Dispensable ribosomal resistance to spiramycin conferred by srmA in the spiramycin producer Streptomyces ambofaciens

Jean-Luc Pernodet1, Anne Gourmelen1, Marie-Hélène Blondelet-Rouault1 and Eric Cundliffe2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptomyces ambofaciens produces the macrolide antibiotic spiramycin, an inhibitor of protein synthesis, and possesses multiple resistance mechanisms to the produced antibiotic. Several resistance determinants have been isolated from S. ambofaciens and studies with one of them, srmA, which hybridized with ermE (the erythromycin-resistance gene from Saccharopolyspora erythraea), are detailed here. The nucleotide sequence of srmA was determined and the mechanism by which its product confers resistance was characterized. The SrmA protein is a methyltransferase which introduces a single methyl group into A-2058 (Escherichia coli numbering scheme) in the large rRNA, thereby conferring an MLS (macrolide–lincosamide–streptogramin type B) type I resistance phenotype. A mutant of S. ambofaciens in which srmA was inactivated was viable and still produced spiramycin, indicating that srmA is dispensable, at least in the presence of the other resistance determinants.

Keywords: macrolide antibiotics, MLS resistance, rRNA methylation, spiramycin, Streptomyces ambofaciens

Abbreviations: MLS, macrolide–lincosamide–streptogramin type B; SAM, S-adenosylmethionine

The EMBL/GenBank accession number for the nucleotide sequence described in this paper is AJ223970.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptomyces spp. are filamentous Gram-positive bacteria that are well-known for producing many important antibiotics. Organisms that synthesize antibiotics to which they are potentially sensitive must have a resistance mechanism to protect themselves, at least during the phase of antibiotic biosynthesis. In these organisms, three different resistance strategies have been reported: modification of the antibiotic target, intracellular inactivation of the antibiotic and limitation of intracellular drug to subinhibitory levels via efflux and/or exclusion (Cundliffe, 1989 ).

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 (macrolide–lincosamide–streptogramin 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 ; O’Hara 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
Strains and plasmids used in this study are listed in Table 1.


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Table 1. Strains and plasmids used in this study

 
Media and microbiological methods.
Hickey–Tressner (HT) (Pridham et al., 1957 ) or NE (Skeggs et al., 1985 ) complete media were used for the growth and sporulation of Streptomyces strains at 30 °C. Liquid culture media for routine growth were YEME (Hopwood et al., 1985 ) or tryptic soy broth (TSB; Difco); MP5 (Pernodet et al., 1993 ) was used for spiramycin production. The culture conditions and the assay for spiramycin production (bioassay and HPLC) were as described elsewhere (Gourmelen et al., 1998 ). E. coli strains were grown at 37 °C, except when harbouring pKC505 or its derivatives (Richardson et al., 1987 ), in which case they were grown at 30 °C. Conjugal DNA transfer from E. coli to Streptomyces was performed essentially as described by Bierman et al. (1992 ) with slight modification (Gourmelen et al., 1998 ). MICs were determined on HT agar plates containing antibiotics, and seeded with 5x104 c.f.u. of spores per plate. Growth was monitored after 3 d incubation. Antibiotics were obtained from the following sources: apramycin, Eli Lilly; ampicillin, Appligene; carbomycin, Pfizer; chalcomycin, erythromycin, geneticin (G418), nalidixic acid, oleandomycin and tylosin, Sigma; hygromycin B, Boehringer Mannheim; josamycin, Pharmuka; rosaramicin, Schering-Plough; nosiheptide, pristinamycin I and spiramycin, Rhône-Poulenc Rorer.

