INTRODUCTION

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Macrolides inhibit protein synthesis by binding to the large ribosomal subunit (14). Several mechanisms of resistance to macrolides have been described. The most widespread consists of target modification by methylation of 23S rRNA (for a review, see reference 45). Resistance can also be the result of antibiotic export (23; reference 41 and references included) or inactivation. Various chemical modifications leading to macrolide inactivation have been described phosphorylation (24, 25), esterification of the lactone ring (2, 26), and glycosylation (11, 21, 33, 38). All these resistance mechanisms have been identified in pathogens and in Streptomyces spp., both those producing and those not producing macrolide antibiotics. Often, macrolide producers have several genes involved in self-resistance. For instance, in Streptomyces fradiae, the tylosin producer, four resistance genes have been cloned (4, 9, 35, 47).

Streptomyces ambofaciens produces the macrolide antibiotic spiramycin (31). Spiramycin is composed of a 16-atom lactone ring with two amino sugars and one neutral sugar attached. The mycelium is sensitive to spiramycin during exponential growth phase and then becomes resistant, with the concomitant production of spiramycin, during stationary phase. In S. ambofaciens, at least two resistance mechanisms are present (29). Several resistance determinants have been cloned (34, 39). One of them, srmA, encodes a monomethyltransferase acting at position A2058 of 23S rRNA (28), thereby conferring resistance to macrolides-lincosamides-streptogramin B (type I resistance phenotype [30]). Here we report the cloning, sequencing, and characterization of gimA, a new macrolide resistance gene located downstream of srmA.

	MATERIALS AND METHODS

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Strains and culture conditions. Strains and plasmids used in this study are listed in Table 1. Streptomyces strains were maintained on Hickey-Tressner (HT) medium at 30°C (32). For spiramycin production, S. ambofaciens strains were grown in MP5 liquid medium (29) at 27°C. Streptomyces lividans strains were maintained on R2YE medium (17) at 30°C. AS1 medium (3) was used for conjugation experiments, and cultures were grown at 37°C (see below). S. lividans strains were grown in tryptic soy broth medium (TSB; Difco) at 30°C for preparation of S30 cellular extracts. To ensure maintenance of plasmids carrying the thiostrepton resistance gene (tsr), nosiheptide (NO) was added to the solid medium at 40 µg/ml and to the liquid medium at 10 µg/ml for S. lividans cultures, and at 200 µg/ml to the solid medium for S. ambofaciens cultures.

Antibiotics. The antibiotics were obtained from the following sources: hygromycin B (HM), Boehringer (Mannheim, Germany); ampicillin, Appligene (Illkirch, France); chalcomycin, tylosin, geneticin (GN), erythromycin, oleandomycin, and nalidixic acid, Sigma (St. Louis, Mo.); carbomycin and azithromycin, Pfizer (Orsay, France); josamycin, Pharmuka (Genevilliers, France); rosaramicin, Schering-Plough Corporation (Union, N.J.); neospiramycin, forocidin, spiramycin, NO, and CG, Rhône-Poulenc Rorer (Paris, France); angolamycin, lankamycin, methymycin, and pikromycin, E. Cundliffe (Leicester University, Leicester, United Kingdom).

DNA manipulations and bacterial transformation. DNA extraction and manipulation and transformations of Escherichia coli and Streptomyces were performed according to standard protocols (17, 36).

DNA sequencing. The DNA sequencing protocol was as described by Sanger et al. (37), with modifications as described by Biggin et al. (8). A Deaza T7 sequencing kit was obtained from Pharmacia, and -³⁵S-dATP was obtained from Amersham. Sequences were determined for both strands.

Computer-assisted sequence analysis. Sequence comparisons with databases were performed with the FASTA (27) and BLAST programs (1).

