Institut de Génétique et Microbiologie, UMR CNRS 8621, Bât. 400, Université Paris-Sud XI, F-91405 Orsay Cedex, France1
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK2
Author for correspondence: Jean-Luc Pernodet. Tel: +33 1 69 15 46 41. Fax: +33 1 69 15 72 96. e-mail: pernodet{at}igmors.u-psud.fr
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ABSTRACT |
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Keywords: macrolide antibiotics, MLS resistance, rRNA methylation, spiramycin, Streptomyces ambofaciens
Abbreviations: MLS, macrolidelincosamidestreptogramin type B; SAM, S-adenosylmethionine
The EMBL/GenBank accession number for the nucleotide sequence described in this paper is AJ223970.
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INTRODUCTION |
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Streptomyces ambofaciens produces spiramycin, a macrolide antibiotic consisting of a 16-membered lactone ring with three sugar or amino-sugar residues, which specifically inhibits bacterial protein synthesis by binding to the large subunit of the ribosome (Gale et al., 1981 ; Brisson-Noël et al., 1988
). The most widespread macrolide-resistance mechanism involves the so-called MLS (macrolidelincosamidestreptogramin type B) resistance phenotype (Weisblum, 1995
). Despite structural dissimilarities, antibiotics belonging to these three groups bind to the ribosomes in mutually competitive fashion and inhibit protein synthesis. MLS resistance involves N6-methylation of the amino group of a particular adenosine residue (A-2058, Escherichia coli numbering scheme) in the large rRNA, leading to reduced binding of MLS antibiotics to the ribosome. Such resistance was first encountered in clinical strains of Staphylococcus aureus and has since been detected in a broad range of micro-organisms, including actinomycete producers or non-producers of MLS antibiotics. Two varieties of MLS resistance have been described. The phenotypes resulting from N6-monomethylation of A-2058 (MLS-I phenotype conferred by erm type I genes) or N6,N6-dimethylation (MLS-II phenotype conferred by erm type II genes) differ mainly in the resistance levels to specific macrolides (Pernodet et al., 1996
).
Other macrolide-resistance mechanisms involving export or inactivation of the antibiotics have been characterized, both in actinomycetes and in pathogenic bacteria. Genes conferring macrolide resistance and whose products belong to the superfamily of ATP-dependent transport proteins have been found in pathogenic bacteria such as Staph. aureus (Ross et al., 1990 ) and in several macrolide-producing Streptomyces spp. (Mendez & Salas, 1998
), including S. ambofaciens. Macrolide inactivation can be due to different modifications, for instance phosphorylation (Marshall et al., 1989
; OHara et al., 1989
), glycosylation (Kuo et al., 1989
; Cundliffe, 1992
; Vilches et al., 1992
) or esterification of the lactone ring (Ounissi & Courvalin, 1985
; Arthur et al., 1986
).
S. ambofaciens protects itself against spiramycin via at least two different resistance mechanisms, one of which involves ribosomal modification (Pernodet et al., 1993 ). Among the resistance determinants previously isolated from this strain (Richardson et al., 1987
), one of them, srmB, is supposed to be involved in antibiotic export (Schoner et al., 1992
). We report here the cloning of several resistance determinants from S. ambofaciens, the identification of one that is involved in ribosomal resistance and biochemical characterization of the encoded resistance mechanism.
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METHODS |
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DNA techniques and cloning procedures.
DNA extractions and manipulations were performed according to well-established procedures (Hopwood et al., 1985 ; Sambrook et al., 1989
). For the construction of the S. ambofaciens gene library, genomic DNA of S. ambofaciens ATCC 23877 was partially digested by Sau3AI to yield fragments in the range 3040 kb. Digested genomic DNA (3 µg) was then ligated with 1 µg BamHI-digested cosmid vector pKC505, which confers apramycin resistance in E. coli and Streptomyces. The ligation mixture was packaged in vitro in
heads, using packaging extracts obtained from Amersham, and the resulting phage particles were used for infection of E. coli HB101. About 20000 apramycin-resistant E. coli clones were pooled and their cosmid DNA was extracted.
Standard conditions were used for the transformation of E. coli strains (Sambrook et al., 1989 ). Streptomyces protoplasts were formed, transformed and regenerated as described previously for Streptomyces griseofuscus (Rao et al., 1987
) and Streptomyces lividans (Hopwood et al., 1985
).
DNA sequence determination and analysis.
