hemolysins of strain RC106 (pRC91) compared to ISP479 (pRC91). 3.3. Physical mapping of the sae locus In order to map the sae locus, SmaI digests of the ISP479 chromosome were subjected to pulse-¢eld gradient gel electrophoresis. The digoxigenin-labeled EcoRI-ClaI fragment of pRC9 containing the sae locus was found to hybridize with the band corresponding to the SmaI-D fragment of the S. aureus chromosome (data not shown). 3.4. Sequence analysis of the sae locus Restriction fragments from the 3.4-kb EcoRI-ClaI fragment of pRC9, which was shown to complement the sae: :Tn551 exoprotein de¢ciency, were subcloned and sequenced (Fig. 1B). Sequence data of the subclones of pRC9 revealed that Tn551 was inserted into an open reading frame (ORF) encoding a product that exhibits high homology to the response regulator of bacterial two-component regulatory systems. This gene was designated saeR. A second ORF located downstream of saeR, designated saeS, is transcribed in the same direction and encodes a product with homology to sensor histidine protein kinases (Fig. 1). The GC contents of saeR (33.6%) and saeS (31.2%) are similar to the overall content of the S. aureus chromosome (33%). The saeR coding region,
of 687 bp, begins with an ATG codon, ends with a TAA stop codon and is preceded by a potential Shine-Dalgarno ribosome binding site (AGAGG) at the appropriate position. A putative promoter region is located upstream of the Shine-Dalgarno sequence (Fig. 1B). The saeS gene, of 1062 bp, starts with a GTG codon, is preceded by a putative Shine-Dalgarno sequence located in the saeR ORF and ends with a TAA stop codon. The fact that there are only two nucleotides at the end of the saeR gene and upstream of the coding region of saeS suggests that these two genes are cotranscribed. An incomplete ORF transcribed in the opposite direction was identi¢ed downstream of saeS (Fig. 1B). This sequence, which shows a high homology with the csbB gene of Bacillus subtilis, would have no relation to the sae genes. An inverted repeat (vG = 39 kcal or 337 kJ at 37³C) located in the non-coding region lying downstream of saeS and of the cbs homolog ORF might act as terminator of transcription (Fig. 1B). The saeR and saeS ORFs encode proteins of 228 and 353 amino acids, with predicted molecular masses of 26 959 and 39 504 Da, and isoelectric points of 5.2 and 6.8, respectively. The N-terminal region of the protein encoded by saeR, which is the most conserved among response regulators [20], contains the predicted aspartate phosphorylation site (D at position 51), two aspartate residues (D at positions 8 and 9) and the conserved lysine (K at position 101) (Figs. 1B and 2A). Examination of the hydropathy pro¢le of the N-terminal portion of the SaeS protein reveals two putative transmembrane sequences, spanning amino acids 10^27 and 40^58 (Fig. 1B), that are characteristic of the N-terminal region of sensor proteins [20]. The C-terminal region shows the conserved functional subdomains [20] with the predicted histidine autophosphorylation site (H
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Fig. 2. Alignments of the deduced amino acid sequences of (A) SaeR and (B) SaeS proteins with other bacterial two-component regulatory systems. Sequences of SaeR correspond to N-terminal conserved regions. Sequences of the histidine protein kinase correspond to Cterminal H, N, G1, F and G2 motifs. The residues that are strictly conserved are indicated in boldface, residues that are identical in most members of each group of protein are shaded, and the putative phosphorylation sites are indicated with asterisks. Numbers indicate amino acid positions. Abbreviations of organism names : Sa, S. aureus ; Tm, Thermotoga maritima ; Bs, B. subtilis; Ll, Lactococcus lactis; Ef, Enterococcus faecium; Mt, Mycobacterium tuberculosis; Ec, Escherichia coli. The protein sequences are from GenBank and the accession numbers are indicated in parentheses: DrrA and HpkA (U67196) ; ResD (P35163) ; PhoP (M16775) ; ArcA (AJ001103) ; VanR (Q06239) ; MtrA (Z95121); PhoR (P23545); YkoH (AJ002571) ; VanS (Q06240) ; BaeS (P30847) ; ResE (P35164).
