Characterization of an Inducible, Antibiotic-Resistant Aminoacyl-tRNA Synthetase Gene in Streptomyces coelicolor

Abstract

Streptomyces coelicolor has two genes encoding tryptophanyl-tRNA synthetases, one of which (trpRS1) is resistant to and transcriptionally activated by indolmycin. We found that this gene also confers resistance to chuangxinmycin (another antibiotic that inhibits bacterial tryptophanyl-tRNA synthetases) and that its transcription is not absolutely dependent on either antibiotic.

Streptomyces bacteria and their relatives among the actinomycetes are the producers of nearly two-thirds of the known secondary metabolites with antibiotic properties. As producers of antibiotics, these organisms harbor genes that confer resistance to their toxic metabolites (7, 21). There is considerable evidence that these resistance genes are readily exchanged between streptomycetes and are frequently transferred from streptomycetes to other bacterial genera (9, 10, 27). Although antibiotic resistance can be advantageous to bacteria, there is mounting evidence that antibiotic-resistant bacteria suffer a significant fitness cost (1, 3, 12). Specifically, bacteria that constitutively express antibiotic resistance genes or have resistance-conferring point mutations in essential genes grow poorly compared to their antibiotic-sensitive counterparts (1, 12). Some bacteria circumvent the fitness cost associated with antibiotic resistance because they harbor resistance genes that are expressed only in the presence of antibiotics (11). Presumably, bacteria with inducible antibiotic resistance genes are more fit than those that constitutively express resistance genes or have resistance-conferring point mutations in essential genes.

An intriguing case of antibiotic resistance involves the inducible transcription of a gene encoding an antibiotic-resistant tryptophanyl-tRNA synthetase in Streptomyces coelicolor (19). S. coelicolor has two genes encoding tryptophanyl-tRNA synthetases, whose products are 47% identical (2). Kitabatake et al. reported that one gene, trpRS2 (SCO4839), is constitutively transcribed, while the other, trpRS1 (SCO3334), is induced by indolmycin (19). Indolmycin (Fig. 1), produced by Streptomyces griseus ATCC 12648 (22, 23), is a structural analog of l-tryptophan that competitively inhibits bacterial tryptophanyl-tRNA synthetases (26). In vitro enzymatic assays showed that TrpRS2 is indolmycin sensitive, whereas TrpRS1 is highly resistant to the antibiotic (19). Because S. coelicolor does not produce indolmycin (2, 18), it is presumed that it acquired the indolmycin-resistant tryptophanyl-tRNA synthetase gene (trpRS1) from a streptomycete that produces indolmycin. It is unclear how indolmycin triggers the transcription of the trpRS1 gene in S. coelicolor (19).

FIG. 1.

Structures of indolmycin, tryptophan, and chuangxinmycin. Indolmycin and chuangxinmycin are structural analogs of tryptophan that competitively inhibit bacterial tryptophanyl-tRNA synthetases (5, 26). The indolmycin used in these studies was chemically synthesized by us (see the supplemental material). Chuangxinmycin was provided as a gift from GlaxoSmithKline.

Herein, we describe the construction of trpRS1 and trpRS2 null mutants of S. coelicolor. We show that the trpRS1 null mutant is sensitive to two different antibiotics that inhibit bacterial tryptophanyl-tRNA synthetases and that the trpRS2 null mutant constitutively transcribes trpRS1 when grown in the absence of antibiotics. Thus, trpRS1 is an antibiotic resistance determinant in S. coelicolor, and its transcription is not absolutely dependent on antibiotics. We also report transcriptional analyses that reveal the transcription start sites of both genes and provide new insights into the mode of trpRS1 transcription.