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 30–40 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 {lambda} 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]dATP{alpha}S 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 transcription–translation assay.
Coupled transcription–translation was performed as described previously (Calcutt & Cundliffe, 1989 ). All components of the coupled transcription–translation 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 ).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of spiramycin-resistance determinants and subcloning of srmA
Cosmid DNA was extracted from a pool of about 20000 E. coli clones constituting the S. ambofaciens gene library in pKC505 and introduced by protoplast transformation into S. griseofuscus, a strain naturally sensitive to spiramycin. Transformants containing the vector were selected on apramycin and about 1300 apramycin-resistant colonies were picked onto HT medium containing 5 µg spiramycin ml-1. Several apramycin-resistant colonies also grew in the presence of spiramycin and cosmids extracted from these colonies were used to transform E. coli and S. griseofuscus. Five cosmids able to confer apramycin resistance in E. coli and co-resistance to spiramycin and apramycin in S. griseofuscus were conserved.

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 PstI–KpnI 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 EcoRI–HindIII fragment, yielding pOS41.3, which conferred spiramycin resistance in S. griseofuscus. The restriction map of the PstI–KpnI 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 PstI–KpnI insert were introduced into S. griseofuscus and examined for their ability to confer spiramycin resistance, they precisely located srmA within the BamHI–SplI fragment (Fig. 1a).



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Fig. 1. (a) Restriction map of the chromosomal region of S. ambofaciens ATCC 23877 near srmA. Lines below the restriction map represent the length of the inserts in various plasmids (see also Table 1). R and S indicate if the plasmid conferred spiramycin resistance or not, respectively. (b) Restriction map of the chromosomal region of S. ambofaciens OS41.102 in which srmA has been inactivated by gene replacement. The insert used in gene replacement experiments is represented schematically.

 
Nucleotide sequence of srmA
The nucleotide sequence of the BamHI–SplI fragment of pOS41.3 was determined for both strands. Analysis of the sequence for ORFs presenting the biased codon usage typical of Streptomyces genes (Bibb et al., 1984 ) revealed only one complete ORF, evidently corresponding to srmA, with truncated ORFs on either side. The deduced product of srmA is a protein of 259 aa (molecular mass 28·6 kDa) and sequence comparisons with databases showed end-to-end similarity to several rRNA methyltransferases that confer resistance to MLS antibiotics. For instance, SrmA presents 86% and 75% identity with the Lrm protein (Jenkins & Cundliffe, 1991 ) and ErmSV (Kamimiya & Weisblum, 1997 ), respectively. Also, SrmA contains, between residues 40 and 48, a glycine-rich sequence that is shared by other SAM-dependent methyltransferases (Kagan & Clarke, 1994 ) and has been shown by crystallographic analysis to be part of the SAM-binding site (Schluckebier et al., 1995 ). Since there is only one candidate start codon upstream of this motif in the gene sequence, we were able to assign the start of the coding sequence with confidence.

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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Fig. 2. Dendrogram of the deduced MLS resistance methyltransferase sequences from actinomycetes. The name of the protein, the MLS type (if known), the accession number of the sequence in databases (EMBL/GenBank or PIR), the name of the organism and, for producers of macrolides or lincosamides, the antibiotic produced are indicated.

 
Characterization of the SrmA resistance mechanism
Ribosomes from S. griseofuscus (pIJ487; control) or from S. griseofuscus harbouring pOS41.3 (srmA) were introduced into a coupled transcription–translation system in which all other components came from S. lividans. In this system, the effects of various MLS antibiotics on protein synthesis were studied. Ribosomes from the strain containing the cloned srmA gene were highly resistant to lincomycin and showed significant resistance to macrolides such as spiramycin and carbomycin (Fig. 3). This result clearly demonstrated that SrmA acts by ribosome modification. Experiments were also performed with S. ambofaciens ribosomes. When these came from young mycelium, not yet producing spiramycin, they resembled those from S. griseofuscus harbouring pIJ487, i.e. they were sensitive (data not shown). However, when derived from young mycelium in which resistance had been induced by subinhibitory concentrations of spiramycin (Pernodet et al., 1993 ), or from mycelium producing spiramycin, the ribosomes behaved like those from S. griseofuscus harbouring srmA (data not shown). Such behaviour was characteristic of particles that had been monomethylated by the products of erm type I genes (Pernodet et al., 1996 ).



