Plasmid-borne macrolide resistance in Micrococcus luteusa

Wolfgang Liebl1, Wesley E. Kloos2 and Wolfgang Ludwig3

Institut für Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstr. 8, D-37077 Göttingen, Germany1
Department of Genetics, North Carolina State University, Raleigh, USA2
Lehrstuhl für Mikrobiologie, Technische Universität München, Freising-Weihenstephan, Germany3

Author for correspondence: Wolfgang Liebl. Tel: +49 551 393795. Fax: +49 551 394897. e-mail: wliebl{at}gwdg.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A plasmid designated pMEC2 which confers resistance to erythromycin, other macrolides, and lincomycin was detected in Micrococcus luteus strain MAW843 isolated from human skin. Curing of this approximately 4·2 kb plasmid from the host organism resulted in erythromycin sensitivity of the strain. Introduction of pMEC2 into a different M. luteus strain conferred erythromycin resistance upon this strain. Macrolide resistance in M. luteus MAW843 was an inducible trait. Induction occurred at subinhibitory erythromycin concentrations of about 0·02–0·05 µg ml-1. Erythromycin and oleandomycin were inducers, while spiramycin and tylosin exerted no significant inducer properties. With heterologous expression experiments in Corynebacterium glutamicum, using hybrid plasmid constructs and deletion derivatives thereof, it was possible to narrow down the location of the plasmid-borne erythromycin-resistance determinant to a region of about 1·8 kb of pMEC2. Sequence analysis of the genetic determinant, designated erm(36), identified an ORF putatively encoding a 281-residue protein with similarity to 23S rRNA adenine N6-methyltransferases. erm(36) was most related (about 52–54% identity) to erythromycin-resistance proteins found in high-G+C Gram-positive bacteria, including the (opportunistic) pathogenic corynebacteria Corynebacterium jeikeium, C. striatum, C. diphtheriae and Propionibacterium acnes. This is believed to be the first report of a plasmid-borne, inducible antibiotic resistance in micrococci. The possible role of non-pathogenic, saprophytic micrococci bearing antibiotic-resistance genes in the spreading of these determinants is discussed.

Keywords: erythromycin resistance, erm(36), induction, curing

a The GenBank accession number for the sequence reported in this paper is AF462611.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phylogenetically, Micrococcus species belong to the broad group of Gram-positive bacteria with a high G+C content of their DNA. In a new hierarchical classification system for the class Actinobacteria they have been allocated to the family Micrococcaceae within the order Actinomycetales (Stackebrandt et al., 1997 ). Micrococci, e.g. Micrococcus luteus and Micrococcus lylae, are often isolated from the skin of mammals, where they appear to be saprophytes. Micrococcus species are distinguished by various criteria from the second major group of cocci frequently isolated from skin, i.e. the staphylococci (Kocur et al., 1992 ).

Despite their role as common skin inhabitants, little is known about the occurrence and the mechanism of action of antibiotic-resistance determinants in micrococci. However, recent work has demonstrated that significant numbers of erythromycin-resistant M. luteus strains can be isolated from human skin (Eady et al., 2000 ). We have observed that a plasmid-bearing M. luteus strain designated MAW843 was resistant to erythromycin and other macrolide antibiotics (unpublished data). Plasmids with sizes ranging from 1 to about 90 MDa have been detected in micrococci before, but information regarding their possible functions is very scarce (Mathis & Kloos, 1969 ). Therefore, we decided to investigate if the antibiotic-resistance phenotype of M. luteus MAW843 was linked to its plasmid. Also, it was of interest to compare the resistance gene of this strain with previously characterized erythromycin-resistance determinants. Furthermore, since no cloning systems have been established for Micrococcus, an indigenous M. luteus antibiotic-resistance plasmid may be useful for genetic engineering purposes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and media.
The bacterial strains and plasmids used are listed in Table 1. Micrococcus luteus strain MAW843 was isolated during a survey of the microbial population on the skin of a female person treated with clindamycin for acne. For transformation experiments, M. luteus ATCC 27141 was used. M. luteus strains were propagated at 30 °C using P medium (0·5% peptone, 0·5% sodium chloride, 0·1% glucose) or LB medium (1% peptone, 0·5% yeast extract, 0·5% sodium chloride, pH 7·2). Where appropriate, the media were supplemented with agar (12 g l-1) and/or antibiotics as described in the text. Corynebacterium glutamicum strains containing hybrid plasmids were grown in LB medium supplemented with 20 µg kanamycin ml-1.