Preparation of crude extracts. This preparation was performed essentially as described by Cundliffe (11), except that cell breakage was obtained by ultrasonic treatment. Spores were used to inoculate TSB. Cultures were grown with vigorous shaking for 24 h. Mycelia were harvested by filtration, washed twice with HS buffer (10 mM HEPES-KOH [pH 7.5], 10 mM MgCl₂, 1 M NH₄Cl, 5 mM -mercaptoethanol) and once with HRS buffer (10 mM HEPES-KOH [pH 7.5], 10 mM MgCl₂, 50 mM NH₄Cl, 5 mM -mercaptoethanol). Mycelia were resuspended in HRS buffer prior to cell breakage by ultrasonication, followed by centrifugation at 30,000 × g for 30 min. The supernatant was then dialyzed against HM buffer (10 mM HEPES-KOH [pH 7.5], 5 mM MgCl₂, 5 mM -mercaptoethanol, 10% glycerol), resulting in S30 extracts, and stored as aliquots at 70°C. For S. ambofaciens, cultures were grown in TSB containing spiramycin at 10 µg/ml for 20 h, and 10 µg of spiramycin per ml was again added 90 min before harvesting. Spiramycin was added because the cultivation time was too short to allow production and appearance of the resistance. As the expression of some resistance genes (29), including srmA and probably gimA (15), could be induced by spiramycin, this treatment ensured their early expression.

In vitro inactivation of macrolides. Complete reaction mixtures (total volume, 100 µl) contained dialyzed S30 extract (90 µl), UDP-glucose (1 mM), and macrolide (100 µg/ml) and were incubated at 30°C. At time zero and after various times of incubation, samples were removed and frozen at 20°C to stop the reaction. They were then applied to Whatman AA paper discs (6 mm in diameter), and residual antibiotic activity was examined by bioassay with M. luteus.

Glycosyl transferase assays. The experiments were performed according to the method developed by Cundliffe (11) except that S30 extracts were used instead of S100*. S30 extract was incubated (total volume, 110 µl) with 0.125 µCi of UDP-[¹⁴C]glucose (specific activity, 335 mCi/mmol or 12.4 GBq/mmol) and 50 µg of macrolide antibiotic. The extent of ¹⁴C-glycosylation of the drug was calculated by the subtraction of background values obtained from drug-free control assays. For S. lividans harboring the cloned gene, 10 µl of S30 extracts was used, while for S. ambofaciens, 100 µl was needed for the assays.

Resistance conferred by gimA. Various dilutions of spore suspensions of S. lividans OS456(pOS41.90) or -(pIJ903) (control) were plated on HT medium supplemented with increasing concentrations of various macrolides or without antibiotics. The cultures were then incubated for 48 h before counting was performed.

Spiramycin production assays. For spiramycin production, MP5 was inoculated with spores at a concentration of 2.5 × 10⁶ spores/ml. No pregermination treatment was done before inoculation. The cultures (70 ml in 500-ml baffled flasks) were incubated at 27°C in an orbital shaker at 250 rpm. In order to take in account the variability of individual cultures, six identical cultures of each strain were included in the study. At 0, 32, 45, 54, 72, and 94 h, samples were withdrawn, and supernatants were independently frozen. The presence of spiramycin in the supernatant of each sample was detected and quantified by bioassay against M. luteus and by high-pressure liquid chromatography (HPLC).

Conjugal transfer from E. coli to S. ambofaciens. Conjugal transfer was performed essentially as described by Bierman et al. (7). Spores of S. ambofaciens ATCC 23877 or OS81 (Table 1) pregerminated as described by Hopwood et al. (17) were used. The optimal conditions were found to be 10⁶ S. ambofaciens CFU and 6 × 10⁷ E. coli CFU per plate. Plates were incubated at 37°C. After 18 h, they were washed with LB and gently scraped to remove the E. coli layer. Then they were covered with an overlay containing HM (at a final concentration of 50 µg/ml) to select transconjugants and nalidixic acid (at a final concentration of 50 µg/ml) to inhibit E. coli growth. Incubation was continued for 5 days.

Nucleotide sequence accession number. The nucleotide sequence obtained in this study has been deposited in the GenBank/EMBL database under accession no. AJ223970.