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Fig. 3. Effects of antibiotics on protein synthesis in a coupled transcription–translation system. Ribosomes were from: (a) S. griseofuscus harbouring pIJ487 (control); (b) S. griseofuscus harbouring pOS41.3 (srmA). All other components were derived from S. lividans. Antibiotics added were: no drug ({blacksquare}); 1 µg carbomycin ml-1 ({circ}); 1 µg spiramycin ml-1 ({bullet}); 50 µg lincomycin ml-1 ({blacktriangledown}).

 
Methylation of rRNA
Since SrmA conferred resistance via ribosomal modification and was highly similar to authentic rRNA methyltransferases, we tested directly for the presence of clone-specific rRNA methyltransferase activity, using [methyl-3H]SAM as methyl donor and total rRNA as substrate. Methyltransferase activity was detected in S30 crude extracts and in ribosomal wash fractions from S. griseofuscus harbouring the cloned srmA gene, when total rRNA from S. griseofuscus harbouring pIJ487 (control strain) was used as substrate (Fig. 4). The stoichiometry of methylation was about 0·8. As a negative control total rRNA from S. griseofuscus harbouring srmA was used as substrate, and was not significantly methylated in vitro. Total rRNA from S. griseofuscus harbouring pLST391, a plasmid containing the ermE gene, was also not methylated by extracts containing SrmA. This result suggested that the ErmE and SrmA methyltransferases might act at the same or at closely related sites.



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Fig. 4. Methylation of rRNA in vitro. Extract from S. griseofuscus harbouring pOS41.3 (srmA) was used as the source of methyltransferase together with [methyl-3H]SAM as co-substrate. Total rRNA came from S. griseofuscus harbouring pIJ487 (control) ({blacksquare}), pOS41.3 (srmA) ({bullet}) or pLST391 (ermE) ({blacktriangleup}).

 
Identification of the methylated residue
rRNA was radiomethylated in vitro by extracts containing SrmA and submitted to acid hydrolysis or enzymic digestion, following which the products were separated by various chromatographic methods. When the products of acid hydrolysis (purine bases, pyrimidine-3'-mononucleotides and ribose phosphate) were separated by descending paper chromatography, using 2-propanol/HCl as solvent, a single radioactive spot migrated ahead of adenine (data not shown), together with N6-monomethyladenine and N6,N6-dimethyladenine standards, which are poorly resolved in this chromatographic system. However, when the radiolabelled material was eluted from the paper and analysed again with butanol/ammonia solvent, it co-migrated with the N6-monomethyladenine standard, which was clearly resolved from N6,N6-dimethyladenine (data not shown, but see Fig. 5). To confirm the nature of the modified residue, radiomethylated rRNA was completely digested by nuclease P1 and the nucleoside-5'-monophosphates obtained were separated by two-dimensional TLC. A single radioactive spot was detected, co-migrating with the N6-monomethyladenosine-5'-monophosphate standard (data not shown). Collectively, these data indicated that SrmA was a monomethyltransferase acting on adenine at position N6.



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Fig. 5. Location of the methylated residue within rRNA. Total rRNA was first methylated by extracts containing SrmA, in the presence of [methyl-3H]SAM. Then a large excess of cold SAM was added and incubation was continued after addition of: (a) buffer; (b) extract containing ErmE. The rRNA was then subjected to acid hydrolysis and the products were separated by paper chromatography. m6A, N6-monomethyladenine; A, N6,N6-dimethyladenine.