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Table 1. Strains and plasmids

 
Isolation of plasmid DNA from M. luteus cells.
For large-scale plasmid DNA isolation, cells of M. luteus from a total of 2 litres of P-broth culture were harvested and suspended in 40 ml 50 mM Tris/HCl pH 7, 10 mM EDTA. Cell lysis was achieved by treatment with lysozyme (15 min at 37 °C at a final concentration of 5 mg ml-1) and subsequent addition of 3 ml 0·5 M EDTA pH 8 and 5 ml 10% SDS. The lysate was mixed with 10 ml 5 M sodium chloride, incubated at 0 °C for 120 min, and cleared by centrifugation (23500 g, 30 min, 4 °C). The DNA was precipitated with 0·7 vol. 2-propanol, collected by centrifugation, redissolved in 10 mM Tris/HCl pH 8, 1 mM EDTA (TE), and purified further via caesium chloride/ethidium bromide equilibrium gradient centrifugation (Sambrook et al., 1989 ). Small amounts of plasmid DNA were isolated from M. luteus strains by a rapid boiling method. Cells of a 3–5 ml culture were suspended in 100 µl 40 mM Tris/acetate buffer pH 8, 2 mM EDTA and mixed with 150 µl 1 M sorbitol and 30 µl 100 mg ml-1 lysozyme solution. After 15 min at 37 °C, 25 µl 0·5 M EDTA pH 8 and 50 µl 10% SDS were added. The suspension was boiled for 2 min, cooled on ice, and cleared by centrifugation for 15 min in a table-top centrifuge. The lysate was extracted once with phenol/chloroform (1:1, v/v) and once with chloroform. The aqueous phase was transferred to a fresh tube and the DNA was precipitated by the addition of 0·1 vol. 3 M sodium acetate and 1 vol. 2-propanol (20 °C, 15 min). After centrifugation for 15 min, the DNA was dissolved in TE buffer, reprecipitated (this time with 2 vols ethanol at -20 °C for 2 h), and finally dissolved in TE buffer.

DNA analysis and modification, and construction of hybrid vectors.
Restriction endonucleases and other modifying enzymes were purchased from Pharmacia, Boehringer or New England Biolabs, and used as recommended by the suppliers. In general, DNA sequencing and other modifications were carried out according to standard procedures (Sambrook et al., 1989 ). Computer analysis of nucleotide sequences and the deduced amino acid sequences was performed with the programs FASTA (Pearson & Lipman, 1988 ) and BLAST (Altschul et al., 1990 ). Pairwise alignments for the calculation of protein sequence similarities were created with the program GAP included in the University of Wisconsin Genetics Computer Group (GCG) software package for UNIX (Devereux et al., 1984 ). Multiple amino acid sequence comparison was done with CLUSTAL W (Thompson et al., 1994 ).

The C. glutamicum/E. coli shuttle vector pWST3B was constructed from pWST1 (Liebl et al., 1989b ). First, one BamHI site proximal to the multiple cloning site of pWST1 was removed by BamHI partial digestion, Klenow DNA polymerase fill-in, and religation, yielding pWST1B. Then, pWST1B was converted into pWST3B by deletion of about 3·4 kb of non-essential DNA. This vector was used for the construction of hybrid vectors by fusion with pMEC2.

