MecI represses synthesis from the ß-lactamase operon of Staphylococcus aureus

Richard A. Lewis{dagger} and Keith G. H. Dyke*

University of Oxford, Microbiology Unit, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmid diploids were constructed in Staphylococcus aureus to study the effect of the repressor of methicillin resistance (MecI) on the synthesis of both ß-lactamase and the ß-lactamase repressor (BlaI). MecI-mediated repression of the synthesis of ß-lactamase was shown by reduction in the specific activity of nitrocefinase in bacteria containing a plasmid carrying mecI but not when containing the same plasmid deleted for mecI. Antisera prepared against purified MecI and against purified BlaI were used in Western blots which showed that MecI repressed the synthesis of BlaI in these diploids. The interactions between the mec operon and the bla operon are discussed.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Staphylococcus aureus is an important human pathogen which is often resistant to a wide range of antibiotics. Resistance to ß-lactams is primarily due to the production of a ß-lactamase which inactivates the antibiotic.1 The production of ß-lactamase in S. aureus is specified by blaZ and was believed to be regulated by a repressor.2 Subsequently this was shown to include a signal transduction system specified by blaR1, blaI and also possibly by blaR2.3 blaZ, blaR1 and blaI are located together on the transposon Tn552 which has been completely sequenced.4 It has been shown that blaI directs the synthesis of a protein that binds specifically to two regions of dyad symmetry located between the divergently transcribed blaZ and blaR1 genes.5 Binding of a ß-lactam to the signal transducer BlaR1 is presumed to alter its structure in such a way that a signal is transmitted across the membrane to the cytoplasm. This signal finally results in the removal of the BlaI repressor from the region between blaZ and blaR1 so allowing transcription of blaZ and ultimately synthesis of ß-lactamase.

A second ß-lactam resistance mechanism, usually called methicillin resistance, has been found in S. aureus6 and is due to the production of a penicillin-binding protein 2a (PBP2a, also known as PBP2').7 The gene mecA, which specifies this protein has been sequenced8 and the sequence of the region upstream of this gene is homologous to that upstream of blaZ. It contains a region of dyad symmetry and sequences that specify the proteins MecI and MecR1 which are similar to BlaI and BlaR1.9 It is known that methicillin resistance can be controlled by the blaI/blaR1 regulatory system.10,11 Thus, BlaI represses the synthesis of both PBP2' and ß-lactamase in vivo. BlaI has also been shown in vitro to bind specifically to the dyad upstream of mecA.5

Some methicillin-resistant S. aureus (MRSA) (called pre-MRSA) carry mecA but are sensitive to methicillin.12 These strains are not inducible for the production of PBP2' even when the bla genes are removed.13 The explanation is that MecI is a powerful repressor of the transcription of mecA and that the repression is not removed by the addition of an inducer such as methicillin.14,15 There are no data published that allow an estimate of the prevalence of pre-MRSA strains in nature and so it is possible that they are more common than MRSA strains. It was therefore of interest to determine whether MecI represses synthesis from the ß-lactamase operon. A pre-MRSA strain develops into an MRSA strain when mutations, including deletions, of mecI or the site to which MecI binds occur.12,16 The mecI-inactivated strain then becomes inducible for methicillin resistance in the presence of the bla regulatory system. The regulation of methicillin resistance has been reviewed.12,17,18 In this paper we report the results of experiments designed to investigate whether MecI is a repressor of the bla operon.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General microbiology

S. aureus was grown in casein hydrolysate/yeast extract (CY) medium or on CY agar19 without the addition of glucose or ß-glycerophosphate. Escherichia coli was grown in Luria– Bertani (LB) medium or on LB agar.20 For S. aureus the media contained, as required, 20 mg/L erythromycin and/or 5 mg/L chloramphenicol. 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 of Gotz et al.,21 and E. coli was transformed by the method of Hanahan.22

Recombinant DNA technology

All materials and methods were as described in Sambrook et al.,20 Wizard Plus SV Magic Miniprep kit (Promega, Madison, WI, USA) was used to prepare DNA for cloning. T4 DNA ligase was from Life Technologies Ltd (Paisley, UK) and all restriction enzymes were from New England Biolabs (Hitchin, UK) or Boehringer–Mannheim (Lewes, UK). Oligonucleotides: 5'-TATTAGTACTTAATGACCCGGGCC and 5'-AATAGGCCCGGGTCATTAAGTACT used to generate the DNA linker: were from Valerie Cooper, Oligonucleotide Synthesis Service, Dyson Perrins Laboratory, Oxford, UK. Agarose was from FMC BioProducts (Rockland, ME, USA) and the acrylamide used in protein separation was UltraPure Protogel (National Diagnostics, Atlanta, GA, USA).

