Microbiology Unit, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK1
Author for correspondence: Keith Dyke. Tel: +44 1865 275293. Fax: +44 1865 275297. e-mail: kdyke{at}bioch.ox.ac.uk
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ABSTRACT |
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Keywords: penicillin resistance, promoter strength, transcription
Abbreviations: CAT, chloramphenicol acetyltransferase; CBAP, 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid; MRSA, methicillin-resistant Staphylococcus aureus
a Present address: Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
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INTRODUCTION |
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The region responsible for synthesis of ß-lactamase has been sequenced (Rowland & Dyke, 1989 , 1990
) and shown to consist of blaZ (the gene coding the ß-lactamase), blaRI (the gene encoding a putative transmembrane signal transducer) and blaI (the gene encoding the repressor). The genes blaRI/blaI are believed to be transcribed as a single mRNA in the opposite direction to the transcription of blaZ (Gregory et al., 1997
). This is very similar to the arrangement found in the pen region of Bacillus licheniformis (Kobayashi et al., 1987
) except that the order of transcription of the homologues of blaRI (penJ) and blaI (penI) is reversed, with penI nearest to the promoter in B. licheniformis. This difference in the order of the genes is perhaps the reason for the difference in the kinetics of induction. In S. aureus the ß-lactamase is fully induced in just a few minutes whereas in B. licheniformis full induction does not occur until about 90 min (Collins, 1979
). Transcription of penI and penJ has been studied, and in the first 30 min of induction 2·3 kb transcripts encoding penI and penJ are produced but these are then replaced by shorter transcripts of 0·75 and 0·95 kb that encode penI only (Salerno & Lampen, 1988
). They suggested that PenJ was required early in the induction process. It is not known if different sized mRNAs encoding BlaI are produced on induction of ß-lactamase in S. aureus. The induction of ß-lactamases in bacteria has been reviewed by Bennett & Chopra (1993)
.
The mechanism of induction of the ß-lactamase in S. aureus is beginning to be understood and is based on proteolytic cleavage of the repressor, BlaI, that is dependent on the presence of BlaRI (Gregory et al., 1997 ; Lewis et al., 1999
). The hypothesis is that a ß-lactam binds to the extracellular part of BlaRI and causes a conformational change that activates the intracellular proteolytic activity of BlaRI. The repressor, BlaI, is cleaved by the BlaRI protease so that it no longer binds to the bla operator, thus allowing synthesis of blaZ mRNA and hence ß-lactamase. It is not obvious that there has to be continuous synthesis of BlaI and BlaRI during induction and there is evidence from Western blotting that BlaI is not significantly induced (Lewis et al., 1999
).
The mechanism of induction may also apply to the synthesis of PBP2a from mecA of MRSA. In some MRSA strains the synthesis of PBP2a is inducible and controlled by two genes: mecRI and mecI, which are homologous to blaRI and blaI (Hiramatsu et al., 1992 ). Indeed it has been shown that BlaI can repress the synthesis of MecA (Hackbarth & Chambers, 1993
) and that MecI can repress synthesis of ß-lactamase (Lewis & Dyke, 2000
). Thus any unravelling of the mechanism of induction of ß-lactamase will probably also apply to the mechanism in at least some MRSA strains. In other MRSA strains the blaRI or blaI genes carry inactivating mutations but despite this, in the presence of the bla operon, the synthesis from mecA is inducible. There is an urgent need for new therapies to control infections by MRSA and it is possible that better knowledge of the mecA induction system could eventually lead to the design of alternative therapies.
In this paper the results of experiments designed to discover, in particular, whether or not the synthesis of BlaRI and BlaI is inducible and to find out if they share the same promoter are reported. The techniques used include Northern blotting and the use of the cat gene as a reporter of synthesis from the promoters. The promoters for both blaZ and blaRI/blaI were found to be inducible and evidence is presented to show that the blaZ promoter is about six times stronger than that for blaRI/blaZ.
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METHODS |
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Preparation of RNA.
S. aureus was grown to an OD680 of 0·6 and, if required, induced for various times by continuing growth in the presence of 5 µg 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) ml-1. RNA was extracted from the harvested bacteria by the method of Hart et al. (1993) except that the protoplasts were lysed with 5 µl SDS (2%, w/v) and 50 µl proteinase K (5 mg ml-1). The pellet of RNA was resuspended in 100 µl nuclease-free water. All plastic and glassware used for the preparation and storage of RNA was treated with RNase ZAP (Sigma), rinsed with 0·1% (v/v) diethyl pyrocarbonate and autoclaved at 121 °C, 103·5 kPa for 20 min.
Labelling of oligonucleotides and probes.
The methods described by Sambrook et al. (1989) were used for end labelling oligonucleotides with 32P by the T4 polynucleotide kinase forward reaction and for analysis of RNA by primer extension. The Random Primers DNA Labelling System (Life Technologies) was used to label the probes for Northern blotting.