 
Location of the methylated residue within rRNA
rRNA modified by ErmE was a very poor substrate for SrmA, suggesting that both methyltransferases might act at the same site, although SrmA is a monomethyltransferase and ErmE a dimethyltransferase. ErmE can also introduce a second methyl group at the N6 position of A-2058 that has already been N6-monomethylated. Therefore, to know if SrmA acts on A-2058, a two-step methylation experiment was performed as previously described by Jenkins et al. (1989 ). Total rRNA was first radiolabelled in vitro by extracts containing SrmA, in the presence of [methyl-3H]SAM; then a 150-fold excess of non-radioactive SAM was added and the reaction was divided in two. Extract containing ErmE was added to one half of the reaction and incubation was continued for a further 45 min. As a control, buffer was added to the other half and incubation was carried out under similar conditions. Radiolabelled rRNA was then extracted, submitted to acid hydrolysis and the products were analysed by descending paper chromatography in the butanol/ammonia solvent system (Fig. 5). Since the N6-monomethyladenine produced by SrmA was converted into N6,N6-dimethyladenine after the action of ErmE, a single adenosine is modified by SrmA and this being the one also modified by ErmE, is A-2058 within the large 23S rRNA.

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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Table 2. MICs for various antibiotics against strains of S. lividans

 
Generation of an srmA null mutant in S. ambofaciens
We attempted to determine the role played by srmA in self-protection of S. ambofaciens against spiramycin by inactivating the gene in the S. ambofaciens ATCC 23877 wild-type strain. As shown in Fig. 1(b), the {Omega}hyg cassette (conferring hygromycin resistance in E. coli and Streptomyces) was used to disrupt srmA at the XhoI site within pOS41.102 (a conjugative vector derived from pOJ260, which confers geneticin resistance in E. coli and Streptomyces). Then, pOS41.102 was introduced into S. ambofaciens via conjugal transfer from E. coli S17.1 and hygromycin selection was applied. Five days after conjugation, hygromycin-resistant transconjugants were picked and examined for geneticin resistance. Hygromycin-resistant, geneticin-sensitive clones had presumably undergone a double crossover recombination event, resulting in the replacement of srmA by its disrupted counterpart from pOS41.102. This was confirmed by Southern blot hybridization analysis (data not shown). One such disrupted strain was designated OS41.102. This strain was not affected in its growth or differentiation and, when grown in MP5 medium, it produced spiramycin at a level comparable to the wild-type, as checked by bioassay and confirmed by HPLC (data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
At least five genes conferring resistance to macrolides have been isolated from S. ambofaciens (Richardson et al., 1987 ; Gourmelen et al., 1998 ; present work). The presence of several genes conferring resistance to the autogenous antibiotic is not unusual in antibiotic-producing actinomycetes. Among macrolide producers, pairs of resistance genes have been isolated from the carbomycin producer Streptomyces thermotolerans (Epp et al., 1987 ), and from ‘Micromonospora griseorubida’, the mycinamicin producer (Inouye et al., 1994 ). Four tylosin-resistance genes have been cloned from the tylosin producer Streptomyces fradiae (Birmingham et al., 1986 ; Baltz & Seno, 1988 ; Rosteck et al., 1991 ; Zalacain & Cundliffe, 1991 ), and at least five genes are supposed to be involved in oleandomycin resistance in Streptomyces antibioticus (Hernandez et al., 1993 ; Rodriguez et al., 1993 ; Olano et al., 1995 ; Quiros et al., 1998 ). In contrast, only single resistance genes have been isolated from ‘Streptomyces mycarofaciens’, the midecamycin producer (Hara & Hutchinson, 1990 ), and from the erythromycin producer Sacch. erythraea (Uchiyama & Weisblum, 1985 ; Dhillon & Leadlay, 1990 ).

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.


   ACKNOWLEDGEMENTS
 
We are very grateful to M. Zalacain for discussion, advice and kind help. We thank J. Gagnat for skilful technical assistance. We thank M. Richardson and R. Nagaraja Rao for the gift of cosmids (pKC505 and pKC534), M. Bibb for the gift of the ermE probe, and colleagues in the pharmaceutical industry for the gift of antibiotics. We also thank EMBO for a short-term fellowship awarded to J.-L.P. A.G. received a PhD fellowship from the Ministère de l’Education Nationale, de la Recherche et de la Technologie, and from the Fondation pour la Recherche Médicale.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 18 January 1999; accepted 2 March 1999.