	RESULTS

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Cloning and sequencing of gimA. Downstream of macrolide resistance gene srmA (28), sequence analysis revealed a truncated open reading frame (ORF). The putative GTG start codon of that ORF overlapped the srmA stop codon (GTGA). To investigate a possible role of this second ORF in resistance, the cloning of the complete ORF was undertaken. An S. ambofaciens gene library constructed in cosmid pWED1 (Table 1) was probed by colony hybridization with a DNA fragment containing srmA and the truncated ORF. This led to the isolation of cosmid pOS41.78, from which a 3.6-kb BamHI fragment containing srmA and 1.7 kb of downstream DNA sequence was subcloned into pUC19 to yield pOS41.80. The sequence of the region downstream of srmA was determined. It revealed the presence of a 1,254-bp ORF which could encode a 45-kDa protein (data not shown). Its GTG start codon was preceded, 8 bp upstream, by the sequence GAGGAG, which could constitute a good Shine-Dalgarno sequence. This ORF showed a typical Streptomyces codon usage (46). The analysis of the deduced protein sequence revealed high degrees of similarity to proteins deduced from mgt of S. lividans (82.5% identity) (20) and oleD of Streptomyces antibioticus (80.9% identity) (16). Mgt from S. lividans is a glycosyl transferase inactivating macrolides by the addition of a glucose residue (11, 20). The gene mgt is also cited as mgtA in the literature. S. lividans is not known to produce any macrolide. In S. antibioticus, producer of the macrolide oleandomycin, OleD is a glycosyl transferase inactivating several macrolides including oleandomycin (33). Because of the strong similarity of the product of the ORF downstream of srmA to Mgt from S. lividans, we hypothesized that this product might also be a glycosyl transferase inactivating macrolides. Therefore, the ORF was called gimA.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Map of the chromosome of S. ambofaciens in the region of srmA and gimA. The inserts found in the plasmid constructions used for this study are indicated. The names of the corresponding vectors are in parentheses. The srmA-gimA chromosomal regions of S. ambofaciens strains, in which gimA has been inactivated by gene replacement, are also presented.

Activity of the gimA product. In a first approach, experiments were performed to test if the gimA product could inactivate macrolides. As S. ambofaciens possesses several other resistance genes, this was done with the cloned gimA gene. pOS41.90 was introduced into S. lividans OS456 (Table 1), a mutant strain of S. lividans in which the resistance genes lrm and mgt have been inactivated, providing a host strain highly sensitive to macrolides and with no background Mgt activity (30). Crude S30 extracts were prepared from OS456(pOS41.90) and from OS456(pIJ903) (control strain containing the vector without insert) and used for various tests: inactivation of macrolides followed by biological assay of residual antibiotic activity with M. luteus as the indicator strain and glycosylation tests using UDP-[¹⁴C]glucose.

View larger version (127K): [in this window] [in a new window]   FIG. 2.   In vitro inactivation of chalcomycin by S30 extracts from S. lividans OS456(pOS41.90). Paper disks impregnated with samples from inactivation assays were used to test residual antibiotic activity on M. luteus. Samples came from complete reaction mixture: S30 extract, chalcomycin, and UDP-glucose (A); reaction mixture without UDP-glucose (B); and reaction mixture without S30 (C).

Substrate specificity of GimA. The radiotransfer assay allows quantification of the GimA activity. This assay was used to study the substrate specificity of GimA, in order to compare it with those of other known macrolide-inactivating glycosyl transferases from S. lividans (11), S. antibioticus (33), and Streptomyces hygroscopicus (38). Moreover, the radiotransfer assay should indicate if GimA could glycosylate spiramycin precursors in the biosynthetic pathway. This last point could not be studied by inactivation, because some of the precursors of spiramycin lacked antibiotic activity.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Glycosylation of macrolide antibiotics with extracts from S. lividans OS456(pOS41.90). Reaction mixtures contained crude GimA together with 5 µl of UDP-[14C]glucose and 5 µl of macrolide solution in dimethyl sulfoxide (at 10 mg/ml) as described in Materials and Methods. (A) Glycosylation of various macrolides. The same results were obtained with erythromycin, angolamycin, and lankamycin; only those for lankamycin are shown. (B) Glycosylation of spiramycin and its precursors compared with glycosylation of chalcomycin.

Resistance profile conferred by gimA. It was unknown whether a macrolide-inactivating glycosyl transferase could confer a resistant phenotype or not. To answer this question, the resistances of S. lividans OS456(pOS41.90) to various macrolides were compared to those of S. lividans OS456(pIJ903) (control strain). This study could only be performed with the cloned gimA gene and not with S. ambofaciens, which possesses several resistance mechanisms.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Resistance conferred by gimA. Spores from S. lividans OS456(pOS41.90) and OS456(pIJ903) (control) were plated on HT medium without or with different macrolides at various concentrations. The survival rates at various macrolide concentrations, each calculated as the ratio of the number of CFU on the medium with the macrolide to the number of CFU on the same medium without the antibiotic (log scale), are presented for OS456(pOS41.90) (white squares) and OS456(pIJ903) (black squares).

