A Novel Tryptophanyl-tRNA Synthetase Gene Confers High-Level Resistance to Indolmycin

Bacterial strains and culture conditions.The strains used in this work are provided in Table 1. Escherichia coli strains DH5α and ET12567/pUZ8002 were grown in Luria-Bertani medium at 37°C for routine subcloning (30). Streptomyces species were grown at 30°C on mannitol soya flour medium (SFM), Difco nutrient agar medium, yeast extract-malt extract medium (YEME), or minimal liquid medium (NMMP) (20). SFM was used for conjugations between S. coelicolor and E. coli and for generating spore suspensions. For transcriptional analysis, S. coelicolor and Streptomyces avermitilis strains were grown in NMMP. The indolmycin producer S. griseus ATCC 12648 was grown in NMMP without polyethylene glycol (PEG) for transcriptional analysis and the liquid chromatography-mass spectrometry (LC-MS) analysis of culture supernatants. For selecting E. coli, ampicillin, apramycin, chloramphenicol, hygromycin, and kanamycin were employed at 100, 50, 25, 80, and 50 μg/ml, respectively. Nalidixic acid was used at 20 μg/ml to counterselect E. coli in conjugations with S. coelicolor. Apramycin and hygromycin were used at 50 μg/ml for selecting S. coelicolor.

Plasmids and primers.Standard cloning procedures were employed in generating the plasmids listed in Table 2 (30). The site-specific integrating vector pMS81 (10) was used for genetic complementation as previously described (36). pIJ10257 was used for overexpression in S. coelicolor (12). DNA sequencing was performed by Davis Sequencing (Davis, California). Table 3 shows the primers used in this work, all of which were synthesized by Invitrogen. PCR was performed with Taq (Invitrogen) and Pfu (Stratagene, Agilent Technologies). All PCRs were performed with 5% (vol/vol) dimethylsulfoxide (DMSO) (20).

Indolmycin and chuangxinmycin.(±)-Indolmycin was chemically synthesized according to the procedure developed by Hasuoka et al. (11). The ¹H and ¹³C nuclear magnetic resonance spectra, as well as mass spectra, of the synthetic material were identical to those reported for authentic indolmycin (11). All amounts of (±)-synthetic indolmycin described herein are corrected to reflect only the active enantiomer. Chuangxinmycin was generously provided as a gift from Richard L. Jarvest of GlaxoSmithKline (3).

Selection of indolmycin-resistant mutants.An S. coelicolor B725 (trpRS1::apr; indolmycin sensitive) spore suspension containing 10¹⁰ spores was spread over SFM supplemented with 1 μg/ml indolmycin. Several spontaneous low-level indolmycin-resistant mutants arose after incubation at 30°C for 7 days. Fifteen mutants were randomly harvested, and the trpRS2 locus of each mutant was analyzed by PCR using genomic DNA as the template with primers 15 and 16 (Table 3). Four low-level indolmycin-resistant mutants were chosen for selection for higher-level resistance (strains B730 to B733) (Table 1). A suspension containing 5 × 10⁹ spores of each strain was spread over SFM supplemented with 10 μg/ml indolmycin; spontaneous high-level indolmycin-resistant mutants arose from each strain after incubation at 30°C for 7 days (strains B734 to B737) (Table 1).

Site-directed mutagenesis.Mutations were created in the S. coelicolor trpRS2 open reading frame (ORF) using the Stratagene QuikChange site-directed mutagenesis kit by following the manufacturer's protocol. Using plasmid pJS330 (Table 2) as the template with primers 23 and 24 (Table 3), the TrpRS2 [G(−13)T/L9F] locus was created. The TrpRS2[G(−13)T/H48Q] locus was created using pJS330 as the template with primers 27 and 28 (Table 3). Plasmid pJS329 (Table 2) was used as the template with primers 25 and 26 (Table 3) to create the TrpRS2(L9F/Q13K) ORF, and the TrpRS2(L9F/H48Q) ORF was created using pJS329 as the template with primers 27 and 28. The TrpRS2(Q13K) ORF was created using plasmid pJS328 (Table 2) as the template with primers 25 and 26.

MIC assays.All MIC assays were performed on Difco nutrient agar medium supplemented with the indicated concentrations of indolmycin. Growth was assessed after incubation at 30°C for 48 h.