Antibiotic susceptibility tests.
The MICs of antibiotics were determined by inoculation of 2 ml aliquots of serial twofold dilutions of antibiotics in P broth (final concentrations from 0·06 to 1024 µg ml-1) with 2x106 cells. The cultures were incubated with shaking at 30 °C and monitored for 3 days. For agar diffusion tests, cells of M. luteus strains were evenly spread onto P or LB agar plates either by a top agar technique or via plating in order to obtain a confluent lawn of bacterial growth. Then, filter disks containing defined amounts of various antibiotics were placed on the surface of the plates and incubation was carried out at 30 °C for 2–5 days. For the detection of inducer properties of antibiotics, filter disks soaked with possible inducer antibiotics were placed in the centre of plates seeded with M. luteus cells. After incubation at 32 °C for 3 h, disks soaked with other antibiotics were arranged around the central disk, and incubation was continued for 2–3 days. The distortion of the growth inhibition halo around a satellite disk in the direction facing the central disk was an indication that the latter disk contained an inducing antibiotic.

Plasmid curing, transformation, and spiramycin filter strip test.
A fresh overnight culture of the plasmid-bearing M. luteus strain MAW843 was diluted 1:5000 in several tubes with P-broth containing ethidium bromide at various concentrations (0·3–50 µg ml-1). The tubes were incubated on a rotary shaker at 30 °C for about 20 h. An aliquot was drawn from the culture with the highest ethidium bromide concentration where visible growth had occurred. Appropriate dilutions were plated onto LB agar plates and after growth were replica-plated onto plates supplemented with 2·5 µg erythromycin ml-1 in order to screen for erythromycin-sensitive isolates. Transformation of C. glutamicum with hybrid plasmids and deletion derivatives was carried out as described before (Liebl et al., 1989a ; Yoshihama et al., 1985 ). Transformants were selected with kanamycin (20 µg ml-1).

For electrotransformation experiments with M. luteus cells, a method originally developed for C. glutamicum (Liebl et al., 1989a ) was used except that M. luteus was grown in brain heart infusion broth (Difco) instead of LB. In some cases, the washing buffer was modified by substituting Tris for HEPES buffer and/or 10% (v/v) glycerol for 10% sucrose. After electroporation, the cells were diluted 1:10 into P broth supplemented with 0·04 µg erythromycin ml-1, incubated at 30 °C for 3 h, and plated onto P agar plates containing 2 or 5 µg erythromycin ml-1. Unfortunately, after 4–5 days at 30 °C, numerous spontaneous (chromosomal) erythromycin-resistant mutants that did not contain a plasmid grew on the selection plates. In order to screen for true pMEC2 transformants, a simple spiramycin agar diffusion method was developed that allows discrimination between a constitutive resistance phenotype typical for the spontaneous mutants and the inducible resistance phenotype expected for transformants bearing pMEC2. The method was based on the observation that M. luteus MAW843 cells induced with erythromycin were resistant to spiramycin, but spiramycin itself does not induce resistance development. Strains to be tested were inoculated as parallel streaks onto the surface of two P agar plates, one of which contained a subinhibitory concentration (0·05 µg ml-1) of erythromycin. After 4–6 h at 30 °C, a 6 mm wide strip of filter paper soaked with 100 µl of a 1 mg ml-1 spiramycin solution was laid perpendicular to the bacterial streaks on each plate. After 2 days at 30 °C, the strains with the inducible resistance phenotype (pMEC2-bearing strains) revealed a broad zone of growth inhibition next to the antibiotic strip on the plate without erythromycin but not on the other plate with 0·05 µg erythromycin ml-1. With plasmid-free sensitive strains or spontaneously constitutive chromosomal mutants, the growth inhibition zones next to the spiramycin strip or the lack thereof, respectively, were identical on both parallel plates.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Inducible macrolide resistance in M. luteus MAW843
The isolate MAW843 was identified as M. luteus by the following criteria (see Kocur et al., 1992 ): macroscopic colony characteristics, microscopic morphology, and various physiological characteristics (data not shown). The G+C content of strain MAW843 chromosomal DNA was 68·5 mol% (data not shown). The strain was found to be resistant not only to clindamycin, but also to other macrolide and lincosamide antibiotics (Fig. 1). The MICs of erythromycin, oleandomycin and lincomycin for M. luteus MAW843 were 512 µg ml-1, >1024 µg ml-1 and 1024 µg ml-1, respectively. Strain MAW843 was susceptible to tylosin (MIC 1 µg ml-1) but developed increased resistance to this antibiotic in the presence of subinhibitory concentrations of erythromycin (MIC 4 µg ml-1 when grown in the presence of 0·05 µg erythromycin ml-1). Strain MAW843 was moderately resistant to spiramycin (MIC 64 µg ml-1), but the MIC increased to 1024 µg ml-1 in the presence of 0·05 µg erythromycin ml-1. These results showed that the resistance phenotype of M. luteus strain MAW843 was an inducible trait. This was also observed with an agar diffusion technique on nutrient agar plates seeded with M. luteus MAW843 cells (see Methods). In this test, diffusion of an inducing antibiotic from a filter disk on the agar surface caused the distortion of the growth inhibition halo around a neighbouring disk soaked with one of the antibiotics named above (Fig. 2). In this way, erythromycin and oleandomycin, both 14-membered-ring macrolides, were identified as potent inducers of macrolide resistance in M. luteus MAW843. The 16-membered-ring macrolides tylosin and spiramycin were not inducers.