Gene replacement inactivating gimA in S. ambofaciens. In order to determine the role played by gimA in the self-protection of S. ambofaciens from spiramycin, its inactivation was attempted both in the ATCC 23877 wild-type strain and in the non-spiramycin-producing mutant OS81 (Table 1). As shown in Fig. 1, an SplI fragment internal to gimA was deleted and replaced by the hyg cassette, generating pOS41.98 (in pUC19) and then pOS41.99 (in conjugative vector pOJ260; conferring GN resistance) (Table 1). Then, pOS41.99 was introduced into S. ambofaciens via conjugal transfer from E. coli S17.1 (40) and HM selection was applied. Five days after conjugation, Hm^r transconjugants were picked and examined for their GN resistance. Hm^r Gn^s clones were obtained from both strains. Such clones had presumably lost the pOJ260 part by a double-crossover recombination event. The replacement of gimA by its disrupted counterpart from pOS41.99 was confirmed by Southern blot analysis (data not shown). For instance, the gimA probe revealed that the hybridizing BamHI fragment had increased from 3.6 kb in the wild type to 6.1 kb in the mutant strains. Hybridization with hyg confirmed its presence in the mutants and its absence in the wild-type and OS81 strains. One mutant strain with gimA interrupted derived from the wild-type strain was designated OS41.99. One equivalent mutant derived from OS81 was designated OS41.99NP. These two strains were not affected in their growth or sporulation.

Effect of gimA on spiramycin production. The issue investigated was the ability of strain OS41.99 to produce spiramycin, and the possible effect of gimA disruption or overexpression on the level of spiramycin production. OS41.99 was first grown in MP5 production medium, and the supernatant activity was tested by a bioassay using M. luteus Cg^r (Table 1) and further characterized by HPLC. It was found that OS41.99 could still produce spiramycin, indicating that gimA is not a resistance gene essential for the survival of the strain, even under antibiotic production conditions.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Effect of gimA expression on spiramycin production in S. ambofaciens. Six identical independent cultures of each strain were grown in MP5 medium. Samples were withdrawn at 0, 32, 45, 54, 71, and 94 h. Spiramycin was detected and quantified by HPLC. For each strain, the curve was drawn from the averages of the six independent values obtained for each time. wt, wild type.

Evidence for the existence of another glycosyl transferase inactivating macrolides in S. ambofaciens. S30 extracts were prepared from the S. ambofaciens wild-type strain and the OS41.99 mutant. Surprisingly, extracts from both strains inactivated chalcomycin in the presence of UDP-glucose (data not shown). Moreover, these extracts were used in radiotransfer assays, and a comparison of the results presented in Fig. 3A and 6A shows that the substrate specificity of GimA was clearly different from that of the activity detected in OS41.99. For instance, lankamycin, which was a very poor substrate for GimA (Fig. 3A), was found to be a very active substrate for the activity detected in both S. ambofaciens strains (Fig. 6). The activity observed in the wild-type strain (Fig. 6B) could result from the addition of both activities. A comparison of results presented in Fig. 6A and 6B shows that lankamycin was glycosylated to comparable levels with extracts from both strains but that gimA inactivation led to a decrease in the glycosylation levels of chalcomycin, tylosin, and methymycin, which were good substrates for GimA. These observations indicated the existence of another macrolide-inactivating glycosyl transferase activity, with a substrate specificity different from that of GimA.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Glycosylation of macrolides with S30 extracts from S. ambofaciens. Reaction mixtures contained crude extract together with 5 µl of UDP-[14C]glucose and 5 µl of macrolide solution in dimethyl sulfoxide (at 10 mg/ml) as described in Materials and Methods. (A) Extracts from OS41.99 (gimA-null mutant); (B) extract from the wild-type strain.

	DISCUSSION

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The mechanism of macrolide inactivation by glycosylation at the C-2' position has been most often described for Streptomyces: this glycosyl transferase activity was first described for Streptomyces vendargensis acting on erythromycin A (21) and seems widespread among Streptomyces strains (38). It was studied for strains not producing macrolides such as S. lividans (11), and S. hygroscopicus (38). In S. antibioticus, producer of the macrolide oleandomycin, two glycosyl transferases (OleD and OleI) and a glycosidase (OleR) are involved in oleandomycin modification during its biosynthesis (33).