RNA isolation and RT-PCR.The transcriptional analyses of the S. coelicolor trpRS1 and trpRS2 genes were performed as described previously (36). To analyze the transcription of the tryptophanyl-tRNA synthetase genes in other Streptomyces species, two NMMP cultures were inoculated for each organism to test transcription in the presence or absence of indolmycin. Strains were grown for 21 h prior to treatment with 40 μg/ml indolmycin for 3 h. Cells were washed once with 10.3% (wt/vol) aqueous sucrose, resuspended in L (lysis) buffer (20), and incubated for 15 min at 30°C. Total RNA then was isolated using the RNeasy Mini kit (Qiagen) by following the manufacturer's protocol. The concentration of each purified, DNA-free total RNA isolate was measured with a NanoDrop ND-1000 spectrophotometer. An equal quantity of total RNA (1.8 μg) was employed in all reverse transcription-PCRs (RT-PCRs). RT-PCR was performed with the OneStep RT-PCR kit (Qiagen) according to the manufacturer's protocol for transcripts with high GC content (which requires the use of the Q-solution). Primers used for RT-PCR analyses are listed in Table 3 (primers 1 to 14). The PCR program used for the detection of all transcripts was 50°C for 30 min, 95°C for 15 min, N cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 60 s, and a final elongation at 72°C for 10 min. For the analysis of SAV3417, 35 cycles was employed. All other genes were analyzed at 25 cycles except SAV4725 and SGR3809, which were analyzed at 20 cycles. No signals were detected in control experiments with Pfu polymerase, indicating that the RT-PCR signals correspond to the amplification of transcripts.

Detection of indolmycin in culture media by LC-MS.A published procedure was adapted for the isolation and detection of indolmycin (37). For the LC-MS analysis of secreted indolmycin in culture supernatants, S. griseus ATCC 12648 was grown in NMMP without PEG. Culture filtrates then were adjusted to pH 3 with 4N HCl and extracted twice with 20 ml ethyl acetate. The organic phases were combined and extracted with 4 ml of a 10% (wt/vol) Na₂CO₃ solution and then dried over Na₂SO₄. Solvent was removed under reduced pressure to leave residues that were purified twice by solid-phase extraction using strata-x 60-mg, 3-ml columns (part no. 8B-S100-UBJ-TN; Phenomenex) by following the manufacturer's protocol. Purified residues were dissolved in 100 μl methanol for LC-MS analysis. LC-MS analysis was performed with a Thermo LCQ DECA XP MAX ion trap mass spectrometer (C₁₈ column; gradient: 5 to 65% mobile phase B for 30 min, where mobile phase B is acetonitrile plus 0.1% formic acid and mobile phase A is water plus 0.1% formic acid; Waters part no. WAT058965).

Sequence accession numbers.All sequences described in this work are available at the NCBI database under the accession numbers listed in Table S1 in the supplemental material. The nucleotide sequences of both tryptophanyl-tRNA synthetase genes from the indolmycin producer S. griseus ATCC 12648 were deposited in the GenBank database under accession numbers FJ744752 (SGI3809) and FJ744751 (SGItrpRS2).

An auxiliary tryptophanyl-tRNA synthetase gene from S. griseus NBRC 13350 encodes an indolmycin-resistant enzyme.Bioinformatic analysis revealed that Streptomyces griseus NBRC 13350 has a unique auxiliary tryptophanyl-tRNA synthetase gene (SGR3809) (Fig. 2A). The tryptophanyl-tRNA synthetase encoded by SGR3809 is only 34% identical in amino acid sequence to TrpRS1, a known indolmycin resistance determinant from S. coelicolor. Curiously, auxiliary tryptophanyl-tRNA synthetases homologous to SGR3809 were observed in several bacterial genera (Fig. 2B). We detected a cDNA 93% identical in sequence to SGR3809 by the RT-PCR analysis of RNA from the indolmycin producer, Streptomyces griseus ATCC 12648 (see Fig. S1 in the supplemental material). The indolmycin producer is classified distinctly from S. griseus NBRC 13350, which does not produce indolmycin.

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FIG. 2.