Northern blotting.
RNA samples were heated at 65 °C for 5 min, cooled on ice and then loaded onto a 1·5% (w/v) agarose-MOPS [20 mM MOPS, 5 mM sodium acetate, 0·5 mM EDTA] denaturing gel containing 6·5% (w/v) formaldehyde, and electrophoresed at 80 V. After electrophoresis, the RNA was transferred to a positively charged nylon membrane by capillary blotting in 10x SSC. The RNA was fixed to the membrane by UV crosslinking. The membrane was pre-hybridized at 42 °C for 2 h in 20 ml pre-warmed hybridization buffer [50% (w/v) deionized formamide, 5x SSC, 2% (w/v) blocking agent (Boehringer Mannheim), 0·1% (w/v) N-laurylsarcosine, 0·02% (w/v) SDS, 0·1 mg boiled calf thymus DNA ml-1].
Hybridization with heat-denatured radiolabelled probe was for 18 h in 2 ml hybridization buffer. The membrane was washed twice at room temperature in 100 ml membrane wash (2x SSC; 0·1%, w/v, SDS) for 5 and 15 min consecutively. Bands were visualized by autoradiography.
DNA sequencing.
Sequenase version 2.0 DNA sequencing kit (USB) was used for manual DNA sequencing. The M13Forward primer (Table 2) was used to produce a sequence to locate the transcription start sites for blaZ and blaRI/blaI.
Construction of cat gene reporter plasmids.
Plasmids in which the cat gene was driven by either the blaZ or blaRI/blaI promoter were constructed to allow comparison of both the strengths of the promoters and their responses to induction. Plasmid pOX690 has a cat gene driven by the blaRI/blaI promoter (Gregory et al., 1997 ) but does not have the blaRI and blaI genes that are essential to study the induction from the blaRI/blaI promoter. To provide BlaRI and BlaI, pOX491 (Fig. 1
) was digested with AvaII/AccI, the ends filled in with the Klenow polymerase reaction, religated and used to transform E. coli JM109 with selection for ampicillin resistance. The resulting plasmid (pSRC900) was then digested with PstI and the 3·6 kb piece containing the three bla genes was ligated into PstI-digested pOX690 and used to transform E. coli JM109 with selection for ApR to give pSRC910. A similar cloning strategy, but starting with pOX617 (Gregory et al., 1997
) in which the bla operator had been cloned into the pUC18 SmaI site, was followed to produce a plasmid which contains the three bla genes and in which the cat gene is driven by the blaZ promoter. First, both pOX617 and pRB394 (Bruckner, 1992
) were cut with EcoRI/PstI, ligated and used to transform E. coli to chloramphenicol resistance. A clone that had the operator region ligated into pRB394 so that the blaZ promoter drives the cat gene was chosen and the plasmid called pSRC920. DNA of pSRC920 was cut with PstI and ligated to the 3·6 kb PstI fragment from pSRC900. The resulting plasmid is pSRC921 and contains a complete bla operon in addition to the blaZ promoter that drives the cat gene. Restriction enzyme maps of pSRC910 and pSRC921 are shown in Fig. 2
. So that the plasmids can replicate in S. aureus, both pSRC910 and pSRC921 were digested with BglII and separately ligated to BamHI-digested pT181MCS (Augustin et al., 1992
). The ligation mixtures were used to transform E. coli JM109 with selection for ampicillin resistance and the resulting plasmids were then used to transform S. aureus RN4220 to tetracycline resistance. A plasmid from each transformation which had pT181MCS inserted in the same orientation was chosen and designated pSRC910::pT181MCS and pSRC921::pT181MCS respectively.
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RESULTS AND DISCUSSION |
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The results for blaZ (Fig. 3) show that the start site for mRNA synthesis is the same for PS80d (lane 1), NCTC9789(pI9789) (lane 2) and 3796(pI3796) (lane 3), and is at a thymidine as shown in Fig. 4
. This thymidine is one base away from the site identified for blaZ mRNA by an in vitro transcription system using E. coli RNA polymerase (McLaughlin et al., 1981
). The -10 sequence for the blaZ promoter is likely to be TATTAT which is precisely in the expected position and the -35 sequence is probably TTGACA which is separated from the -10 sequence by 18 bp. The untranslated part of this mRNA contains the Z dyad which is one of the sites on the DNA to which BlaI is known to bind (Gregory et al., 1997
). The second dyad (RI dyad) to which BlaI binds lies between the -35 sequence and the -10 sequence, and partly overlaps the latter (Fig. 4
). These data fit with the hypothesis that binding of the repressor BlaI at this dyad will prevent the binding of RNA polymerase and with the experimental observation that deletion of the Z dyad increases transcription from the blaZ promoter (S. R. Clarke, unpublished results). PS80d does not produce ß-lactamase but it must possess at least the first section of the blaZ gene including the promoter. It therefore seems likely that there is a mutation that prevents the synthesis of active ß-lactamase. The data for 3796(pI3796) are the same as for NCTC9789(pI9789) and the pI3796 DNA used here was found not to have the deletion at the beginning of the blaZ gene. The reason for the discrepancy with the results of East & Dyke (1989)
is unknown since the earlier reported sequence was not found with the cultures now available.
