Analysis of mutations in the Staphylococcus aureus clfB promoter leading to increased expression

Fionnuala M. McAleese and Timothy J. Foster

Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland

Correspondence
Timothy J. Foster
tfoster{at}tcd.ie


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A clfB : : tetK reporter was constructed in Staphylococcus aureus strains Newman and 8325-4, whereby the level of tetracycline resistance reflected the activity of the clfB promoter. Wild-type strains carrying a single copy of this construct exhibited a low level of tetracycline resistance, suggesting that the clfB promoter is weak. Spontaneous mutants that grew at higher tetracycline concentrations were isolated. Some were due to point mutations in the clfB promoter that led to increased expression of the tetK gene. The clfB promoter was identified by primer extension analysis and -35 and -10 elements were assigned. The promoter regions from the tetracycline-resistant mutants were sequenced and several had base changes within or adjacent to the -35 box. Three created the consensus -35 sequence TTGACA. The mutant clfB promoters were fused to lacZ. ß-Galactosidase activity was six- to ninefold higher in the mutant strains compared to the wild-type. The wild-type clfB gene was placed under the control of the mutant promoters. ClfB expression was higher than the corresponding wild-type strains and the protein was present on bacteria from the stationary phase instead of being confined to the exponential phase. Therefore, mutations in the clfB promoter that cause changes in the -35 region produce a stronger promoter that is capable of increased transcription and, as a result, increased expression of ClfB.

Abbreviations: Cm, chloramphenicol; Em, erythromycin; Tc, tetracycline


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Staphylococcus aureus is an important human pathogen that causes both community-acquired and nosocomial infections, ranging from skin abscesses to more serious invasive diseases including endocarditis, osteomyelitis and septicaemia (Waldvogel, 1995). Furthermore, S. aureus is a major cause of disease associated with indwelling medical devices. S. aureus expresses a number of surface proteins that promote adherence to components of the host extracellular matrix, such as fibrinogen, collagen, fibronectin and elastin (Höök & Foster, 2000). These proteins belong to the MSCRAMM family of surface proteins (microbial surface components recognizing adhesive matrix molecules) (Patti et al., 1994).

S. aureus expresses several surface proteins that bind to fibrinogen. The dominant factors responsible for bacterial adherence to immobilized fibrinogen are the structurally related proteins ClfA and ClfB (McDevitt et al., 1994; Ní Eidhin et al., 1998). In addition, the fibronectin-binding proteins FnBPA and FnBPB also bind to fibrinogen (Wann et al., 2000). We have shown that the expression of ClfB is confined to the exponential phase of growth (Ní Eidhin et al., 1998; McAleese et al., 2001). The ClfB protein is also subject to proteolytic cleavage and inactivation by the staphylococcal metalloprotease aureolysin. Cleavage occurs at a motif, SLAVA, in the ligand binding A region resulting in the loss of the N-terminal N1 domain (McAleese et al., 2001). Transcription of the clfB gene monitored using a clfB : : lacZ fusion ceased towards the end of the exponential phase (McAleese et al., 2001). Furthermore, expression of clfB was approximately fivefold lower than that of spa, which encodes protein A, suggesting that the clfB promoter is weaker than that of spa or that these promoters are regulated differently.

Promoters from Gram-positive bacteria have not been characterized as well as those from Gram-negative bacteria, although work with Bacillus subtilis has suggested that the sequences involved in Gram-positive bacteria are similar to those in Escherichia coli. E. coli promoters recognized by RNA polymerase with the {sigma}70 sigma factor contain the consensus sequences TTGACA and TATAAT, which are located -35 and -10 bp, respectively, from the transcription start site (Pribnow, 1975; Schaller et al., 1975; Takanami et al., 1976). These sequences are highly conserved among E. coli promoters (Hawley & McClure, 1983). The major vegetative B. subtilis sigma factor, {sigma}A, also recognizes promoters with this consensus sequence (Moran et al., 1982). In general there are 16–19 bp of DNA between the two regions, the most common spacer length being 17 bp (Harley & Reynolds, 1987). The length of the spacer is critical for promoter function. Signals located upstream and downstream from the core promoter may also be functionally important (Ross et al., 1993; Brunner & Bujard, 1987).

Base substitutions that reduce the homology of the promoter to the consensus generally reduce the rate of transcription initiation, whereas mutations that increase homology to the consensus sequence cause increased transcription (Hawley & McClure, 1983). Mutations affecting the spacing between the -35 and -10 regions also affect promoter function (Hawley & McClure, 1983). Promoters were found to be stronger if the spacing was closer to 17 bp. Certain promoters do not contain an obvious -35 sequence. The sed promoter of S. aureus has a consensus -10 sequence but a much less conserved -35 region. A TG dinucleotide is present at position -14/-13 and may compensate for this deficiency (Ponnambalam et al., 1986; Barne et al., 1997; Zhang & Stewart, 2000). Alternatively, sequence elements upstream from the -35 region may be important for promoter function in the absence of a consensus -35 sequence (Record et al., 1996). The effects of AT-rich tracts of DNA may allow DNA bending to occur, thus promoting the formation of a closed complex with RNA polymerase (Busby & Ebright, 1994).

