University of Oxford, Microbiology Unit, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK
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Abstract |
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Introduction |
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The purified repressor (BlaI) of ß-lactamase production has been shown to bind specifically to two regions of dyad symmetry, known as operators,5 which are located between the divergently transcribed ß-lactamase structural gene (blaZ) and the gene (blaR1) encoding the putative transmembrane sensor protein. In the proposed model, dimeric BlaI binds independently but with similar affinities to the two operators. The repressor is thought to consist of a C-terminal DNA-binding domain and an N-terminal dimerization domain.5 Binding of a ß-lactam to BlaR1, the signal transducer, is presumed to alter its conformation so that a signal is transmitted across the bacterial membrane and this signal renders BlaI unable to bind to operator DNA, thus allowing transcription of blaZ, and hence production of ß-lactamase.
The organization of the mec operon responsible for the synthesis of PBP2' is similar to that of the bla operon.6 The repressor, MecI, binds to the operator preventing the transcription of mecA, the gene for PBP2'. It has been suggested that MecI undergoes extensive oligomerization,7 although Lewis8 hypothesized that, in solution, the predominant form is a dimer but that it may exist as a tetramer when bound to the operator. In many methicillin-resistant S. aureus (MRSA) strains mecA is inducible in the presence of the blaRI/blaI regulatory system.9,10 BlaI represses transcription of mecA and the repression is relieved in the presence of ß-lactams. It has also been shown that MecI represses the synthesis of ß-lactamase.11
The aims of the experiments reported here were: to examine whether MecI bound to the same sites on the bla operator as BlaI; to determine whether co-operative interactions occur between BlaI molecules bound at the two bla operators; and to discover any changes in the geometry of the inter-dyad DNA on binding of BlaI. With the aid of the reporter gene for chloramphenicol acetyltransferase (CAT) the role of the sequence between the promoter and start site of blaZ has been investigated.
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Materials and methods |
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The bacteria and plasmids used are listed in Table I. S. aureus was grown in casein hydrolysate (Oxoid, Basingstoke, UK)/yeast extract (Oxoid) (CY) medium or on CY agar19 without the addition of glucose or ß-glycerophosphate. Escherichia coli was grown in LuriaBertani (LB) medium (Oxoid) or on LB agar.20 For S. aureus the media contained, as required: 20 mg/L erythromycin, 5 mg/L chloramphenicol, 5 mg/L tetracycline. For E. coli, the media contained, as required, 100 mg/L ampicillin or 50 mg/L chloramphenicol. S. aureus was grown at 30°C and E. coli at 37°C. S. aureus was transformed by the method described by Gotz et al.,21 and E. coli was transformed by the method of Hanahan.22
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BlaI was prepared from the GSTBlaI fusion protein.5 MecI was also prepared from a GSTMecI fusion protein as described by Lewis.8
Recombinant DNA technology
All materials and methods were as in Sambrook et al.20 Wizard Plus SV Miniprep kit (Promega, Madison, WI, USA) was used to prepare DNA for cloning. T4 DNA ligase and all restriction enzymes were obtained from New England Biolabs (Hitchin, UK) or Boehringer-Mannheim (Lewes, UK). Oligonucleotides OT (5'-ATTATTGATTTATAA) and OB (5'-GTACTTATAAATCAATAAT) were obtained from Genosys (Cambridge, UK). DNA for footprinting and gel retardation was radioactively labelled by the method of Gregory et al.5
RepressorDNA binding reactions
Binding reactions were set up by preparing a reaction mix containing an amount of DNA appropriate for the footprinting reaction, 1 mg of non-specific DNA competitor [poly(dIdC)] in 10% (w/v) glycerol, 10 mM Trisglycine, 1 mM EDTA and 10 mM NaCl. This mix was dispensed into Eppendorf tubes, BlaI added and the reaction mixture was incubated at 37°C for 10 min. Samples were stored on ice and then loaded on to a pre-run acrylamide Trisglycine gel (30 mM Tris, 10 mM glycine) and electrophoresed at a constant 300 V at 4°C. The position of the complexes was determined using StrataMarkers (Stratagene, La Jolla, CA, USA) and autoradiography.