(A) Alignment of trpRS1 with other indolmycin resistance genes. The aligned sequences show the region surrounding the HIGH signature consensus of class I aminoacyl-tRNA synthetases. Positions found to correlate to indolmycin resistance in this study are shaded yellow, and the class I aminoacyl-tRNA synthetase signature sequence HIGH is shaded blue. trpRS1 is highly similar in sequence to the indolmycin-resistant tryptophanyl-tRNA synthetase gene SAV4725 from S. avermitilis but is dissimilar to the indolmycin-resistant tryptophanyl-tRNA synthetase gene SGR3809 from S. griseus NBRC 13350 (SGR3809). NCBI accession numbers are provided in the supplementary information (see Table S1 in the supplemental material). (B) Translated sequences of the trpRS1 and SGR3809 genes aligned with homologs from various bacterial species. NCBI accession numbers are provided in the supplementary information (see Table S1 in the supplemental material).

The observation of an SGR3809 homolog in the indolmycin producer and the robust growth of S. griseus NBRC 13350 in media supplemented with inhibitory concentrations of indolmycin suggested that SGR3809 encodes an antibiotic-resistant enzyme. S. griseus NBRC 13350, like all other indolmycin-resistant streptomycetes, also grows in the presence of chuangxinmycin (Fig. 3); these are the only bacteria known to be resistant to chuangxinmycin. To determine if SGR3809 is an antibiotic resistance gene, it was heterologously expressed in Streptomyces coelicolor B725, the indolmycin-sensitive trpRS1 null strain (Table 1) (36). In this genetic background, its capacity to confer indolmycin resistance was compared to that of trpRS1 and its homolog in S. avermitilis (SAV4725). The indolmycin MIC for S. coelicolor B725 increased from 0.25 to 15 μg/ml upon the expression of the novel tryptophanyl-tRNA synthetase gene SGR3809 (Table 4). A similar MIC elevation was observed upon the expression of trpRS1 in this background (Table 4). Thus, SGR3809 encodes an antibiotic-resistant tryptophanyl-tRNA synthetase.

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FIG. 3.

Various Streptomyces species grown in the presence of inhibitory concentrations (100 μM) of indolmycin and chuangxinmycin. SAV, Streptomyces avermitilis ATCC 31267; SCO, Streptomyces coelicolor M600; SGI, Streptomyces griseus ATCC 12648; SGR, Streptomyces griseus NBRC 13350; SLI, Streptomyces lividans TK24; SVZ, Streptomyces venezuelae ATCC 10712.

Antibiotic-resistant tryptophanyl-tRNA synthetase genes are transcribed differently.The transcription of the S. coelicolor trpRS1 gene is induced in the presence of indolmycin and chuangxinmycin (22, 36). Transcriptional analyses of the antibiotic-resistant tryptophanyl-tRNA synthetase genes in other streptomycetes were performed to determine if they are regulated in a similar fashion. Using RT-PCR analysis, we found that the transcription of the trpRS1 ortholog in S. avermitilis (SAV4725) also is induced by indolmycin (Fig. 4A). In contrast, SGR3809 in S. griseus NBRC 13350 and its ortholog in the indolmycin producer were constitutively transcribed (Fig. 4B). Curiously, the transcript of the SGR3809 ortholog from the indolmycin producer was present when secreted indolmycin could not be detected by LC-MS (Fig. 4B; also see Fig. S5 in the supplemental material). This observation is noteworthy because in antibiotic-producing organisms, resistance genes often are cotranscribed with genes encoding enzymes for antibiotic biosynthesis (13, 33).

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FIG. 4.

RT-PCR analyses of the tryptophanyl-tRNA synthetase genes from indolmycin and chuangxinmycin-resistant Streptomyces species. (A) RT-PCR analyses of tryptophanyl-tRNA synthetase genes in S. avermitilis and S. coelicolor. The transcription of the trpRS1 homolog SAV4725 also is induced in S. avermitilis upon exposure to indolmycin. (B) RT-PCR analyses of tryptophanyl-tRNA synthetase genes in the streptomycin producer S. griseus NBRC 13350 and in the indolmycin producer S. griseus ATCC 12648. The transcription of these genes was not influenced by indolmycin.

Selection of indolmycin-resistant mutants yields point mutations in the tryptophanyl-tRNA synthetase ORF and promoter.We used the indolmycin-sensitive S. coelicolor trpRS1 null strain B725 to select for spontaneous indolmycin-resistant mutants. We sought to compare the identities of resistance-conferring substitutions to the sequences of the native indolmycin-resistant enzymes. This strain lacks trpRS1 but has the trpRS2 gene that encodes an indolmycin-sensitive tryptophanyl-tRNA synthetase. In the initial selection, the indolmycin-sensitive trpRS1 null mutant (MIC, 0.25 μg/ml) was grown on solid medium supplemented with 1 μg/ml of indolmycin. Spontaneous indolmycin-resistant mutants were observed at a frequency of approximately 10⁻⁸. Fifteen indolmycin-resistant mutants were isolated, and their TrpRS2 loci were cloned and sequenced. Only 1 of the 15 strains had a mutation in the trpRS2 locus; mutations in the other strains were not identified. This strain (B730; see Table S1 in the supplemental material) had a single point mutation in the trpRS2 ORF that changed leucine at position 9 to phenylalanine [TrpRS2(L9F)].