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Fig. 1. Agar diffusion assay for the qualitative detection of antibiotic resistance in M. luteus MAW843 and its plasmid-cured derivative MAW843-46,1. Note the reduction of the inhibition zone diameter around the disks with the antibiotics clindamycin (C, 2 µg), lincomycin (L, 2 µg) and spiramycin (S, 100 µg) with strain MAW843 upon growth in the presence of a subinhibitory concentration of erythromycin (central plate) as compared to the plate without erythromycin (left plate). Similar inhibition zone diameters were observed on the MAW843-seeded plates around the disks containing oleandomycin (O, 15 µg), an inducing macrolide antibiotic. Loss of the plasmid pMEC2 in the cured strain MAW843-46,1 resulted in increased sensitivity to macrolide antibiotics, which is reflected by large zones of growth inhibition (right plate). Disks with the non-macrolide antibiotics polymyxin B (P, 300 µg) and thiamphenicol (T, 30 µg) were included on each plate as controls.

 


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Fig. 2. Inducers and non-inducers of macrolide resistance in M. luteus strain MAW843. Abbreviations: E, erythromycin; O, oleandomycin; S, spiramycin; T, tylosin. The amount of antibiotic on the central filter disks was 7·5 µg (spiramycin and tylosin) or 30 µg (erythromycin and oleandomycin); the surrounding disks contained 75 µg (spiramycin and tylosin) or 250 µg (erythromycin and oleandomycin).

 
Identification of a plasmid associated with erythromycin resistance in M. luteus MAW843
A 4·2 kb plasmid was detected in M. luteus strain MAW843 and designated pMEC2. In order to find out if this plasmid was associated with the erythromycin-resistance phenotype of the strain, ethidium-bromide-assisted plasmid curing was carried out as described in Methods. A strain, designated MAW843-46,1 was isolated which lacked a plasmid but was indistinguishable from the parent strain in all other phenotypic traits checked. The cured strain was sensitive to macrolide antibiotics and lincomycin (see Fig. 3). The MIC of erythromycin for strain MAW843-46,1 was 0·5 µg ml-1, in contrast to the MIC of 512 µg ml-1 determined for the parent strain MAW843. A detailed map of restriction endonuclease cleavage sites of pMEC2 is shown in Fig. 4.