DNase I footprinting
A binding reaction mix containing at least 400 counts per second (cps) of the DNA probe per reaction was set up. The DNase I enzyme stock solution was diluted to c. 75 mg/L with a 0.9% NaCl solution, and 2 µL of this was added to 33 µL of a freshly prepared solution of 5 mM CaCl2 and 5 mM MgCl2. Ten microlitres of the binding reaction mix was then added and incubated at room temperature for 20 s, before addition of 50 µL of equilibrated phenol, vigorous vortexing for 20 s and storage on ice until all reactions were completed. Ten microlitres of chloroform was added, the tube was then briefly vortexed and microfuged at 12 000g for 15 min at 4°C. The aqueous layer was recovered and a 1/10 volume of 3 M sodium acetate and 2.5 vols of fresh ethanol were added, and the tube kept at 70°C overnight. Precipitated DNA was pelleted by microcentrifugation and resuspended in sequencing gel loading buffer. The samples were resolved on 15% acrylamide gels.
Copperphenanthroline footprinting
Reaction conditions were adapted from the method of Sigman et al.23 BlaIDNA binding reactions were electrophoresed through 6% (w/v) polyacrylamide gels, and complexes visualized by autoradiography. Excised gel fragments were immersed in 100 µL of 50 mM TrisHCl pH 8.0. Ten microlitres of OP-Cu mix (2 mM 1,10-phenanthroline, 0.45 M CuSO4) and 10 µL of 58 mM mercaptopropionic acid were added and the mixture incubated for 15 min at room temperature. The reaction was stopped by addition of 20 µL 2,9-dimethyl-1,10-phenanthroline and SDS to a final concentration of 0.2%. The DNA was eluted, ethanol precipitated and resolved on a denaturing acrylamide gel.
Dimethyl sulphate footprinting in vitro
Reactions were carried out essentially as described by Sasse-Dwight & Gralla.24 Probe DNA (c. 500 cps) was dissolved in 100 µL of Trisglycine binding buffer containing c. 5 µg BlaI. Samples without added protein were made up to the final volume with 1:1 phosphate-buffered saline and glycerol. After incubation at 37°C for 20 min, 6.7 µL of 150 mM dimethyl sulphate (DMS) was added and the mixture incubated for 5 min at 37°C. The reaction was stopped by the addition of 200 µL of cold stop buffer (3 M ammonium acetate, 1 M 2-mercaptoethanol, 20 mM EDTA, 250 mg/L yeast tRNA). The DNA was precipitated by addition of 600 µL of cold absolute ethanol and incubation of samples at 70°C for 30 min, followed by centrifugation. The pellets were washed in 70% (v/v) ice-cold ethanol and dried. The DNA was then cleaved by an alkaline method adapted from Strauss & Orkin.25 Deionized water was mixed with c. 500 cps of labelled DNA to a final volume of 50 µL. An equal volume of 20 mM NaH2PO4 was added, the reaction incubated at 90°C for 15 min and then transferred to ice. Twenty microlitres of 1 M NaOH was added and the reaction was incubated at 90°C for a further 30 min. The solution was neutralized with 20 µL of 1 M HCl and the DNA precipitated in 0.3 M sodium acetate and 2.5 vols of absolute ethanol. The DNA was collected after incubation at 70°C for 1 h by centrifugation at 12 000g for 30 min at 4°C, dried and resuspended in formamide loading buffer before electrophoresis in a denaturing polyacrylamide gel.