This report describes the construction of a clfB : : tetK reporter fusion that allowed a promoterless tetK gene to be placed under the control of the clfB promoter. The level of tetracycline (Tc) resistance reflected the activity of the clfB promoter. It was possible to isolate spontaneous mutants with increased resistance to Tc, some of which were likely to carry promoter mutations. The clfB transcription start site was identified by primer extension analysis and the -35 and -10 sequences were identified. The promoters of mutant strains with increased Tc resistance were sequenced. The effects of mutations in the clfB promoter on both transcription and ClfB expression were examined.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
The bacterial strains and plasmids used in this study are listed in Tables 1 and 2, respectively. E. coli strains were grown routinely in L-broth or on the respective agar at 37 °C. S. aureus was grown either in trypticase soy broth (TSB; Oxoid) or on the respective agar (TSA), or in brain–heart infusion (BHI; Oxoid) broth or on the respective agar at 37 °C. For expression studies, bacteria were grown for 16 h in 2 ml TSB. Cells were washed twice in PBS (0·145 M NaCl, 0·15 M sodium phosphate) and diluted 1 : 100 into 50 ml BHI broth in a 250 ml conical flask. Cultures were grown to exponential phase (OD600 0·4–0·8) or stationary phase (OD600 8–12) at 37 °C, with shaking at 200 r.p.m. The following antibiotics were incorporated into the media when appropriate: ampicillin, 100 µg ml-1; erythromycin (Em), 5 or 10 µg ml-1; chloramphenicol (Cm), 5 or 10 µg ml-1; Tc, 2–30 µg ml-1.


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Table 1. Bacterial strains used in this study

 

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Table 2. Plasmids used in this study

 
DNA manipulations.
These were performed by standard methods (Sambrook et al., 1989). Primers are listed in Table 3. Sequencing of DNA inserts was carried out in both directions by MWG Biotech-AG.


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Table 3. Primers used in this study

 
Transduction and transformation.
Plasmid DNA was transformed into E. coli XL-1 or T61 cells made competent by CaCl2 treatment and into S. aureus RN4220 by electroporation. Transduction was performed as described by Foster (1998) using bacteriophage 85 at an m.o.i. of 1·0.

Construction of a clfB : : tetK reporter fusion.
Two clfB fragments, ClfB1 and ClfB2, of 812 and 818 bp, respectively, were amplified from within region A of clfB using pCU1-clfB+ DNA as the template and the primers ClfBF1, ClfBR1, ClfBF2 and ClfBR2. Fragments had restriction sites incorporated at both ends, to facilitate cloning together into pBluescript-SK cut with HindIII and BamHI. A 1390 bp tetK fragment was amplified using the primers TetF1 and TetR1, which incorporated EcoRI sites. This fragment included the entire coding sequence and ribosome-binding site for tetK but not its promoter. A stop codon, TAA, was engineered at the 5' end of the primer ClfBR1, to prevent read-through translation from clfB into tetK. The tetK fragment was ligated between the ClfB1 and ClfB2 fragments at the EcoRI restriction site that separated them. Plasmid pTS2, carrying a mutation in the rep gene that results in temperature-sensitive replication in S. aureus, was subsequently joined to this plasmid by insertion into a unique XbaI site, resulting in plasmid pFMA3. This plasmid was electroporated into S. aureus RN4220 and selected for on Cm agar (5 µg ml-1) at 28 °C, and subsequently transduced into S. aureus strains Newman and 8325-4. Phage plate stocks and cultures were maintained at 28 °C at all times. Plasmid integration by a single crossover homologous recombination event was selected by plating onto Cm agar at the non-permissive temperature of 42 °C. To stimulate plasmid excision and allelic replacement, several temperature shifts were performed. Briefly, a culture of a single crossover integrant was grown at 28 °C in the absence of antibiotic, diluted 1 : 100 into fresh broth and grown at 42 °C for 8 h. It was then diluted 1 : 100 again and grown at 28 °C. This cycle was repeated three times without any antibiotic selection. The culture was diluted and plated onto TSA without antibiotic. Four-hundred colonies were screened for Cm sensitivity. The Cm-sensitive colonies were tested for the presence of the tetK gene by PCR using primers TetF1 and TetR1. Putative double crossovers in strains Newman and 8325-4 were identified at this point and confirmed by Southern blotting using XbaI- and BamHI-cut genomic DNA probed with a digoxigenin (DIG)-labelled fragment complementary to the A region of clfB.

Selection of mutants with increased Tc resistance.
The MIC (minimum inhibitory concentration) values for the clfB : : tetK strains were determined by spotting 10 µl volumes of an overnight culture onto agar containing Tc (0·5, 1·0. 2·5, 5·0, 7·5, 10·0, 15·0, 20, 25 and 30 µg ml-1). The MIC was defined as the lowest concentration of Tc required for complete inhibition of growth. Spontaneous mutants with increased resistance were isolated at a concentration of 10 µg Tc ml-1 and a single colony was purified on 5 µg Tc ml-1. The frequency of transduction of the increased Tc resistance with the clfB : : tetK fusion was determined using a Tc concentration of 2 µg ml-1. A higher concentration was not used to avoid the possibility of selecting further mutations that conferred increased resistance to Tc. A lower concentration was insufficient to inhibit the growth of the recipient strain.