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Identification of mRNA for BlaRI and BlaI by Northern blotting
The probe for blaRI mRNA was made by PCR with the primers 5106SRC (51065087 on the Tn552 sequence) and 3720SRC (37043720 on the Tn552 sequence), and with pOX491 as the template, which results in a 1403 bp probe entirely from within the blaRI sequence. The RNA was prepared from S. aureus RN4220(pOX491) which contains an intact bla operon (Fig. 1) and from S. aureus RN4220(pOX492) which was derived from pOX491 by cutting with SnaBI, ligating and then transforming into E. coli JM109 with selection for chloramphenicol resistance. The plasmid was then transformed into S. aureus RN4220 with selection for erythromycin resistance. pOX492 lacks the end of the blaRI gene and the part of Tn552 that encodes blaI, binL and (P480). The results of the Northern blot (Fig. 5
) show that only a small amount of message for BlaRI is produced by RN4220(pOX491) unless the bacteria are induced and that the message is about 2100 nt long. The mRNA from pOX492 is about 1700 nt, which is about the predicted size provided that there is a suitable termination signal shortly after the SnaBI site in pOX492. Since blaI has been deleted it is to be expected that similar amounts of mRNA will be produced with and without inducer for pOX492 and this is seen (Fig. 5
). Since no other bands were seen, the Northern blot provides evidence that a single mRNA is produced from the blaRI/blaI promoter and that it is inducible.
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Reporter gene assays
S. aureus RN4220(pSRC910::pT181MCS) and RN4220(pSRC921::pT181MCS) were grown at 30 °C for 18 h in CY medium containing 5 µg tetracycline ml-1, diluted 1:100 in the same medium and growth continued at 30 °C to an OD680 of 0·5. The gratuitous inducer CBAP (Leggate & Holms, 1968 ) was added to a final concentration of 5 µg ml-1 and samples taken every 2 min for the assay of CAT activity. The results for the expression of CAT in the absence of inducer (results not shown) are the same as those for the zero time points for addition of inducer (Table 3
) so that it is concluded that, in the absence of inducer, the blaZ promoter is about six times more active than the blaRI/blaI promoter. There is rapid induction of CAT in S. aureus from both promoters (Table 3
) and it seems that a balance between synthesis and degradation of CAT is attained after about 8 min induction. The reporter system used may not accurately mimic the wild-type position; for example, there are two copies of the operator region in the bacterium but only one set of active blaRI/blaI genes. However, it provides excellent evidence that synthesis from the blaRI/blaI promoter is inducible and supports the Northern blot results. A further problem could be associated with variation of copy numbers of the plasmids. In both pSRC910 and pSRC921 the copy number is controlled by the pT181MCS plasmid and the plasmids are of similar size. Care was taken to harvest the bacteria at the same density (OD680 0·6) to avoid differences in copy number at different growth phases. Thus comparison of the strengths of the two promoters is taken as representative of the state in the wild-type. However, the activity of the blaZ promoter in the absence of inducer is somewhat higher than was predicted.
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It is probable that the results that apply to the regulation of blaZ will also apply to the regulation of mecA in cases where mecRI and mecI are not intact. Because of the extensive homology between the proteins it also seems likely that an analogous system will apply when the synthesis of mecA is regulated by MecRI and MecI. In the latter case, chemicals that interfered with the binding of the inducer to MecRI would inhibit the synthesis of MecA and hence inhibit the expression of methicillin resistance by the bacterium.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Augustin, J., Rosenstein, R., Weiland, B., Schneider, U., Schnell, N., Engelke, G., Entian, K. & Gotz, F. (1992). Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants. Eur J Biochem 204, 1149-1154.[Abstract]
Bennett, P. M. & Chopra, I. (1993). Molecular basis of ß-lactamase induction in bacteria. Antimicrob Agents Chemother 37, 153-158.[Medline]
Bruckner, R. (1992). A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene 122, 187-192.[Medline]
Bruns, O., Bruns, W. & Pulverer, G. (1997). Regulation of ß-lactamase synthesis as a novel site of action for suppression of methicillin resistance in Staphylococcus aureus. Zentbl Bakteriol 285, 413-430.
Collins, J. F. (1979). ß-Lactamases of Staphylococcus aureus. In ß-Lactamases , pp. 351-368. Edited by J. M. Hamilton-Miller & J. T. Smith. London: Academic Press.