DMS footprinting in vivo
The method was based on that of Sasse-Dwight & Gralla.24 Cells to be footprinted were grown overnight in L-broth [16 g/L tryptone (Oxoid), 10 g/L yeast extract, 5 g/L NaCl] with appropriate antibiotic selection and the culture diluted 1:100 the next morning. At an A675 of c. 0.6, 10 mL of culture were induced (where required) with 2-(2'- carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) at 5 mg/L, and the induction was allowed to proceed for 10 min. Cultures were then treated with DMS at a final concentration of 4 mM for 5 min with shaking at 30°C.
Following treatment, the cells were removed to 20°C, while DNA was isolated using the Wizard Plus SV DNA purification system. DNA samples were processed as in the alkaline denaturation primer extension method.24
Piperidine cleavage of DNA
Modified DNA was suspended in 100 µL of 1 M piperidine and placed at 90°C for 30 min. Butan-1-ol (1 mL) was added, the mixture vortexed and centrifuged at 12 000g for 5 min. The supernatant was removed and the DNA resuspended in 100 µL of 0.5% SDS. One millilitre of butan-1-ol was added and the DNA precipitated as before. The DNA was resuspended in formamide loading buffer for electrophoresis on a denaturing gel.
Construction of a plasmid to investigate the effect of the blaZ dyad on expression from the blaZ promoter
Oligonucleotides OT and OB were annealed, mixed with pUC18 that had been digested with SspI and Asp718, ligated and used to transform E. coli JM109 with ampicillin selection. The resultant plasmid (designated pSRC500) was digested with SspI, mixed with SspI-digested pOX617, ligated and used to transform E. coli JM109 to ApR. The plasmid with the 0.6 kb SspI fragment from pOX617 inserted into pSRC500 in the required orientation was designated pSRC501 (Figure 1). Figure 2
shows the sequences of the operator regions of the wild type and the clone with the Z-dyad deleted.
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To enable pSRC520 and pSRC920 to replicate in S. aureus, a staphylococcal plasmid had to be inserted. BglII-digested pSRC520 and pSRC920 were separately mixed with BamHI-digested pT181MCS,17 ligated and used to transform E. coli JM109 to CmR. The required plasmids were designated pSRC522 and pRSC922 and were separately transformed into a strain that does not contain the bla operon (S. aureus RN4220) with selection for TcR.
Construction of plasmids to investigate the effect of BlaI on CAT production
To construct a plasmid from which the bla genes could be easily removed as a unit, pOX491 was digested with AvaII and AccI, the ends filled using Klenow polymerase and dNTPs and ligated. The ligation mix was used to transform E. coli JM109 to ApR and the resulting plasmid designated pSRC900. The bla genes next had to be inserted into the plasmids carrying the bla-derived promoters downstream of the cat reporter gene. The 3.6 kb PstI fragment of pSRC900 containing the bla genes was purified and cloned into PstI-digested pSRC920 and pSRC520. The ligation mixtures were transformed into E. coli JM109 with selection for CmR. The new plasmids were designated pSRC921 and pSRC521, respectively (Figure 4). To provide staphylococcal replicons, pT181MCS was ligated into the BglII site of both plasmids, to make pSRC923 and pSRC523, which were separately transformed into S. aureus RN4220 with selection for TcR.
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Reaction conditions used were adapted from those of Tomizawa.26 Cells (c. 500 µL from a mid-log phase culture) were harvested and resuspended in 500 µL of 0.1 M Tris HCl pH 8.0. Twenty microlitres of lysis buffer (0.1 M EDTA, 100 mM dithiothreitol, 50 mM TrisHCl pH 8.0) and a drop of toluene were added and the mixture vortexed. The sample was incubated at 30°C for 30 min and 10 µL of the mixture were incubated with 100 µL of 100 µM chloramphenicol and 2 µL of [1-14C]acetyl-coenzyme A (60 mCi/mmol) at 30°C for 10 min. The reaction was terminated by addition of 200 µL of toluene and the amount of radiolabelled acetylchloramphenicol partitioned into the toluene was measured in a Beckman LS 5000TD scintillation counter (Palo Alto, CA, USA).