Generation of lacZ fusions to the mutated clfB promoters.
A clfB : : lacZ fusion was constructed by integration of pFMA4 (a derivative of pAZ106 containing a clfB fragment) into clfB via shared homology (McAleese et al., 2001). A phage lysate prepared on strain Newman clfB : : lacZ was used to infect each of the five TcR mutants with point mutations in the clfB promoter regions. Transductants that placed lacZ under the control of the mutated clfB promoters were detected by increased ß-galactosidase production (Fig. 3). Transductants were selected on 5 µg Em ml-1 and X-Gal (40 µg ml-1). Colonies producing a dark-blue colour on X-Gal-containing plates were retained and verified by Southern blotting. Chromosomal DNA was digested with XbaI and BamHI and probed with a DIG-labelled fragment complementary to the 5' end of clfB. ß-Galactosidase assays were carried out as described previously (McAleese et al., 2001).



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Fig. 3. Expression of clfB : : lacZ by strains Newman (a) and 8325-4 (b) with point mutations in the clfB promoter. Cultures were sampled throughout the growth cycle and ß-galactosidase activity was measured using 4-methylumbelliferyl ß-D-galactopyranoside (MUG) as the substrate. The OD600 values for the cultures are represented by open symbols and ß-galactosidase activities are represented by closed symbols. Newman clfB : : lacZ ({circ}, {bullet}); N1 clfB : : lacZ ({triangledown}, {blacktriangledown}); 8325-4 clfB : : lacZ ({lozenge}, {blacklozenge}); 81 clfB : : lacZ ({triangleup}, {blacktriangleup}); 85 clfB : : lacZ ({square}, {blacksquare}). This experiment was repeated three times with similar results.

 
Restoration of the full-length clfB gene in clfB : : tetK strains.
A 2618 bp fragment of clfB was PCR-amplified from pCU1-clfB+ DNA using the primers ClfBF1 and ClfBR4. This fragment included the clfB coding sequence but lacked the promoter and 124 bp at the 5' end. HindIII and EcoRI sites were incorporated at the 5' and 3' ends, respectively. The clfB fragment was cloned into pBluescript-KS cut with HindIII and EcoRI. The temperature-sensitive shuttle plasmid pG+host9 (Maguin et al., 1992) was cut with SalI and ClaI and joined to pBluescript-KS, resulting in plasmid pFMA9 (~9300 bp). pG+host9 was propagated in E. coli T61 to prevent emergence of revertants of the temperature-sensitive mutation in the rep gene. pFMA9 was introduced into S. aureus RN4220 by electroporation and subsequently transduced into each of the 10 TcR mutants and the parental strains, Newman clfB : : tetK and 8325-4 clfB : : tetK, using phage 85 and selection on 100 µg Em ml-1 at 28 °C. Cultures of strains carrying pFMA9 were grown at 28 °C in the presence of 100 µg Em ml-1, diluted to 10-2 or 10-4 and plated in duplicate onto agar containing 100 µg Em ml-1 and incubated at 28 and 42 °C. pFMA9 carries a temperature-sensitive rep gene from pG+host9 and can therefore only replicate at the permissive temperature of 28 °C. At 42 °C, EmR colonies can only arise due to integration of pFMA9 into the chromosome at a site of shared homology. Integration of pFMA9 into the chromosome of S. aureus replaced the clfB : : tetK construct with a full-length functional clfB gene that was under the control of the altered clfB promoters (Fig. 5). Integrants were screened for EmR and TcS, to ensure that pFMA9 had integrated on the desired side of the clfB : : tetK construct. The integrated pG+host9 derivative pFMA9 can be propagated stably at 37 °C (Hartford et al., 2001). pFMA9 integrants were verified by PCR analysis.



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Fig. 5. ClfB expression by strains with mutated clfB promoters. Cultures were grown to exponential phase (OD600 0·4–0·8) and cell-wall proteins were released by lysostaphin digestion in the presence of 30 % raffinose. ClfB was detected by Western immunoblotting using antisera specific to region A. (a) ClfB expression by strains Nwt clfB+ (lane 1), N1 clfB+ (lane 2), N2 clfB+ (lane 3), N3 clfB+ (lane 4), N4 clfB+ (lane 5) and N5 clfB+ (lane 6). (b) ClfB expression by strains 8wt clfB+ (lane 1), 81 clfB+ (lane 2), 82 clfB+ (lane 3), 83 clfB+ (lane 4), 84 clfB+ (lane 5) and 85 clfB+ (lane 6). This experiment was repeated three times with similar results.