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Fig. 3. Spiramycin filter strip test for the qualitative detection of inducible macrolide resistance. A set of representative M. luteus strains, i.e. MAW843 (wild-type containing pMEC2), MAW843-46,1 (cured of pMEC2), ATCC 27141 (wild-type, plasmid-free) and ATCC 27141-5/1 (transformant with pMEC2), were streaked onto two P-agar plates, one of which was supplemented with 0·05 µg erythromycin and challenged with the non-inducing macrolide antibiotic spiramycin as described in Methods by application of a filter paper strip containing 0·5 mg of the antibiotic. Macrolide-sensitive strains (MAW843-46,1 and ATCC 27141) or constitutively resistant chromosomal mutants (not shown) displayed identical zones of growth inhibition or lack thereof, respectively, on both plates, while pMEC2-bearing strains (MAW843 and ATCC 27141-5/1) had no inhibition zones on the plate with a subinhibitory concentration of the inducer erythromycin.

 


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Fig. 4. Restriction map of the M. luteus erythromycin-resistance plasmid pMEC2.

 
Transformation of a plasmid-free M. luteus strain with pMEC2
We attempted to introduce pMEC2 purified from M. luteus MAW843 into a different, plasmid-free M. luteus strain. As a recipient we chose M. luteus ATCC 27141, which can easily be distinguished from strain MAW843 by its purine auxotrophy, level of lysozyme sensitivity and the shade of yellow colour of the colonies. Using electrotransformation, we succeeded in transforming pMEC2 into M. luteus ATCC 27141, albeit with a low efficiency. Transformants were found with the aid of the spiramycin filter strip test described in Methods. This test is a powerful screening method for the detection of pMEC2-bearing strains with an inducible resistance phenotype (see Fig. 3). The transformant strain, which was designated ATCC27141-5/1, had the same colony colour and lysozyme sensitivity as ATCC 27141 and was purine auxotrophic like the parent strain, which clearly demonstrates that the selected strain was an ATCC 27141 derivative. M. luteus ATCC 27141-5/1 contained a plasmid which according to size and restriction endonuclease analysis was identical to pMEC2 (data not shown).

Construction of hybrid plasmids with pMEC2 and heterologous expression of erythromycin resistance in C. glutamicum
The Micrococcus plasmid pMEC2 was linearized with EcoRV or BclI and ligated with the pWST1B (Liebl et al., 1989a ) derivative pWST3B opened with SmaI or BamHI, respectively, thus giving rise to the hybrid plasmids pWMC37, pWMC65, pWMC75 (Fig. 5). The hybrid plasmids and various deletion derivatives thereof (Fig. 5) were transformed into C. glutamicum strain E12. The recombinant strains were inoculated into LB broth supplemented with 20 µg kanamycin ml-1. After growth for 3 h at 30 °C, erythromycin was added at 0·04 µg ml-1, and incubation was continued for 2 h. Then, the strains were streaked onto LB plates containing 0·5 or 1 µg erythromycin ml-1 in addition to 20 µg kanamycin ml-1. All of the full-sized hybrid plasmids, i.e. pWMC37, pWMC65 and pWMC75, conferred erythromycin resistance upon the corresponding C. glutamicum strains, indicating (i) that the M. luteus erythromycin-resistance determinant is functionally expressed in the heterologous host C. glutamicum, and (ii) that the resistance gene had not been inactivated by insertion of the shuttle vector pWST3B at the EcoRV or BclI sites of pMEC2. Some of the deletion derivatives, which lack the 0·7 kb StuI fragment of pMEC2 (pWMC75-StuI-del), the 1 kb PvuII–StuI segment of pMEC2 (pWMC37-PvuII–StuI-del), or the 2·5 kb region between the XhoI and BclI sites of pMEC2 (pWMC75-XhoI-del), also conferred erythromycin resistance upon the heterologous host. On the other hand, deletion of the 0·9 kb SmaI fragment of pMEC2 (pWMC37-SmaI-del) or the 1·2 kb region between the SmaI and BclI sites of pMEC2 (pWMC65-SmaI-del) resulted in C. glutamicum transformants which remained erythromycin sensitive. Taken together, the heterologous expression experiments indicate that the erythromycin-resistance gene is located on an approximately 1·8 kb BclI–XhoI fragment of pMEC2 (see Fig. 4).