Antiserum production

BlaI antiserum was raised in New Zealand White rabbits by Serotec Ltd (Kidlington, UK). MecI antiserum was raised in rabbits by Harlan Sera Lab Ltd (Loughborough, UK).

Preparation of S. aureus lysates and Western blotting

All materials and techniques were as described previously.5

Nitrocefinase assay

The sample of bacterial culture was added to 0.3 mL of 5 mM 5-hydroxyquinolone and stored on ice. One mg of nitrocefin (Oxoid, Unipath Ltd, Basingstoke, UK) was dissolved in 0.1 mL of dimethylsulphoxide (DMSO; Sigma– Aldrich Company Ltd, Poole, UK) and then added to 1.8 mL of 0.1 M potassium phosphate buffer pH 7.0 and diluted 1 to 10 in the same buffer. The solution was warmed to 37°C, the sample (40 µL) was added and the A482 monitored for 15 min. The cuvette containing the sample was kept at 37°C between readings.

ß-Lactamase starch test assay

Five millilitre aliquots of the cultures used in the Western blots were added to CY agar plates containing 1% starch, and 5% NaCl. The plates were incubated for 24 h at 30°C and then flooded with a mixture of 1 g of penicillin G dissolved in 10 mL of 0.05 M sodium phosphate buffer pH 7.0 and 5 mL of 20% KI in 4% iodine.23 The liquid was poured away, the plates left for 10 min and then photographed.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construction of plasmids and diploid strains

In order to investigate the in vivo effects of MecI on the production of ß-lactamase and BlaI it was first necessary to construct the relevant plasmids which could be used in diploid generation in S. aureus RN4220. For the bla plasmids the starting plasmid was pOX457 (Figure 1Go). This plasmid is based on pHSG39824 into which has been inserted the blaI, blaRI and blaZ genes from Tn5524 and the plasmid pE194.25 pOX457 was cut at the BbsI site which lies within blaI, and a BbsI compatible linker, containing a ScaI and a SmaI site, together with two in-frame nonsense codons, was ligated into the cut pOX457 to give pRAL511 (Figure 1Go). This plasmid is potentially able to direct the synthesis of only the 77 N-terminal amino acids of BlaI which is not expected to be a functional repressor. pRAL511 was digested with ScaI, the digestion products ligated and a recombinant plasmid containing a deletion of the ScaI fragment identified. The resultant plasmid pRAL512 carries a truncated version of blaI and is sensitive to chloramphenicol in E. coli (Figure 1Go). There will be read-through past the ScaI site to give a protein consisting of the 77 N-terminal amino acids of BlaI, two amino acids specified by the DNA of the linker and five amino acids specified by DNA of the pHSG398 portion of pRAL512. The basic shuttle vector (pRAL521), which was to act as a negative control, was constructed by ligation of PstI-cut pE194 into similarly cut pHSG398 (Figure 1Go). The plasmids pOX457, pRAL511, pRAL512 and pRAL521 were all separately transformed into S. aureus RN4220 with selection for resistance to erythromycin.



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Figure 1. Restriction enzyme maps of the plasmid constructs. Only relevant restriction enzyme sites and genes are shown. erm, erythromycin resistance gene; rep, gene for Rep protein; cat, gene for chloramphenicol transacetylase; ori, origin of replication; binL, gene for resolvase. bla genes are described in the text.