 
Western immunoblotting.
S. aureus cells were grown to exponential phase (OD600 0·4–0·8) or stationary phase (OD600 10–12) and harvested by centrifugation at 14 000 g for 10 min. Cells were washed twice in PBS and adjusted to an OD600 value of 80 in 30 % (w/v) raffinose, 50 mM Tris (pH 8·0) and 20 mM MgCl2 in a final volume of 100 µl. Samples were incubated with 12 µl lysostaphin (2 mg ml-1; Ambi) and 8 µl protease inhibitors (Complete protease inhibitor cocktail; Roche) for 20 min at 37 °C with occasional shaking. Supernatants containing wall-associated proteins were incubated at 95 °C for 5 min in an equal volume of final sample buffer [0·125 M Tris/HCl, pH 6·8, 4 % (w/v) SDS, 20 % (v/v) glycerol, 10 % (v/v) ß-mercaptoethanol and 0·002 % (w/v) bromophenol blue]. Protein samples were subjected to twofold serial dilutions, to facilitate quantitative comparison where indicated. SDS-PAGE was performed by standard methods (Laemmli, 1970). Following separation, proteins were electrophoretically transferred to PVDF Western blotting membranes (Roche) using the wet-system (Bio-Rad). Membranes were incubated overnight at 4 °C in 10 % Marvel milk powder. Primary anti-rClfB (44–542) antibodies (Ní Eidhin et al., 1998) were used at a dilution of 1 : 5000. Protein A coupled to horseradish peroxidase was used as the secondary antibody. Blots were developed using the chemiluminescent substrate LumiGLO (New England Biolabs), according to the manufacturer's instructions, and exposed to X-ray film (X-Omat; Kodak).

Primer extension analysis.
BHI broth cultures (50 ml in a 250 ml flask) were grown to mid-exponential phase (OD600 0·4–0·8). Cells were washed twice in PBS and 1x109 cells were resuspended in 200 µl RNase-free diethyl-pyrocarbonate-treated water. Total cellular RNA was prepared using the FastPrep system (Bio 101) and the FastPrep FP120 machine (Bio 101), as described by Cheung et al. (1994) and following the manufacturer's instructions. The Primer Extension System (AMV reverse transcriptase) kit from Promega was used for both [{gamma}-32P]dNTP primer labelling and the primer extension reaction according to the manufacturer's instructions. A reverse primer, ClfBp.ext, was designed complementary to the 5' end of the clfB coding sequence and beginning at base position 69. A control primer and molecular mass markers were also end-labelled with [{gamma}-32P]dNTPs. The primer extension reaction was carried out using 25 µg total RNA prepared from strain Newman clfA clfB (pCU1-clfB+). Samples were analysed by PAGE alongside a DNA sequencing reaction carried out using the same oligonucleotide as for the primer extension reaction. The sequencing reaction was carried out using the T7 DNA polymerase Sequencing Kit (USB) according to the manufacturer's instructions with [35S]dATP (Amersham). Two micrograms of Wizard-SV (Promega) purified pCU1-clfB+ plasmid DNA and 10 pmol oligonucleotide (ClfBp.ext) were used for the sequencing reaction. The products from sequencing and primer extension reactions were separated by electrophoresis on an 8·3 M urea, 6 % (w/v) polyacrylamide gel. Once electrophoresis was complete, the gel was transferred to Whatman 3 MM paper, covered in Saran Wrap (Dow-Chemical) and dried under vacuum at 80 °C for 1 h using a Savant SG Speedgel gel-drying system. The dried gel was exposed to X-ray film following overnight incubation at -70 °C to enhance [{gamma}-32P]dNTP detection.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of spontaneous mutants of S. aureus clfB : : tetK with increased resistance to Tc
A clfB : : tetK fusion was constructed in strains Newman and 8325-4, whereby a promoterless tetK gene was placed under the control of the clfB promoter and the levels of Tc resistance reported on the activity of the clfB promoter. The Newman and 8325-4 clfB : : tetK strains expressed a MIC of 2·5 µg Tc ml-1 compared with 1·5 µg Tc ml-1 for the wild-type strains. The MIC was 15 µg ml-1 when a single copy of the tetK gene was expressed from its own promoter (McDevitt et al., 1993). Spontaneous TcR mutants arose at a frequency of 1x10-5 on plates containing 10 µg Tc ml-1. The mutants were presumed to have arisen in a single step. To isolate several independent TcR mutants from strains Newman clfB : : tetK and 8325-4 clfB : : tetK, five cultures of each strain were started from single colonies, grown overnight and plated onto agar containing 10 µg Tc ml-1. Colonies appeared after 48 h incubation at 37 °C. Only one TcR mutant from each culture was retained for further analysis. Mutants were isolated from strains Newman clfB : : tetK and 8325-4 clfB : : tetK and termed N1–N5 and 81–85, respectively.

Characterization of mutants with increased Tc resistance
The MIC values for Tc for the 10 spontaneous TcR mutants were determined (Table 4). MIC values ranged from 15 to 20 µg Tc ml-1 compared to the MIC of 2·5 µg ml-1 for the parental strains Newman clfB : : tetK and 8325-4 clfB : : tetK. To determine whether the mutations were closely linked to clfB, the frequency of transduction of clfB : : tetK with the high level TcR phenotype was measured. Phage lysates were prepared on each of the 10 TcR mutants as well as on the parental strains, Newman clfB : : tetK and 8325-4 clfB : : tetK. It was expected that if the mutations responsible for TcR were associated with the clfB promoter it would be possible to transduce the TcR phenotype into the Newman wild-type. If the mutation was not closely linked to clfB : : tetK, transduction would not occur. Under the conditions employed, it was not possible to transduce the wild-type clfB : : tetK construct from either Newman clfB : : tetK or 8325-4 clfB : : tetK into the Newman wild-type selecting on 2 µg Tc ml-1. Furthermore, it was not possible to transduce the TcR phenotype from three of the mutant strains, N2, N3 and N5. Seven mutants (N1, N4 and 81–85) produced transductants that suggested a link between clfB : : tetK and the mutation responsible for elevated resistance to Tc (Table 4).