Based on heterologous expression of the S. coelicolor trpRS1 gene in Escherichia coli, Kitabatake et al. predicted that it is the indolmycin resistance determinant in S. coelicolor (19). To prove definitively that trpRS1 confers resistance to indolmycin, we used a PCR targeting procedure (13) to replace the trpRS1 gene in wild-type S. coelicolor M600 with an apramycin resistance cassette, yielding trpRS1 null strain B725 (ΔtrpRS1::apr). The disruption of trpRS1 reduced the indolmycin MIC for S. coelicolor from >150 μg/ml to <1 μg/ml. Unlike the wild-type strain, the trpRS1 null mutant was also incapable of growth on Difco nutrient agar (18) containing 100 μM (23 μg/ml) of chuangxinmycin (Fig. 1), an antibiotic produced by Actinoplanes tsinanensis that also competitively inhibits bacterial tryptophanyl-tRNA synthetases (5). Thus, the S. coelicolor trpRS1 gene provides resistance to both indolmycin and chuangxinmycin (Fig. 2) despite the differences in their structures. Although multiple modes of indolmycin resistance are known (4, 15, 17), to our knowledge, this is the first time that resistance to chuangxinmycin has been reported. Apparently, TrpRS1 has unique structural features that render it insensitive to competitive inhibition by both indolmycin and chuangxinmycin. These resistance-conferring structural features do come at a cost to the activity of TrpRS1, which has a fourfold-lower catalytic efficiency than TrpRS2 in vitro (19).

FIG. 2.

Phenotypes of wild-type S. coelicolor M600, S. coelicolor B725 (ΔtrpRS1::apr), and S. coelicolor B728 (ΔtrpRS2::apr). The strains were grown for 2 days at 30°C on solid Difco nutrient agar (18) with and without 100 μM indolmycin or 100 μM chuangxinmycin. See the supplemental material for complete information regarding the construction of these strains.

Because S. coelicolor is resistant to indolmycin and chuangxinmycin, we hypothesized that both antibiotics are inducers of trpRS1 transcription. Indeed, the trpRS1 transcript was detected by reverse transcription-PCR (RT-PCR) in S. coelicolor cultures to which indolmycin or chuangxinmycin had been added to a final concentration of 100 μM (Fig. 3A). Based on those observations, it was possible that both antibiotics directly activate the transcription of trpRS1. Alternatively, the induced transcription of trpRS1 may not have been a response to a specific antibiotic but to a loss of TrpRS2 activity. Consistent with the latter hypothesis was the phenotype of the trpRS2 null mutant (S. coelicolor B728 ΔtrpRS2::apr) that was also constructed by PCR targeting (13). Unexpectedly, the trpRS2 null mutant grew robustly in the absence of indolmycin (Fig. 2). Furthermore, the trpRS1 transcript was detected in RNA isolated from the trpRS2 null mutant grown in the absence of indolmycin (Fig. 3B). Taken together, these observations indicate that the loss of TrpRS2 activity is sufficient for the induction of trpRS1 transcription. It is noteworthy that in NMMP medium (18), the trpRS2 null mutant grows at the same rate as that of the wild-type strain despite the reported low catalytic efficiency of TrpRS1 in vitro (data not shown). To further examine the mechanism of trpRS1 induction, selected protein synthesis inhibitors were tested for their ability to induce its transcription. Interestingly, serine hydroxamate, a known inhibitor of seryl-tRNA synthetase (24, 25), induced trpRS1 transcription in wild-type S. coelicolor. Among three antibiotics tested that directly inhibit the ribosome (i.e., hygromycin, spectinomycin, and thiostrepton) (18), only hygromycin induced trpRS1 transcription (Fig. 3A). The profile of the inducers suggests that a perturbation of translation is the stimulus for trpRS1 transcription.

FIG. 3.