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Results |
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The precise sequences within the bla operator to which BlaI binds have been determined5 by DNase I footprinting.27 To investigate by the same technique whether or not there are differences between the sites on the bla operators that are bound by MecI and BlaI, the purified proteins were bound to the 135 bp DNA bla operator probe, which contains the inter blaZblaR1 sequence. This probe was prepared from plasmid pOX617.5 The results show that the two footprints are indistinguishable (Figure 5). The sequence protected by the repressors is a little longer than the dyads but this could be due to steric hindrance between the repressor and DNase I. The assays for the other strand with the probe derived from pOX61818 gave similar results (data not shown).
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The 135 bp DNA bla operator probes used in the DNase I footprinting experiments were also employed in copper phenanthroline footprinting. The nuclease activity of the copperphenanthroline complex efficiently induces single-strand breaks in the DNA through the minor groove of the double helix,23 but is prevented from doing so when protein is bound. If the DNA is bent in the region between the sites bound to BlaI then the copperphenanthroline is likely to react differently and may produce a different pattern of breaks. The results (Figure 6) show the prevention of breaks where BlaI binds but no alteration in the pattern of breaks in the region between the sites of BlaI binding. It is concluded that, since no change in the geometry of the inter-operator DNA was detected, there is probably no interaction between the repressor molecules bound at the two operator sites.
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The technique of in vivo methylation protection was also used to discover changes in the geometry of inter-operator DNA on induction of the ß-lactamase by whole bacteria. DMS methylates the N-7 of guanine and the N-3 of adenine in DNA provided that the sites are not blocked by protein. The assay was set up with S. aureus RN4220 (pOX491) which has an intact bla operon, and S. aureus RN4220 (pOX637) in which blaI has been deleted. The cultures were either induced with the gratuitous inducer, CBAP or not induced. The results (Figure 7) show that induction exposes guanines at positions 57 and 88 on the bottom strand and 52 and 83 on the top strand. In the culture in which blaI is deleted these bases are exposed to methylation whether or not the culture has been induced. It is noted that the guanine at 54 is not protected in the absence of inducer although it is at the middle of the Z-dyad. Since all four guanines that are affected are within the two operator regions, it is concluded that, in vivo, the repressor BlaI binds to the same sites as it does in in vitro DNase I footprinting experiments. Since there are no changes in band intensity in the inter-operator DNA region, it is also concluded that there is no apparent alteration of the geometry of the DNA in this region on repressor binding.
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To establish whether methylation protection was the same in vitro as in vivo, purified BlaI was mixed with purified pOX491 DNA and then treated with DMS, followed by piperidine cleavage and primer extension. The results (Figure 8) indicate that there is no significant difference between the two, confirming that the in vivo result can be attributed to the binding of BlaI. Enhancements of intensity of certain bases occurred, particularly at positions 55, 56, 62, 63 and 92 on the top strand. It would seem likely that this is due to hydrophobic regions of BlaI causing a funnelling effect for DMS resulting in increased methylation of bases at these positions. A similar phenomenon caused by LacI has been reported.28
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The reason for the presence of two dyads: the Z-dyad nearest to blaZ and the RI-dyad nearest to blaRI (Figure 2), is not obvious. The RI-dyad includes elements of the promoters for both blaZ and blaRI and is presumably essential for expression of this operon. It was therefore decided to investigate the role of the Z-dyad by deleting it.
Assay for CAT in the absence of BlaI
To measure the strength of the wild-type promoter compared with that with the Z-dyad deleted, S. aureus RN4220 (pSRC922) and (pSRC522) were grown to mid-log phase (A680 of c. 0.5) at 30°C, harvested, lysed with toluene and the amount of CAT assayed. The results (Table II) indicate that the removal of the Z-dyad results in a significant decrease in the amount of CAT produced.