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Table 4. MIC values and transduction frequencies

 
Southern blotting was performed on the 10 TcR mutants. Their chromosomal DNA was digested with HindIII and probed with a DIG-labelled fragment complementary to DNA 5' to the clfB gene. A band of ~1200 bp was detected for all 10 mutants and the wild-type controls, suggesting that the clfB promoter region had not undergone a gross genetic rearrangement that might have fused the tetK gene to a strong promoter (data not shown).

Cloning and sequencing of clfB promoter regions from clfB : : tetK mutants with increased resistance to Tc
The region of DNA upstream of clfB was PCR-amplified from chromosomal DNA prepared from each of the 10 TcR mutants (N1–N5 and 81–85) and from the parental strains Newman clfB : : tetK (Nwt) and 8325-4 clfB : : tetK (8wt). The resulting fragments included 395 bp of DNA upstream from the start codon, which was presumed to include the clfB promoter. Only five of the 10 TcR mutants were found to have a base change in the sequence upstream from clfB. Two classes of mutation were identified. Strains N1, 81 and 82 had a T->C transition at a position 60 bp upstream from the start codon. Strain 83 had an A->G transition 65 bp upstream from the start codon and strain 85 had an A->C transversion at the same position (Fig. 1b).



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Fig. 1. Identification of the clfB gene transcription start site by primer extension analysis. (a) PAGE analysis of reverse transcriptase reactions using the primer ClfBp.ext with no RNA template (lane 1), Newman clfA clfB (pCU1-clfB+) mRNA (lane 3) or with a positive control primer and RNA (lane 2). Radiolabelled DNA size standards are indicated. A sequencing reaction performed using pCU1-clfB+ DNA as template and primed with ClfBp.ext is also shown and the relevant lanes are labelled. (b) Sequence of the promoter region of clfB. The -35 and -10 promoter elements are boxed and the consensus E. coli and B. subtilis sequences are shown above. The clfB transcription start site is underlined in (a) and (b). The positions of the N1, 81, 82, 83 and 85 promoter mutations are indicated in lower case. An inverted repeat sequence upstream from the -35 region is shown.

 
Identification of the clfB transcription start site by primer extension analysis
Five of the 10 TcR mutants had mutations within 65 bp of the clfB start codon. It was possible that these mutations occurred within the clfB promoter region allowing increased transcription of the clfB : : tetK fusion and causing a corresponding change in resistance to Tc. The exact location of the clfB transcription start site and the promoter was not known, but they must lie within 500 bp 5' of the start codon (Ní Eidhin et al., 1998). To map the transcription start site of clfB, primer extension analysis was performed using a reverse primer specific to DNA at the 5' end of the clfB coding sequence. A sequencing reaction primed with the same oligonucleotide was analysed by PAGE alongside the cDNA product to determine exactly where the clfB transcript began (Fig. 1a). A control RNA and primer were used to generate a cDNA product of 87 bases (positive control) and no product was obtained in a negative control reaction without RNA (Fig. 1a). Primer extension analysis mapped the clfB transcription start site to 29 bp (±1) upstream from the clfB start codon. The sequence TACAAT, with five matches to the consensus -10 promoter sequences (TATAAT) of B. subtilis and E. coli, began 10 nt upstream from the transcription start site. A -35 element, TTGATA, with five matches to the consensus sequence TTGACA was also identified 17 nt upstream from the -10 region. Assignation of these putative -10 and -35 sequences placed the point mutations identified by sequencing within or adjacent to the -35 box (Fig. 1b). Furthermore, the T->C transition at position -32 in strains N1, 81 and 82 resulted in a -35 sequence identical to the consensus sequence TTGACA.

Construction of a lacZ transcriptional fusion to report on the activity of the mutant clfB promoters
To monitor transcription from the mutated promoters, it was decided to construct fusions with a promoterless lacZ gene. The construction of a clfB : : lacZ (EmR) fusion where the production of ß-galactosidase reflects the activity of the wild-type clfB promoter has been described previously (McAleese et al., 2001). This fusion was transduced into the clfB promoter mutant strains N1, 81 and 85. Strains 81 and 85 represent both classes of promoter mutations isolated in 8325-4. Phage 85 was used to infect the donor strain RN4220 clfB : : lacZ (EmR). Phage heads can package ~44 kb of bacterial DNA, so most containing clfB : : lacZ are also likely to include the wild-type clfB promoter. It is therefore likely that transduction of clfB : : lacZ into the clfB : : tetK mutant strains will replace the mutant promoter with the wild-type clfB promoter from the donor strain. In a small number of cases a transductant will arise where the promoterless lacZ gene is under the control of the mutated promoter. This will occur if homologous recombination between the chromosome of the recipient strain and the DNA released from the phage occurs at a position downstream from the mutated promoter (Fig. 2). Transductants were selected on plates containing Em and incorporating the ß-galactosidase substrate X-Gal. The appearance of pale-blue colonies signified transductants where lacZ remained under the control of the wild-type clfB promoter. Transductants that placed lacZ under the control of the mutated promoters caused increased transcription of lacZ and darker blue colonies in the presence of X-Gal. Dark-blue colonies arose at a frequency of approximately 2x10-2. The fidelity of these transductants was verified by Southern blotting (data not shown).