Single-stranded DNA templates were sequenced by the chain-termination technique using restriction fragments cloned in M13 mp18/19. We used a Deaza T7 sequencing kit from Pharmacia and [35S]dATPS from Amersham. Sequence was analysed with the frame program (Bibb et al., 1984
) and the GCG package (Wisconsin Package version 9.1, Genetics Computer Group). Sequence comparisons with databases used the fasta (Pearson & Lipman, 1988
) and blast programs (Altschul et al., 1997
).
Preparation of mycelial extracts and salt-washed ribosomes.
Crude mycelial extracts (30000 g supernatant, S30) and salt-washed ribosomes were prepared as described elsewhere (Skeggs et al., 1985 ). Ribosomal wash fraction was prepared as previously described (Zalacain & Cundliffe, 1989
). In S. ambofaciens, expression of some resistance genes, probably including srmA, could be induced by spiramycin (Pernodet et al., 1993
). Therefore, strains harbouring the cloned srmA gene were grown in the presence of 5 µg spiramycin ml-1 to select for the presence of the cloned gene and to ensure its expression.
Coupled transcriptiontranslation assay.
Coupled transcriptiontranslation was performed as described previously (Calcutt & Cundliffe, 1989 ). All components of the coupled transcriptiontranslation system except the salt-washed ribosomes were prepared from S. lividans. Ribosomes were prepared from S. griseofuscus containing various plasmids. One microgram of the plasmid pUC18 was used as template. Protein synthesis was measured as incorporation of [35S]methionine into TCA-precipitable material as described by Thompson et al. (1984
).
rRNA methylation in vitro.
Total rRNA was prepared by extraction of 70S salt-washed ribosomes with LiCl and urea (Fahnestock et al., 1974 ). Methylation assays were carried out at 30 °C in a final volume of 100 µl and contained 40 pmol rRNA as substrate, 55 µl S30 extract or ribosomal wash fraction as methyltransferase enzyme, 2·5 µCi S-adenosyl[methyl-3H]methionine ([methyl-3H]SAM; 500 mCi mmol-1; 18·5 GBq mmol-1) as methyl donor cofactor, in buffer containing 50 mM HEPES/KOH (pH 7·5 at 20 °C), 7 mM MgCl2, 37·5 mM NH4Cl and 3 mM ß-mercaptoethanol. Methylation of rRNA was measured as incorporation of [3H]methyl groups into TCA-precipitable material as previously described by Skeggs et al. (1985
). To allow identification of the radiolabelled residue, the assay mixture was scaled up by a factor of three, and 15 µCi [methyl-3H]SAM with higher specific activity (15 Ci mmol-1; 555 GBq mmol-1) was used. Incubation was for 45 min at 30 °C and the labelled rRNA was recovered by phenol extraction and ethanol precipitation.
Identification of the methylated residue.
Radiolabelled rRNA obtained after bulk methylation was hydrolysed with 1 M HCl at 100 °C for 60 min and the products of hydrolysis (purine bases, pyrimidine-3'-mononucleotides and ribose phosphate) were separated by descending chromatography on Whatman 3MM paper using as solvent either 2-propanol/HCl/water (170:41:39, by vol.) or butanol/water/ammonia (95:14:5, by vol.), as previously described (Zalacain & Cundliffe, 1989 ). After drying, markers were detected under UV light, the chromatogram tracks were cut into strips 1 cm wide and radioactivity was measured by liquid-scintillation spectrometry. To confirm the identification radiolabelled rRNA was digested with nuclease P1 to generate nucleoside-5'-monophosphates, which were analysed by two-dimensional TLC, as previously described by Jenkins et al. (1989
).
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RESULTS |
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As we had shown previously that a ribosomal resistance mechanism was present in S. ambofaciens (Pernodet et al., 1993 ), the conserved cosmids were screened by hybridization analysis for the presence of MLS-resistance genes, using as the probe ermE from Saccharopolyspora erythraea, which produces the macrolide antibiotic erythromycin (Uchiyama & Weisblum, 1985
). This gene had been successfully used as a probe in the cloning of ermSF (synonym, tlrA) from Streptomyces fradiae, which produces the macrolide tylosin (Kamimiya & Weisblum, 1988
). One of the cosmids, pOS41.1, hybridized with ermE (data not shown), suggesting that the resistance determinant carried by pOS41.1 could also encode an rRNA methyltransferase. Accordingly, this resistance determinant was subcloned and the resistance mechanism was investigated.