A number of high-level resistant strains were selected from the low-level resistant strains (i.e., strain B730 and strains B731 to B733 with unidentified resistance-conferring mutations). The selected high-level resistant strains had point mutations in the trpRS2 ORF and/or upstream of the trpRS2 transcription start site. Two strains selected at 10 μg/ml had single point mutations in the trpRS2 ORF that resulted in substitutions of the histidine at position 48 of the synthetase; one strain (B734) had TrpRS2(H48N) and the other (B735) had TrpRS2(H48Q).

To enable the direct comparison of the trpRS2 point mutants and the trpRS1 and SGR3809 genes, they were cloned into pIJ10257 for expression under the control of the constitutive ermE* promoter (ermE*p) and introduced into the S. coelicolor trpRS1 null strain (B725). TrpRS2 L9F increased the MIC for the null strain 20-fold (Table 5); by comparison, TrpRS1 increased the MIC 40-fold (Table 4). It is remarkable that TrpRS2(H48N) and TrpRS2(H48Q) increased the MIC for the null strain more than 300-fold (Table 5), far more than the native resistance genes (Table 4).

Two spontaneous high-level indolmycin-resistant strains had point mutations immediately upstream of the trpRS2 ORF. These mutations were predicted to lie within the trpRS2 promoter region (36) (Fig. 5). One strain (B736) (Table 1) had a point mutation (G to T) 13 nucleotides upstream of the predicted trpRS2 start codon. This mutation changed the sequence of the predicted −10 region (GAGAAT to TAGAAT; the change is in boldface) such that it matched the −10 consensus (T-A-G-purine-purine-T) for streptomycete promoters (31) (Fig. 5). The other strain (B737) (Table 1) had a point mutation (C to A) in the spacer region between the −10 and −35 regions of the trpRS2 promoter, as well as a mutation in the ORF that substitutes phenylalanine for leucine at position 9 (Fig. 5). To compare the resistance-conferring promoter mutations, trpRS2 loci with either the mutation in the −10 region or the mutation in the spacer region were cloned and introduced into the S. coelicolor trpRS1 null strain B725 via the integrative plasmid pMS81. The G-to-T mutation in the −10 region increased the MIC 20-fold, while the C-to-A mutation in the −10 and −35 spacer increased the MIC only 8-fold (Table 5). The results of these selections demonstrate that point mutations in the trpRS2 promoter can contribute significantly to indolmycin resistance.

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FIG. 5.

Mutations in the trpRS2 promoter that confer resistance to indolmycin. Brackets mark the boundaries of the trpRS2 promoter. The −10 and −35 regions are in boldface, and the spontaneous mutation sites are shaded gray.

Combinations of mutations in the trpRS2 locus synergistically enhance indolmycin resistance.We used site-directed mutagenesis to determine if specific combinations of mutations in the trpRS2 locus would act synergistically with respect to indolmycin resistance. Indeed, promoter mutations and ORF mutations enhanced indolmycin resistance synergistically (Table 5). We also examined combinations of resistance-conferring point mutations in the trpRS2 ORF. The mutation causing the L9F substitution was combined with a mutation that changes glutamine at position 13 to lysine (Q13K). The former mutation increased the MIC for the null strain 20-fold (Table 5). The latter mutation was reported to confer weak resistance to trpRS2 (22), and in our studies it enhanced the MIC for the null strain only 12-fold (Table 5). These mutations acted synergistically; the MIC for the null strain expressing the double mutant was 32-fold higher than that for the parent (Table 5). It is interesting that this TrpRS2 (L9F/Q13K) double mutant confers nearly as much indolmycin resistance as TrpRS1 (Tables 4 and 5). The combination of L9F and H48Q did not have an obvious effect on indolmycin resistance below 300 μg/ml (Table 5), but the strain expressing this double mutant grew poorly compared to the growth of the wild-type strain (data not shown).