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Fig. 2. Construction of lacZ transcriptional fusions to the mutated clfB promoters. The clfB gene is represented by solid boxes and the mutant clfB promoters are marked with asterisks. Em resistance (EmR), Tc resistance (TcR) and ß-galactosidase (LacZ) activity, as detected using the substrate X-Gal, are indicated. Recombination during transduction resulting in an EmR phenotype can occur at positions 1 and 3 or 2 and 3. The outcome of a double crossover at positions 2 and 3 is shown here. tetK encodes a Tc-resistance gene; erm specifies resistance to Em; lacZ encodes the enzyme ß-galactosidase; P, clfB promoter.

 
Analysis of clfB transcription from mutant promoters
The expression of clfB : : lacZ fusions from wild-type and mutant clfB promoters was analysed throughout the growth cycle. A sixfold increase in clfB : : lacZ expression was observed for strain N1 clfB : : lacZ compared to Newman clfB : : lacZ with the wild-type clfB promoter (Fig. 3a). A six- to ninefold increase in clfB : : lacZ expression was detected for strains 81 and 85 compared to 8325-4 clfB : : lacZ (Fig. 3b). These results confirm that the increased expression of TcR strains with a mutated clfB promoter can be linked to increased transcription driven by these promoters. The clfB : : lacZ expression detected for strains 81 and 85 was not completely reduced in the stationary phase, unlike expression of clfB : : lacZ by strain N1 and the wild-type strains Newman and 8325-4.

Linkage of clfB to the mutant promoters
To examine the effects of point mutations within the clfB promoter on ClfB expression, it was necessary to restore a full-length copy of clfB in the TcR clfB : : tetK strains. To facilitate this, the clfB gene from strain Newman was cloned into a temperature-sensitive vector. The resulting plasmid, pFMA9, was introduced into each of the 10 TcR strains and the corresponding parental strains Newman clfB : : tetK and 8325-4 clfB : : tetK by transduction at the permissive temperature of 28 °C. This plasmid had the potential to integrate into the S. aureus chromosome at two places when cultures were grown at the non-permissive temperature of 42 °C. Integration to the right of the promoterless tetK cassette would not produce a functional clfB gene and high levels of Tc resistance would be maintained (Fig. 4b). Integration 5' to the tetK cassette would place a full-length copy of clfB under the control of the mutated promoters and Tc resistance would be lost (Fig. 4a). There is greater homology between pFMA9 and the chromosomal clfB locus 3' to the tetK gene. Therefore, TcR integrants occurred at a lower frequency than those that were TcS. pFMA9 integrants were screened for resistance to Tc and only the TcS integrants were retained for further study. The following nomenclature was used to describe the resulting strains, Nwt clfB+, N1 clfB+–N5 clfB+, 8wt clfB+ and 81 clfB+–85 clfB+,to indicate strains where the full-length clfB gene was placed under the control of the wild-type or mutated promoter sequences. The integration events were verified by PCR (data not shown).



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Fig. 4. Schematic representation of pFMA9 integration into the clfB gene. pFMA9 was introduced into the promoter mutant strains by transduction at the permissive temperature (28 °C). Following a shift to the non-permissive temperature (42 °C), pFMA9 could integrate to the left-hand or the right-hand side of the tetK gene, generating an EmR TcS construct (a) or an EmR TcR construct (b). Pmut, mutant clfB promoter; erm encodes resistance to Em; tetK encodes resistance to Tc.

 
Effects of promoter mutations on ClfB expression
The expression of ClfB by strains with a restored clfB gene was examined by Western immunoblotting. These strains included mutants N1 clfB+–N5 clfB+ and 81 clfB+–85 clfB+, five of which have mutations in the clfB promoter, and the corresponding strains with wild-type clfB promoters, Nwt clfB+ and 8wt clfB+. These strains were grown to exponential phase and cell-wall proteins were solubilized by lysostaphin digestion in the presence of 30 % raffinose. ClfB was detected by Western immunoblotting using anti-ClfB region A antibodies. The amount of ClfB detected for strain N1 clfB+ was approximately fourfold greater than that detected for either strain Nwt clfB+ or strains N2 clfB+–N5 clfB+ (Fig. 5a). Strain N1 has been shown previously to have a point mutation in the -35 region of the clfB promoter. Likewise, the four 8325-4 strains with point mutations in the clfB promoter, 81 clfB+, 82 clfB+, 83 clfB+ and 85 clfB+, expressed more ClfB than their corresponding parent strain or the 84 clfB+ strain, which did not have a mutation in this region (Fig. 5b). As reported previously, 8325-4 strains expressed a full-length and truncated form of ClfB (McAleese et al., 2001).

