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
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ABSTRACT |
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Keywords: erythromycin resistance, erm(36), induction, curing
a The GenBank accession number for the sequence reported in this paper is AF462611.
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INTRODUCTION |
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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a plasmid-bearing M. luteus strain designated MAW843 was resistant to erythromycin and other macrolide antibiotics (unpublished data).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.
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METHODS |
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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 25 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 23 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 (0·350 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 45 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 46 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.
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RESULTS |
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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 PvuIIStuI segment of pMEC2 (pWMC37-PvuIIStuI-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 BclIXhoI fragment of pMEC2 (see Fig. 4
).
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DISCUSSION |
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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 stemloop 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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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 0·51 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.
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ACKNOWLEDGEMENTS |
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Received 21 December 2001;
revised 29 April 2002;
accepted 1 May 2002.
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