at position 131), the highly conserved asparagine residue (NA motif at position 251), the two glycine-rich G1 and G2 motifs (DXGXG and GXG at positions 281 and 313) and the conserved phenylalanine (F at position 295) (Figs. 1B and 2B). The sequences of putative SaeR and SaeS proteins were analyzed for homology to other sensor histidine protein kinases and regulatory proteins of two-component systems. The SaeR sequence shows the highest homology to DrrA of Thermotoga maritima and ResD and PhoP of B. subtilis with 39% identity and 60% similarity over the entire length of the protein (Fig. 2A). All these proteins show the C-terminal e¡ector domain characteristic of the OmpR-PhoB subfamily of response regulators. The C-terminal portion of the SaeS protein, which is the conserved region of the histidine protein kinases, shows the highest homology to PhoR and YkoH of B. subtilis and VanS of Enterococcus faecium with about 27% identity and 47% amino acid similarity, within an overlap of 250 and 300 amino acids (Fig. 2B).
3.5. sae and other S. aureus regulatory loci In this work we show that sae is a two-component regulatory locus. Two-component regulatory systems have been shown to play a crucial role in the signal transduction implicated in sensing di¡erent environmental conditions. Many of these two-component systems control the expression of virulence determinants. Two other two-component regulatory systems have been identi¢ed in S. aureus. One of them corresponds to lytS and lytR genes which control the rate of autolysis by a¡ecting the expression of murein hydrolases [21]. The other involves the agrA and agrC genes which belong to the agr locus. A low homology was detected between the proteins of these two regulatory two-component systems and the corresponding ones of the sae system. AgrC-AgrA and SaeR-SaeS are two di¡erent sensor-e¡ector systems that modulate exoprotein synthesis. In the agr system the response regulator pro-
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tein AgrA would be controlling exoprotein synthesis indirectly, by activating RNAIII transcription from agr P3 promoter, probably by an as yet unknown interaction with SarA [6]. AgrA would be activated through the interaction of AgrC with an autoinducing peptide encoded by agrD [4]. Northern blot analysis has shown that sae is necessary for transcription of hla, hlb and coa genes and that its inactivation does not exert any e¡ect on agr or sarA transcription [8]. From these observations we could infer that SaeR exerts its e¡ect by direct interaction with the target genes which are also subject to regulation by agr. However, a direct interaction between sae and agr cannot at present be excluded. Whatever the mechanism, sae and agr would be acting, directly or indirectly, on the expression of several common target genes (hla, hlb, nuc and coa). This action would result in additive, synergistic or epistatic e¡ects, as has in fact been observed for several exoproteins in studies with a double sae agr null mutant [11]. Additive e¡ects were observed with the sae agr null double mutant in relation to diminished virulence, which could result from the reduction in the production of several exoproteins and cell-bound proteins involved in pathogenicity [11]. Further work will be necessary to understand the regulation of sae expression during the life cycle and under di¡erent environmental conditions, as well as its interactions with other regulatory systems.
Acknowledgements Thanks are due to Dr. T. Foster for the generous gift of the V genomic library of S. aureus. A.G., A.A.C. and R.N. are Career Investigators of the Consejo Nacional de Investigaciones Cient|¨¢cas y Te¨cnicas (CONICET). This work was supported by grants from CONICET, CONICOR, and UNRC (Argentina). References [1] Kornblum, J., Kreiswirth, B.N., Projan, S.J., Ross, H. and Novick, R.P. (1990) agr: a polycistronic locus regulating exoprotein synthesis in Staphylococcus aureus. In: Molecular Biology of the Staphylococci (Novick, R.P., Ed.), pp. 373^402. VCH Publishers, New York.
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[18] Altschul, S.F., Madden, T.L., Scha«¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^ 3402. [19] Shaw, J.H. and Clewell, D.B. (1985) Complete nucleotide sequence of macrolide-lincosamide-streptogramin B-resistance transposon Tn917 in Streptococcus faecalis. J. Bacteriol. 164, 782^796.
[20] Stock, J.B., Surette, M.G., Levit, M. and Park, P. (1995) Twocomponent signal transduction systems : structure-funcion relationships and mechanisms of catalysis. In: Two-Component Signal Transduction (Hoch, J.A. and Silhavy, T.J., Eds.), pp. 25^51. ASM Press, Washington, DC. [21] Brunskill, E.W. and Bayles, K.W. (1996) Identi¢cation and molecular characterization of a putative regulatory locus that a¡ects autolysis in Staphylococcus aureus. J. Bacteriol. 178, 611^618.
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