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Fig. 5. Construction of hybrid plasmids with pMEC2 and deletion derivatives. Plus symbols indicate the ability of the plasmids to confer increased erythromycin resistance upon the heterologous host bacterium C. glutamicum. Abbreviations: amp, ampicillin-resistance gene; kan, kanamycin-resistance gene; ori_ec, origin for replication in E. coli; rep_cg, putative C. glutamicum replication gene.

 
Sequence analysis of the erythromycin-resistance determinant of pMEC2
The nucleotide sequence of the pMEC2 erythromycin-resistance gene and flanking regions was determined. The sequenced 1163 bp BclI–ClaI fragment of pMEC2 had a G+C content of 64 mol%, which is about 5% lower than the G+C content of M. luteus MAW843 chromosomal DNA (68·5 mol%). An ORF was identified whose 281-residue deduced amino acid sequence was similar to 23S rRNA adenine N6-methyltransferases encoded by erm genes of various bacteria (Fig. 6). The closest relatives found among known protein sequences in the available databases were the corresponding enzymes from Corynebacterium diphtheriae (53% identity; encoded by plasmid pNG2; Serwold-Davis & Groman, 1988 ), Propionibacterium acnes (53% identity; identical amino acid sequence to the C. diphtheriae pNG2-encoded determinant; GenBank accession no. AF411029), Corynebacterium striatum (previously classified as C. xerosis) (53% identity; ermCX gene of the composite transposon Tn5432; almost identical amino acid sequence to the C. diphtheriae pNG2-encoded determinant; Tauch et al., 1995 , 2000 ), Corynebacterium jeikeium (51% identity; chromosomal determinant; Rosato et al., 2001 ). Further sequences with high similarity to the M. luteus erythromycin-resistance ORF were the macrolide-resistance proteins from other high-G+C Gram-positive bacteria, including various Streptomyces species and Aeromicrobium erythreum (previously classified as Arthrobacter sp.). In agreement with the current classification proposed for erm genes by M. C. Roberts (University of Washington; see http://faculty.washington.edu/marilynr/), the designation erm(36) is proposed for the new erythromycin-resistance determinant from M. luteus plasmid pMEC2.



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Fig. 6. Alignment of amino acid sequences of selected erythromycin-resistance methylases. Abbreviations: ermA_Cd, resistance determinant encoded by Corynebacterium diphtheriae plasmid pNG2 (X51472; Serwold-Davis & Groman, 1988 ; Hodgson et al., 1990 ), which is identical to ermX from Tn5432 found in Propionibacterium acnes (AF411029) and nearly identical to ermCX from Tn5432 of Corynebacterium striatum (gi:709806; formerly C. xerosis; Tauch et al., 1995 ); erm_Sv, resistance determinant from Streptomyces venezuelae (AF079138; Xue et al., 1998 ); erm(36)_Ml, resistance determinant of Micrococcus luteus plasmid pMEC2 (this work); ermC_Sa, resistance determinant encoded by ermC of Staphylococcus aureus plasmid pE194 (J01755; Horinouchi & Weisblum, 1982 ).

 
A small 14-residue ORF and several inverted repeat sequences were detected in the approximately 150 bp sequence region upstream of the erm(36) methylase ORF. These structures may be involved in erm(36) regulation (see Discussion).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the erythromycin-resistance determinant of plasmid pMEC2 and its regulation
The occurrence of plasmids with sizes ranging from about 1 to 90 MDa in strains of the genus Micrococcus has been described before (Mathis & Kloos, 1969 ). We have now for the first time been able to unequivocally assign a function to a M. luteus plasmid. Four lines of evidence demonstrate the presence of a plasmid-borne macrolide- and lincosamide-resistance determinant in M. luteus strain MAW843: (i) ethidium-bromide-assisted curing of the plasmid pMEC2 resulted in an erythromycin-sensitive strain; (ii) transformation of the plasmid isolated from strain MAW843 into a different, plasmid-free M. luteus strain conferred the resistance phenotype upon this strain; (iii) cloning of pMEC2 or parts thereof into a C. glutamicum/E. coli shuttle vector led to hybrid plasmids which conferred elevated levels of erythromycin resistance upon the heterologous host C. glutamicum; (iv) finally, an ORF for a polypeptide similar to known erythromycin-resistance proteins was identified by sequence analysis.