Dyke, K. G. H. & Gregory, P. D. (1997). Resistance to ß-lactam antibiotics: resistance mediated by ß-lactamase. In The Staphylococci in Human Disease , pp. 136-157. Edited by K. B. Crossley & G. L. Archer. Edinburgh: Churchill Livingstone.
East, A. K. & Dyke, K. G. H. (1989). Cloning and sequence determination of six Staphylococcus aureus ß-lactamases and their expression in Escherichia coli and S. aureus. J Gen Microbiol 135, 1001-1015.[Medline]
Gotz, F., Ahrne, S. & Lindberg, M. (1981). Plasmid transfer and genetic recombination by protoplast fusion in staphylococci. J Bacteriol 145, 74-81.[Medline]
Gregory, P. D., Lewis, R. A., Curnock, S. P. & Dyke, K. G. H. (1997). Studies of the repressor (BlaI) of ß-lactamase synthesis in Staphylococcus aureus. Mol Microbiol 24, 1025-1037.[Medline]
Hackbarth, C. J. & Chambers, H. F. (1993). blaI and blaRI regulate ß-lactamase and PBP2' production in methicillin resistant Staphylococcus aureus. Antimicrob Agents Chemother 37, 1144-1149.[Abstract]
Hackbarth, C. J., Miick, T. C. & Chambers, H. F. (1994). Altered production of penicillin-binding protein 2a can affect phenotypic expression of methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother 38, 2568-2571.[Abstract]
Hanahan, D. (1983). Studies on the transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[Medline]
Hart, M. E., Smeltzer, M. S. & Iandolo, J. J. (1993). The extracellular protein regulator (xpr) affects exoprotein and agr mRNA levels in Staphylococcus aureus. J Bacteriol 175, 7875-7879.[Abstract]
Hiramatsu, K., Asada, K., Suzuki, E., Okonogi, K. & Yokota, T. (1992). Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). FEBS Lett 298, 133-136.[Medline]
Horinouchi, S. & Weisblum, B. (1982). Sequence of a small staphylococcal plasmid, pE194. J Bacteriol 150, 804-814.[Medline]
Kobayashi, T., Zhu, Y. F., Nicholls, N. & Lampen, J. O. (1987). A second regulatory gene, blaRI, encoding a potential penicillin-binding protein required for induction of ß-lactamase in Bacillus licheniformis. J Bacteriol 169, 3873-3878.[Medline]
Kreiswirth, B. H., Lofdahl, S., Betley, M. J., OReilly, M., Schleivert, P. M., Bergdoll, M. S. & Novick, R. P. (1983). The toxic shock syndrome exotoxin is not detectably transmitted by a prophage. Nature 305, 709-712.[Medline]
Leggate, J. & Holms, W. H. (1968). Gratuitous synthesis of ß-lactamase in Staphylococcus aureus. J Bacteriol 96, 2110-2117.[Medline]
Lewis, R. A. & Dyke, K. G. H. (2000). MecI represses synthesis from the ß-lactamase operon of Staphylococcus aureus. J Antimicrob Chemother 45, 139-144.
Lewis, R. A., Curnock, S. C. & Dyke, K. G. H. (1999). Proteolytic cleavage of the repressor (BlaI) of ß-lactamase synthesis in Staphylococcus aureus. FEMS Microbiol Lett 178, 271-275.[Medline]
McLaughlin, J. R., Murray, C. L. & Rabinowitz, J. C. (1981). Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus ß-lactamase gene. J Biol Chem 56, 11283-11291.
Novick, R. P. (1963). Analysis by transduction of mutants affecting penicillinase formation in Staphylococcus aureus. J Gen Microbiol 33, 121-136.
Rowland, S.-J. & Dyke, K. G. H. (1989). Characterization of the staphylococcal ß-lactamase transposon Tn552. EMBO J 8, 2761-2773.[Abstract]
Rowland, S.-J. & Dyke, K. G. (1990). Tn552, a novel transposable element from Staphylococcus aureus. Mol Microbiol 4, 961-975.[Medline]
Salerno, A. J. & Lampen, J. O. (1988). Differential transcription of the bla regulatory region during induction of ß-lactamase in Bacillus licheniformis. FEBS Lett 227, 61-65.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sohail, M. & Dyke, K. G. H. (1996). Suppression of the thermosensitive replication phenotype of the derivative plasmid of pI9789::Tn552 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome. FEMS Microbiol Lett 136, 129-136.[Medline]
Tomizawa, J.-I. (1985). Control of ColE1 plasmid replication: initial interaction of RNA I and the primer transcript is reversible. Cell 40, 527-535.[Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]
Received 30 August 2000;
revised 17 October 2000;
accepted 5 December 2000.