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S. aureus RN4220 (pSRC923) and (pSRC523) were grown to mid-log phase (A680 c. 0.5) at 30°C. CBAP (5 mg/L) was added to half the cultures and samples taken every minute for 10 min. The samples were lysed with toluene and the amount of CAT present assayed. The production of CAT is induced by CBAP (Figure 9). There was a similar lag of about 4 min in both induced cultures before CAT was detected and more CAT was produced when the Z-dyad was present (i.e. with pSRC923).
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Discussion |
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The lack of any evidence for a change in the geometry of inter-dyad DNA as measured by in vitro copperphenanthroline footprinting and in vivo DMS footprinting, suggests that no significant bending or looping of the bla operator DNA occurs on binding of repressor. This, coupled with the observations of Gregory et al.,5 strongly suggests that BlaI, presumably in the form of dimers, binds to each inverted repeat independently. These data are especially important in light of the suggestion that MecI forms protein multimers when bound to a large mec operator molecule opening the possibility of proteinprotein interactions between repressor molecules bound to the two separate dyads.7 Both the copperphenanthroline and the DMS experiments produced negative results so that the conclusion that there is an absence of bending or looping must be tentative.
Previous studies5,7 have relied upon electrophoretic mobility shift assays of repressorDNA complexes to study binding. The use of this experimental technique has not proved entirely reliable for studying proteinDNA interactions; indeed the simultaneous binding of LacI to two operator regions (O1 and O3) in the Lac operon of E. coli, was only detected in vivo on negatively supercoiled DNA molecules.24,28 Hence it seemed possible, especially given the very high apparent dissociation constant for the purified form of BlaI, that it may bind differently in vivo with a different ionic environment and a supercoiled DNA molecule. The results obtained with the in vivo system (Figure 7, lanes 5 and 6) are not significantly different from those obtained with the in vitro system (Figure 8
, lanes 5 and 6). Thus it is concluded that the in vitro system reflects the situation found within the bacteria. On the bottom strand the guanine at position 54 (Figure 7
, lanes 1 and 2) is not protected by the bound repressor although it is in the middle of the Z-dyad. This is also seen with the equivalent in vitro experiment (data not shown) so is not a specific feature of the in vivo position. One possibility is that a monomer binds to each half of the Z-dyad in such a way as to allow space for the methylating agent to attack the guanine.
The precise site for the initiation of blaZ mRNA has been determined by primer extension analysis (S. R. Clarke & K. G. H. Dyke, unpublished) and is indicated in Figure 2. This allowed the removal of the Z-dyad from operator DNA without loss of the wild-type transcription start site. The Z-dyad plus a further 27 bp was deleted and the resulting DNA was fused to the cat reporter gene precisely as for the construct including the wild-type operator/ promoter region. The deletion resulted in decreased synthesis of CAT (Table II
). This could be explained by lower mRNA synthesis, greater instability of the mRNA or by poorer translation efficiency. One possibility is that the stemloop structure that may be present when the Z-dyad is transcribed stabilizes the mRNA. In the absence of the stemloop, RNase E or some similar RNase would hydrolyse the mRNA. It is known that the half-life of mRNAs can be decreased by the removal of 5' stemloop structures at the 5' untranslated region of transcripts.29,3032 It is also possible that the presence of a stemloop structure in the mRNA near to the ribosome binding site could increase the efficiency of initiation of translation.
The presence of the genes specifying BlaI and BlaRI on the same plasmid resulted in repression of the synthesis of CAT. This repression was relieved about 4 min after addition of an inducer (Figure 9) and the proportion of the cell protein that was CAT was about three times higher in the presence of the Z-dyad plus an additional 27 bp untranslated section. This agrees with the results obtained in the absence of BlaI (Table II
) and suggests that the system is fully induced. Further experiments are necessary to determine whether or not it is the Z-dyad that has this effect rather than the downstream 27 bp and to discover, for example, whether or not BlaI can bind to the upstream section of the blaZ mRNA.
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Acknowledgments |
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Notes |
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Present address. Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
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References |
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Received 2 August 2000; returned 16 October 2000; revised 8 November 2000; accepted 27 November 2000