There are three principal mechanisms of erythromycin resistance: (i) modification of the target of the antibiotic (the ribosomal 50S subunit), (ii) expulsion of internalized antibiotic molecules via energy-dependent efflux pumps, or (iii) modification of the antibiotic (Ross et al., 1990 ; Sutcliffe et al., 1996 ; see Roberts et al., 1999 , for an overview). In the case of the M. luteus strain MAW843 studied here, erythromycin resistance was unequivocally shown to be mediated by a new erm gene, erm(36), whose deduced amino acid sequence was most closely related to rRNA methylases from high-G+C Gram-positive bacteria of the genera Corynebacterium, Propionibacterium, Aeromicrobium (formerly classified as Arthrobacter sp.) and Streptomyces. The mechanisms leading to the resistance phenotype in other erythromycin-resistant strains of M. luteus isolated from the skin surface of human patients (Eady et al., 2000 ; Luna et al., 1999 ) have not been studied in detail.

The expression of erythromycin-resistance determinants is often regulated. In most cases, such as Staphylococcus aureus ermC, control occurs posttranscriptionally by translational attenuation: a constitutively synthesized mRNA takes an inactive conformation in the absence of inducer; in the presence of erythromycin, ribosome stalling during translation of a short ORF in the mRNA 5'-region leads to the rearrangement of secondary structures in this region, which again results in the demasking of the translation initiation sequences of the methylase structural gene (see reviews by Dubnau, 1984 ; Horinouchi et al., 1983 ; Weisblum, 1983 , 1984 ). In search of DNA sequences that may be involved in regulation of erm(36), we found the following features in the region upstream of the methylase ORF: (i) inverted repeat sequences that could form mutually exclusive secondary structures, and (ii) a small ORF for a 14-residue peptide that overlaps with the largest inverted repeat (Fig. 7). Thus, ribosomes stalled by erythromycin during translation of this small ORF theoretically could disrupt the secondary structure in this part of the control region and thereby trigger a stem–loop redistribution in the leader region of the erm(36) transcript. However, since the putative regulatory region of erm(36) is separated from the ribosome-binding sequence and start codon of the methyltransferase ORF by about 40 nucleotides, we do not assume an ermC-like classical translational attenuation for erm(36) induction. We rather prefer the idea of a transcriptional attenuation mechanism. Regulation of macrolide resistance by transcriptional attenuation has been proposed before for ermK of Bacillus licheniformis (Choi et al., 1997 ) and tlrA of Streptomyces fradiae (Kelemen et al., 1994 ). Further efforts are necessary to prove this hypothesis and unravel the induction mechanism of erm(36).



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Fig. 7. DNA sequence upstream of the erm(36) ORF. A possible leader ORF and inverted sequence repeats, which may be involved in the regulation of erm(36) induction, are indicated as arrows. The {Delta}G values (determined with the program FOLDRNA) for the three putative hairpin structures are (from left to right) -21·7, -16·8 and -14·5 kJ mol-1.

 
Only certain macrolide antibiotics (erythromycin, oleandomycin) were efficient inducers for erm(36) expression, whereas others (the 16-membered-ring macrolides spiramycin and tylosin) did not exert a significant induction effect. Differences in the induction specificity of macrolide-lincosamide-streptogramin B (MLS) resistance genes, i.e. differences in the subsets of MLS antibiotics that induce the expression of the genes, have been observed before (Dubnau, 1984 ; Mayford & Weisblum, 1990 ). In the case of ribosome-mediated regulation, where ribosomes themselves act as regulators and are directly involved in the induction process, this phenomenon appears to reflect differences in the antibiotics’ modes of interaction with the ribosome (see Mayford & Weisblum, 1990 ).