 
For mecI, plasmids were based on pRIT526 which is compatible with pE194-based plasmids and so can be used to construct S. aureus merodiploids. It carries an S. aureus cat gene and an amp gene which expresses ß-lactamase in E. coli but not in S. aureus. In addition, a truncated staphylococcal protein A gene is present with its own promoter (spa) and its own signal peptide sequence. The plasmid pRI14 consists of pRIT5 with a HindIII fragment inserted so that the truncated mecR1 gene and intact mecI are expressed from the spa promoter. pRI{triangleup}I was constructed by cutting pRI with PstI and religating to delete the PstI fragment.14 This removes all except 33 bases of mecI and was used as a mecI-deficient control. The plasmids pRIT5, pRI and pRI{triangleup}I were transformed into S. aureus RN4220 with selection for chloramphenicol resistance.

Diploid strains were constructed by transforming RN4220(pRIT5), RN4220(pRI) and RN4220(pRI{triangleup}I) separately with pOX457, pRAL521, pRAL511 and pRAL512 with selection for resistance to erythromycin. The copy numbers of the two plasmids were approximately equal in all diploid strains.

Determination of nitrocefinase activity

Nitrocefinase assay showed that S. aureus RN4220 (pRIT5), (pRI), (pRI{triangleup}I) and (pRAL521) produced no detectable enzyme confirming that the amp gene present on the pRIT5-based plasmids is not expressed in S. aureus. In the absence of a second plasmid, pRAL512 directed the constitutive synthesis of nitrocefinase since it possesses an intact blaZ and a partially deleted blaI. The presence of pRI, which codes for a functional repressor (MecI), repressed the production of nitrocefinase from blaZ to about 35% (TableGo). Neither pRI{triangleup}I nor pRIT5 caused repression of nitrocefinase production.


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Table. Synthesis of nitrocefinase by S. aureus RN4220 (pRAL512) and its diploids
 
Production of BlaI and MecI in the diploid strains

Polyclonal antisera prepared against purified BlaI and MecI were used in a series of Western blotting experiments to determine whether or not MecI represses the synthesis of BlaI. Figure 2Go(a) shows a Western blot of lysates prepared from RN4220 (pRAL521) and its diploids. The MecI antiserum detected the MecI produced from pRI (Figure 2aGo, lanes 3 and 4). None of the cultures shown in Figure 2Go(a) produced BlaI (data not shown).



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Figure 2. Western blots of lysates of induced and uninduced bacteria electrophoresed in SDS–polyacrylamide and treated with MecI antiserum (Figure 2a, b and dGo) or with BlaI antiserum (Figure 2c and eGo). (a) All lysates are of RN4220 (pRAL521) with, in addition: lanes 1 and 2, no second plasmid; lanes 3 and 4, pRI; lanes 5 and 6, pRI{triangleup}I; lanes 7 and 8, pRIT5. Lysates in lanes 2, 4, 6 and 8 were from induced bacteria. (b) and (c) All lysates are of RN4220(pRAL512) with, in addition: lanes 1 and 2, no plasmid; lanes 3 and 4, pRI; lanes 5 and 6, pRI{triangleup}I; lanes 7 and 8, pRIT5. Lysates in lanes 2, 4, 6 and 8 were from induced bacteria. (d) and (e) Lysates are: lanes 1 and 2, RN4220 (pOX457); lanes 3 and 4, RN4220 (pOX457/pRI); lanes 5 and 6, RN4220 (pRAL511); lanes 7 and 8, RN4220 (pRAL511/pRI); lanes 9 and 10, RN4220 (pRI). Lysates in lanes 2, 4, 6, 8 and 10 were from induced bacteria.