In S. ambofaciens, the gimA gene was found immediately downstream of srmA (28), a gene encoding a methyltransferase conferring macrolide resistance by target modification. As shown in Fig. 7, macrolide-inactivating glycosyl transferase genes are associated with methyltransferase genes in S. ambofaciens, in S. lividans, and probably in Streptomyces viridochromogenes. The last two strains are not known to produce any macrolide antibiotics. For S. antibioticus, no methyltransferase gene is found upstream of oleD or oleI. This is in agreement with the observation that ribosomes from S. antibioticus are sensitive to oleandomycin, even during production (13). Interestingly, the two ORFs upstream of oleD have high degrees of similarity to those upstream of srmA. This could indicate that some deletion or insertion events occurred at the corresponding loci in S. antibioticus or in S. ambofaciens. In S. lividans, the region located upstream of lrm is completely different from those found in the three other strains, indicating transfer may have occurred in this region.

View larger version (11K): [in this window] [in a new window]   FIG. 7.   Comparison of the genetic environments around macrolide resistance genes in various Streptomyces species. gimA, oleD, and mgt gene products have about 80% identity. srmA, lrm, and ermSV encode monomethyltransferases with about 80% identity. The deduced products of orf1SA and orf2SA from S. ambofaciens and of orf1 and orf2 from S. antibioticus have, respectively, 75 and 76% identity. The deduced product of the end of an ORF upstream of ermSV has 69% identity with the deduced products of orf2 and orf2SA. The deduced product of the short sequence downstream of ermSV is similar to glycosyl transferases inactivating macrolides. The GenBank/EMBL accession numbers are AJ223970 for gimA, Z22577 for oleD, M74717 for mgt, and U59450 for ermSV.

The substrate specificities of macrolide-inactivating glycosyl transferases from S. lividans, S. antibioticus, and S. hygroscopicus and that of GimA from S. ambofaciens are different. The size of the lactone ring does not seem to be a factor discriminating the poor and good substrates as discussed by Cundliffe (11). As in other cases, the sugar glycosylated by GimA is probably the one attached to the C-5 position of the 16- and 14-member lactone rings (21) (corresponding to C-3 in the 12-member lactone ring of methymycin). On this sugar, the position modified is probably that of the 2'-OH group. As expected in this case, angolamycin, which lacks this hydroxyl group, was revealed to be a very poor substrate of GimA. Our results, with the exception of those for tylosin, are in agreement with the suggestion that macrolides monosubstituted at position C-5 are better substrates than those disubstituted (43).

The ability of glycosyl transferases inactivating macrolides to confer resistance in vivo has never been investigated until now. This was done with the cloned gene gimA, introduced into a strain susceptible to macrolides, S. lividans OS456. Resistance was indeed observed, but the levels of resistance were very low, in particular, much lower than those conferred by target modifying enzymes: those encoded by either tlrD, ermE (30), or srmA (28). The low level of resistance was probably not due to a low level of expression of gimA. Indeed, cellular extracts prepared from OS456(pOS41.90), the strain used for the whole-cell resistance study, showed greater glycosyl transferase activity than the ones prepared from the S. ambofaciens wild-type strain. Although the level of resistance conferred by GimA was low, GimA nevertheless conferred resistance to chalcomycin and tylosin, while SrmA was unable to confer any resistance to these two macrolides.

In a strain producing a macrolide antibiotic, a gene encoding a glycosyl transferase inactivating macrolides might play a role in the self-protection of the strain, as oleI does in S. antibioticus. Nevertheless, gene gimA could be inactivated in S. ambofaciens without any deleterious effect, even during production of spiramycin. But S. ambofaciens possesses additional macrolide resistance mechanisms (including a second macrolide-inactivating glycosyl transferase), which might provide protection against the produced antibiotic.

In S. antibioticus, oleI is located among oleandomycin biosynthetic genes and its product is supposed to be the enzyme responsible for oleandomycin glycosylation during biosynthesis. oleD is not located in the oleandomycin cluster and might play a more general role, one not directly involved in oleandomycin biosynthesis (33). In S. ambofaciens, gimA is dispensable; it is probably not located in the spiramycin cluster, and its product is not very active on spiramycin. A comparison of genetic organization and protein sequences indicated that gimA is more related to mgt and oleD than to oleI. Therefore, gimA could be involved in the resistance to exogenous macrolides rather than in self-protection against spiramycin during biosynthesis. However, the constitutive overexpression of gimA led to a decrease in spiramycin production. This effect could be due to the action of GimA on spiramycin itself, or more probably on spiramycin precursors such as forocidin. These glycosylated precursors might not be substrates for later steps of the biosynthetic pathway, or the glycosylated spiramycin produced might not be reactivated.