Heterologous expression of erm(36) and potential of pMEC2 as a cloning vector
On the basis of the following criteria, plasmid pMEC2 may be suitable for use as a cloning vector for M. luteus: (i) it is a small plasmid (4·2 kb) with a moderate copy number; (ii) it carries a genetic marker which can be selected for in M. luteus; (iii) a detailed restriction endonuclease map is available; (iv) there are several unique restriction sites that may be useful as insertion loci for foreign DNA fragments. However, it will be necessary to define those regions of the plasmid that are essential for replication and stability.

Interestingly, the pMEC2 erythromycin-resistance determinant was expressed in C. glutamicum, which belongs to the same bacterial phylum as the original host M. luteus, i.e. the high-G+C Gram-positive bacteria. However, the level of erythromycin resistance reached by C. glutamicum transformants bearing hybrid pMEC2-pWST3B plasmids was rather low (resistant to 0·5–1 µg ml-1 on solid media; no growth at 3 µg ml-1; data not shown) as compared with M. luteus strain MAW843 (resistant to >50 µg ml-1 on agar plates). The low level of resistance reached in the heterologous host probably precludes the use of this M. luteus determinant as a selection marker in C. glutamicum cloning vectors. Possible reasons for the low resistance level observed in C. glutamicum include the inefficient expression of the determinant at the transcriptional and/or translational level [suboptimal expression signal structures (promoter, ribosome-binding site), codon usage, inefficient induction]. However, since the erythromycin-resistance gene of M. luteus studied here encodes a 23S rRNA N-methyltransferase, another likely explanation could be that the C. glutamicum 23S rRNA is a poor substrate for the enzyme. It has been demonstrated for the ermC-encoded methylase from Staphylococcus aureus that 23S rRNAs isolated from different genera are not equally efficient substrates for in vitro methylation by the purified enzyme (Denoya & Dubnau, 1987 ).

Possible role of non-pathogenic micrococci in the spreading of antibiotic-resistance genes
M. luteus is generally regarded as being non-pathogenic for non-immunocompromised individuals. Nevertheless, the observation of antibiotic-resistance determinants in strains of this species (Eady et al., 2000 ; Luna et al., 1999 ; this work) may be of clinical importance because these determinants may be transferred to other bacteria. The fact that the protein encoded by erm(36) was found to be most similar to erythromycin-resistance determinants found in pathogenic and opportunistic strains of Corynebacterium and Propionibacterium indicates that non-pathogenic skin micrococci may share a resistance gene pool with other high-G+C Gram-positive bacteria. However, the degree of relatedness between erm(36) and other erm ORFs (up to about 53% identity at the level of amino acid sequences) was too low to suggest a recent gene-transfer event in this particular case. Nevertheless, it is easily conceivable that plasmid-bearing strains of saprophytic skin bacteria could serve as a natural reservoir for resistance genes. Via intergeneric gene transfer such strains could contribute to the spreading of antibiotic-resistance determinants to medically important organisms. In this context it is interesting to note that naturally transformable strains of M. luteus have been described (Kloos, 1969 ; Kloos & Schultes, 1969 ). Thus, some M. luteus strains may acquire resistance factors via natural transformation.

To our knowledge, this is the first detailed characterization of an inducible antibiotic-resistance gene from the genus Micrococcus. It will be interesting to compare other erythromycin-resistance genes from micrococci with determinants found in other high-G+C Gram-positive bacteria from microbial communities on the skin.


   ACKNOWLEDGEMENTS
 
Expert technical assistance by B. Schumacher and U. Ludwig is gratefully acknowledged. The authors are grateful to Dr M. Roberts (University of Washington) for suggestions concerning the nomenclature of the erythromycin-resistance determinant described here.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 21 December 2001; revised 29 April 2002; accepted 1 May 2002.



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