The expression of ClfB by strains 81 clfB+ and 85 clfB+, which represent both classes of clfB promoter mutation, was compared to ClfB expression by strain 8wt clfB+. Cells were grown to either exponential or stationary phase and concentrated to a standard OD600 value of 80. Exponential phase cells from the mutant strains were then subjected to twofold serial dilutions. Cell-wall proteins were solubilized and examined by Western immunoblotting. The 8325-4 strains with a point mutation at position -32 or -37 produced approximately eightfold more ClfB in the exponential phase than the same strain with a wild-type clfB promoter (Fig. 6b, c). Two forms of ClfB were expressed by 8325-4 strains in the exponential phase and only the truncated form was detected in the stationary phase, as observed previously (McAleese et al., 2001). ClfB expression by strains Nwt clfB+ and N1 clfB+ was also compared. Strain N1 clfB+ expressed four- to sixfold more ClfB than strain Nwt clfB+ in the exponential phase (Fig. 6a). Furthermore, all three promoter mutants, and in particular the 8325-4 mutants, expressed more ClfB in the stationary phase compared to their parental strains (Fig. 6). This difference in ClfB expression is consistent with a higher level of transcription of clfB by strains with a promoter mutation (Fig. 3). These results demonstrate that a single base change within the -35 region of the clfB promoter is sufficient to cause increased expression of ClfB. In the case of strains N1, 81 and 82 this is probably due to increased transcription of clfB driven by promoters with increased homology to the consensus -35 sequence.



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Fig. 6. ClfB expression by strains Nwt clfB+ and N1 clfB+ (a), 8wt clfB+ and 81 clfB+ (b) and 8wt clfB+ and 85 clfB+ (c). Lanes 1 and 7 represent ClfB expressed by strain Nwt clfB+ or strain 8wt clfB+ in exponential phase or stationary phase, respectively. Lanes 2–6 represent ClfB expressed by strain N1 clfB+, strain 81 clfB+ or strain 85 clfB+ in exponential phase at various cell concentrations; lane 8 represents ClfB expressed by the same strains in stationary phase. Cell-wall proteins were solubilized with lysostaphin and ClfB was detected by Western immunoblotting using antibodies specific to region A of ClfB. This experiment was repeated three times with similar results.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A clfB : : tetK fusion was constructed in strains Newman and 8325-4, whereby the levels of Tc resistance reflected the activity of the clfB promoter. Strains Newman clfB : : tetK and 8325-4 clfB : : tetK exhibited an MIC of 2·5 µg Tc ml-1 compared with 15 µg ml-1 when tetK was under the control of its own promoter (McDevitt et al., 1993), suggesting that the clfB promoter does not promote high level transcription. Five independent spontaneous mutants with increased Tc resistance (MIC values of 15–20 µg ml-1) were isolated from strains Newman clfB : : tetK and 8325-4 clfB : : tetK. It was possible to transduce the clfB : : tetK fusion expressing increased Tc resistance into a wild-type strain for seven of the 10 mutants, suggesting that the mutations were linked to clfB. A deletion or inversion could have placed tetK under the control of a stronger promoter but Southern blotting analysis of DNA immediately upstream of clfB suggested that gross genetic rearrangements had not occurred. It was considered that the altered phenotype of these seven mutants might have arisen due to a mutation within the clfB promoter that led to increased expression of the clfB : : tetK fusion and increased resistance to Tc.

The exact location of the clfB transcription start site was identified by primer extension analysis. The clfB transcript begins 29 bp upstream from the start codon. A putative -10 sequence (TACAAT) was identified which began at position -10 and had 5/6 matches to the consensus E. coli and B. subtilis sequence TATAAT. A -35 sequence (TTGATA) beginning at -31 was identified which had 5/6 matches to the consensus sequence TTGACA. The spacer region between the -10 and -35 sequences was 17 bp long. This is the consensus spacer length for E. coli promoters as determined by Hawley & McClure (1983). A 10 bp inverted repeat was identified that began at position -41 and which may represent a binding site for a protein involved in regulating clfB expression. A 10 bp inverted repeat upstream from the -35 sequence in the cap8 promoter was found to be essential for promoter activity and most likely serves as a regulatory protein binding site (Ouyang et al., 1999).

S. aureus expresses two sigma factors that can associate with the core RNA polymerase (RNAP) to recognize promoters and initiate transcription. SigA ({sigma}A) is the major vegetative sigma factor, while SigB ({sigma}B) is the stationary phase sigma factor (Deora & Misra, 1996; Deora et al., 1997). SigA is responsible for the expression of housekeeping genes. The alternative sigma factor SigB may play a role in altering bacterial gene expression in response to changing environmental signals (Kullik et al., 1998; Chan et al., 1998). The clfB promoter shares the same consensus sequence as the SigA-dependent promoters of B. subtilis and {sigma}70-dependent E. coli promoters. It is assumed therefore that the clfB promoter is dependent on {sigma}A–RNAP for its activity.