Within pOS41.1, ermE hybridized with a single PstI fragment, a single KpnI fragment, and a single shorter PstIKpnI fragment. This latter fragment (4·8 kb) was ligated into pTZ19R to give pOS41.2 and then introduced into the Streptomyces cloning vector pIJ486 as an EcoRIHindIII fragment, yielding pOS41.3, which conferred spiramycin resistance in S. griseofuscus. The restriction map of the PstIKpnI fragment (Fig. 1a) resembled strongly the one obtained previously for srmA (Richardson et al., 1987
), with the exception of a single PvuII site that we did not detect, perhaps reflecting the use of different S. ambofaciens strains. Moreover, hybridization experiments (data not shown) confirmed that the DNA fragment we had cloned was similar to the one previously isolated by these authors. Therefore, the resistance determinant present in pOS41.3 was designated srmA. When restriction fragments derived from the PstIKpnI insert were introduced into S. griseofuscus and examined for their ability to confer spiramycin resistance, they precisely located srmA within the BamHISplI fragment (Fig. 1a
).
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By alignment of SrmA with other deduced Erm-type protein sequences from actinomycetes, the dendrogram presented in Fig. 2 was constructed. SrmA is most closely related to Lrm and ErmSV, which monomethylate rRNA (Jenkins et al., 1989
; Kamimiya & Weisblum, 1997
), but as previously noted, it was not possible to distinguish between monomethyltransferases (conferring the MLS type I resistance phenotype) and dimethyltransferases (conferring the MLS type II resistance phenotype) on the basis of sequence comparison (Pernodet et al., 1996
). Further investigations were necessary to determine the precise mechanism by which SrmA confers resistance.
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Resistance phenotype conferred by srmA
These experiments utilized S. lividans OS456, a specifically deleted, antibiotic-sensitive strain that was used previously to define the MLS-I and MLS-II resistance phenotypes (Pernodet et al., 1996 ). For comparison, pOS41.44 containing srmA and pLST391 containing ermE were introduced separately into strain OS456 and the MIC values of various MLS antibiotics were determined. As seen in Table 2
, srmA conferred the MLS-I resistance phenotype characterized by high-level resistance to lincomycin, moderate resistance to some macrolides and streptogramin B antibiotics, and lower resistance to chalcomycin, tylosin and erythromycin. In comparison, ermE, which specifies the MLS-II phenotype, conferred high resistance to all MLS antibiotics.
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DISCUSSION |
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The gene srmA encodes a monomethyltransferase acting on A-2058 in the large rRNA. Other macrolide-producing actinomycetes in which genes conferring resistance to the autogenous antibiotic have been sought also contain at least one erm-type gene (see Fig. 2), except in the case of S. antibioticus, the oleandomycin producer. In this organism, no ribosomal resistance mechanism seems to be present (Fierro et al., 1987
). Therefore, although many macrolide producers modify their ribosomes, at least during the antibiotic-production phase, this is often not the only protection mechanism used, and ribosomal resistance is not compulsory in all macrolide producers. Here, when srmA was inactivated in S. ambofaciens, the resulting strain was viable and produced spiramycin, revealing that this particular resistance gene is dispensable. However, in addition to srmA, S. ambofaciens possesses another erm-type gene, srmD, which also confers ribosomal resistance to MLS antibiotics (unpublished data). Therefore, the inactivation of srmA does not imply that S. ambofaciens ribosomes are sensitive during the spiramycin-production phase.
The MLS-I resistance phenotype is characterized by a high level of resistance to lincosamides and variable resistance to macrolides, lower than that observed with the MLS-II phenotype. Type I erm genes are found in strains producing lincosamides or macrolides such as carbomycin or spiramycin (Fig. 2; also Calcutt & Cundliffe, 1990
), where the monomethylation of A-2058 appears to confer an adequate level of resistance. In contrast, type II erm genes are found in strains producing tylosin and erythromycin, against which monomethylation of A-2058 does not confer high-level resistance. In summary, although the paradigm for MLS resistance involves ermC, encoding an MLS-II phenotype in Staph. aureus, most erm-type genes yet characterized in strains that produce macrolides or lincosamides encode type I mechanisms. Interestingly, no erm-type gene (of either variety) has been isolated from streptogramin producers.
S. ambofaciens is not the only macrolide producer that harbours multiple erm-type genes. In the tylosin producer S. fradiae, ribosomes are constitutively monomethylated by the product of tlrD (Zalacain & Cundliffe, 1991 ), and the appearance of glycosylated macrolides induces expression of the erm type II gene tlrA (Kelemen et al., 1994
). In contrast, A-2058 is not constitutively methylated in S. ambofaciens and the interplay between srmA and srmD remains to be elucidated.
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ACKNOWLEDGEMENTS |
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Received 18 January 1999;
accepted 2 March 1999.