 
Western blots of the strains involving pRAL512 are shown in Figure 2Go(b and c). The results obtained with the MecI antiserum are very similar to those shown in Figure 2Go(a). The results obtained using BlaI antiserum illustrate that, in the diploid RN4220 (pRAL512/pRI), no BlaI was detected either in the presence or absence of inducer (Figure 2cGo, lanes 3 and 4). Similar results (Figure 2d and eGo) were obtained for RN4220 (pRAL511/pRI) where synthesis of the truncated version of BlaI is largely repressed, although the results for RN4220 (pOX457/pRI) indicated that repression was not as complete in this case as for the blaI diploid strains.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In pre-MRSA strains, the presence of the mecR1/mecI system results in repression of PBP2' synthesis. PBP2' production is not induced by methicillin whether or not the strains also contain the blaR1/blaI regulatory genes.14 In other MRSA strains with functional mecR1/mecI genes the induction of PBP2' is slow with full induction of methicillin resistance taking up to 48 h.27,28 It is concluded that MecI is a powerful repressor of expression of mecA. If the blaI/blaR1 system is present in MRSA the synthesis of PBP2' is readily and rapidly inducible by CBAP.11 It is not known if MecI is a repressor of the bla operon and it is this question that the experiments reported in this paper were designed to answer.

It was desirable to be able to measure the amount of ß-lactamase, BlaI, BlaR1 and MecI present in cells. Antiserum raised to purified BlaI was available and antisera were prepared against MecI and a portion of BlaR1. Unfortunately the anti-BlaR1 serum proved to be insufficiently sensitive to be used in Western blots and so studies were limited to measurement of the in vivo effects of MecI on synthesis of ß-lactamase, as determined by nitrocefinase assay, and on synthesis of BlaI detected by Western blots. The plasmid pRAL512 carries intact copies of blaZ and blaR1 and a truncated version of blaI predicted to direct the synthesis of a protein consisting of 84 amino acids largely corresponding to the N terminus of BlaI. Expression of these genes resulted in constitutive production of nitrocefinase, a result that confirmed that the truncated BlaI is an ineffective repressor of blaZ. The fact that BlaI itself is not overexpressed in RN4220 (pRAL512) suggests that either BlaI is not autoregulatory or an additional, as yet uncharacterized, regulatory mechanism is involved in control of BlaI synthesis. When present in the same bacterium as pRI, pRAL512 specified the synthesis of very little ß-lactamase, presumably because MecI bound the blaZ dyad sequence in the bla operator region and so prevented transcription of blaZ. ß-Lactamase synthesis was not induced by CBAP probably because synthesis of functional MecR1 is not specified by the truncated version of the gene carried by pRI and although pRAL512 carries an intact copy of blaR1 it is likely that only a very small quantity, if any, of BlaR1 is produced in the pRAL512/pRI diploid (see below). It is also likely that the amount of MecI present in these strains is somewhat larger than that produced in pre-MRSA and the repressive effect is thus enhanced. It is concluded that MecI is an effective repressor of blaZ.

Antisera raised to MecI and BlaI are specific and do not cross-react on Western blots, so allowing the two repressors to be distinguished. This and other data (unpublished) relating to the structure/function of the repressors suggest that BlaI and MecI are not as similar as their sequences suggest. It is clear from the pRAL512/pRI Western blots that MecI repressed the synthesis of the truncated BlaI (Figure 2b and cGo). This was also demonstrated by the pRAL511/pRI Western blots. Presumably MecI also repressed the synthesis of BlaR1, whose gene is co-transcribed with that of BlaI. The repression was not as complete for pOX457/pRI (Figure 2eGo). It may be that interactions between BlaI and MecI, possibly resulting in inactive heterodimer formation, are responsible for reduced repression of blaI in this strain. Addition of CBAP failed to lift MecI-conferred repression of blaI presumably for the same reasons given above concerning blaZ repression.

Thus both BlaI and MecI are implicated in the regulation of synthesis of ß-lactamase, BlaR1, BlaI, PBP2', MecR1 and MecI and it is probable that Song et al.,8 were correct in their view that the two systems are evolutionarily related and are controlled in a coordinate manner.


    Acknowledgments
 
We thank Mr S.P. Curnock for technical assistance, and Professor K. Hiramatsu and Dr S. Farmer for gifts of pRI and pRIT5, respectively. We are grateful to the BBSRC for an award to RAL and to the Edward Penley Abraham Fund for general support.


    Notes
 
{dagger} Present address. School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

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* Corresponding author. Tel: +44-1865-275293; Fax: +44-1865-275297; E-mail: kdyke{at}bioch.ox.ac.uk

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    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 21 April 1999; returned 11 August 1999; revised 15 September 1999; accepted 28 September 1999