The clfB promoter regions of the 10 clfB : : tetK mutants with increased resistance to Tc were sequenced. Five were found to have a single base change within or adjacent to the -35 sequence of the clfB promoter. These were among the seven where the TcR phenotype was transducible. In order for high-level TcR to be transducible the mutation must be closely linked to tetK. Two classes of promoter mutation were identified. Strains N1, 81 and 82 had a single T->C transition at position -32. This base change produced the consensus -35 sequence of TTGACA. Strain 83 had an A->G transition at position -37 and strain 85 had an A->C transversion at the same position. It has previously been shown that promoter mutations that increase homology to the consensus sequence increase promoter function (Hawley & McClure, 1983; Record et al., 1996). This could explain the increased expression of tetK. Similarly, the creation of a consensus -35 promoter sequence upstream from the sed gene resulted in increased transcription (Zhang & Stewart, 2000). Furthermore, a T->C transition in the -35 sequence of the bacteriophage {lambda} PRM promoter caused an increase in transcription (Meyer et al., 1980), as was observed here for clfB transcription by strains N1, 81 and 82, which have an identical mutation. It is not immediately obvious why a base change beside the -35 region should cause an increase in activity from the clfB promoter, as observed for strains 83 and 85. However, similar mutations do have a significant effect on promoter strength. In contrast to the T->G or T->C substitutions observed here, a C->T transition at position -37 in the lac promoter reduced expression 10- to 20-fold (LeClerc & Istock, 1982). Similarly, a C->T substitution just upstream from the -35 hexamer of the rrnB P1 promoter of E. coli, encoding an rRNA, was found to reduce the activity of this promoter (Josaitis et al., 1990). This effect was attributed to the introduction of an unfavourable DNA bend immediately adjacent to the -35 region. In the cases of strains 83 and 85, the T->G or T->C changes may remove such a bend leading to an increase in transcription. In any case, it is clear that increased expression of Tc resistance by the five strains N1, 81, 82, 83 and 85 is due to mutations leading to a stronger clfB promoter.

Promoter mutations have previously been associated with an increase in antibiotic resistance by S. aureus. For example, an increase in methicillin resistance has been linked with point mutations in the promoter region of the mecA gene (encoding the penicillin-binding protein PBP-2A), which prevent the repressor protein MecI from binding (Kobayashi et al., 1998). In addition, increased resistance to fluoroquinolone antibiotics is associated with mutations in the spacer region of the norA promoter leading to increased expression or a multi-drug efflux transporter (Kaatz et al., 1993).

The expression of clfB was monitored in the promoter mutant strains using a clfB : : lacZ transcriptional fusion. A six- to ninefold increase in ß-galactosidase expression was observed when lacZ was under the control of the mutated promoters compared with the wild-type clfB promoter. Transcription peaked in mid- to late-exponential phase, suggesting that the mutated clfB promoter was still subject to temporal control. However, clfB : : lacZ expression by strains 81 and 85 did not reduce completely in stationary phase unlike clfB : : lacZ expression by strain N1. This suggests that strain-dependent differences in regulation may exist or alternatively that ß-galactosidase is more stable in the former strains. An increase in ClfB expression by the mutants was also observed when the full-length clfB gene replaced clfB : : tetK, following integration of pFMA9. Protein was detectable on cells from stationary phase, in contrast to ClfB expression by strains with a wild-type clfB promoter. More ClfB was detected on the surface of strains 81 and 85 compared with strain N1 in the stationary phase, in agreement with the transcription data described above.

Only five of the 10 spontaneous mutants isolated had base changes within the clfB promoter that could account for the increased Tc resistance. For three of these it was not possible to transduce Tc resistance, suggesting that the mutation responsible was not linked to the clfB gene. It is possible that a mutation had occurred in a gene involved in negative regulation of clfB. If this were the case, an increase in ClfB expression compared to the wild-type would be expected following integration of the ClfB+ plasmid, pFMA9. However, no difference in ClfB expression by strains Nwt clfB+, N2 clfB+, N3 clfB+ and N5 clfB+ was observed. It was possible to transduce the increased Tc-resistant phenotype for two of the five strains lacking promoter mutations. This may be due to a mutation that affects clfB mRNA stability or increases function of the TetK protein.

The low level of clfB transcript produced in wild-type strains has previously been ascribed to a weak clfB promoter (McAleese et al., 2001). Optimal clfB expression driven by the wild-type promoter requires activation by the SarA homologue, Rot (McNamara et al., 2000), in the exponential phase (Salim et al., 2003). Recent studies in this laboratory have suggested that the SarS protein (Tegmark et al., 2000; Cheung et al., 2001) is also involved in activating the clfB transcription in the exponential phase (unpublished observations). It is possible that the effect of Rot on clfB expression is mediated through SarS, as Rot is required for expression of the sarS gene (Salim et al., 2003). Further work is required to determine if SarS or Rot bind directly to the clfB promoter and whether the mutant promoters still require these proteins for maximum activity.


   ACKNOWLEDGEMENTS
 
The authors would like to thank Orla Keane and Denise Waldron for their assistance with manual DNA sequencing and the Health Research Board of Ireland for funding.


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Received 28 June 2002; revised 20 August 2002; accepted 16 September 2002.



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