(A) Induction of trpRS1 transcription by protein synthesis inhibitors. The 914-bp and 427-bp RT-PCR products correspond to the trpRS1 and trpRS2 transcripts, respectively. RT-PCR was used to analyze 1.8 μg of RNA isolated from shaken liquid cultures of wild-type S. coelicolor M600 grown in NMMP (18) or in NMMP supplemented with one of the following: 100 μM indolmycin (25 μg/ml), 100 μM chuangxinmycin (23 μg/ml), 25 mM serine hydroxamate (3 mg/ml), 95 μM hygromycin (50 μg/ml), 404 μM spectinomycin (200 μg/ml), or 30 μM thiostrepton (50 μg/ml). All RNA samples were isolated 3 h after the addition of the antibiotics to a stationary-phase culture. The primers (see Table S3 in the supplemental material) and RT-PCR conditions (recommended by manufacturer [Qiagen]) used are indicated in the supplemental material. (B) Induction of trpRS1 transcription in the trpRS2 null mutant. RT-PCR analysis of 1.8 μg of RNA isolated from S. coelicolor B728 (ΔtrpRS2::apr) cells grown for 24 h as a shaken culture in liquid NMMP medium without antibiotics at 30°C yielded a 914-bp product corresponding to the trpRS1 transcript. This transcript was not detected in 1.8 μg RNA isolated from wild-type S. coelicolor grown under the same conditions. The primers (see Table S3 in the supplemental material) and RT-PCR conditions (recommended by manufacturer [Qiagen]) used are indicated in the supplemental material.

To further investigate the transcription of trpRS1 and trpRS2, we used 5′ rapid amplification of cDNA ends (5′-RACE) experiments to map the transcription start sites of both genes (Fig. 4A) (20). Curiously, we found that the trpRS2 transcription start site was 6 bp downstream of the bioinformatically proposed ATG translation start codon (Fig. 4B). Based on alignments of TrpRS protein sequences, it is likely that the true ATG start codon coincides with the transcription start site such that trpRS2 is transcribed as a leaderless message (16). On the other hand, the transcription of trpRS1 was mapped to two sites 157 bp and 173 bp upstream of its predicted translational start site (Fig. 4C). This result was observed in two separate 5′-RACE experiments with different primer sets.

FIG. 4.

(A) Mapping of the transcriptional start sites of trpRS1 and trpRS2 via 5′-RACE. The primers (see Table S3 in the supplemental material) and PCR conditions (recommended by manufacturer [Invitrogen]) used are indicated in the supplemental material. Lane 1 contained 5 μl of Bioline Hyperladder I (Bioline). Lane 2 contained the PCR product amplified from cosmid StE7 using primers MK54 and SCO3334 GSP2D. It spans the region from −14 to +262 bp relative to the trpRS1 translational start site. Lane 3 contained the 5′-RACE product amplified from trpRS1 cDNA modified with a 3′ oligo(dC) tail using primers AAP and SCO3334 GSP2D. This band is a mixture of 5′-RACE products with sequences identical to the region at −173 to +262 bp or −157 to +262 bp relative to the trpRS1 translational start site. Lane 4 contained the PCR product amplified from cosmid StE7 using primers MK54 and SCO3334 GSP2E. It spans the region at −14 to +237 bp relative to the trpRS1 translational start site. Lane 5 contained the 5′-RACE product amplified from trpRS1 cDNA modified with a 3′ oligo(dC) tail using primers AUAP and SCO3334 GSP2E. This band is a mixture of 5′-RACE products with sequences identical to the region at −173 to +237 bp or −157 to +237 bp relative to the trpRS1 translational start site. Lane 6 contained the PCR product amplified from cosmid St5G8 with primers SCO4839 FOR and SCO4839 GSP2A. It spans the region at +1 bp to +526 bp relative to the trpRS2 translation start site. Lane 7 contained the 5′-RACE product amplified from trpRS2 cDNA modified with a 3′ oligo(dC) tail using primers AAP and SCO4839 GSP2A. Its sequence is identical to the region at +7 bp to +526 bp relative to the trpRS2 translational start site. Differences between the sizes of the PCR products (lanes 2, 4, and 6) and the sizes of the corresponding 5′-RACE products (lanes 3, 5, and 7) reflect the lengths of the transcripts' 5′ untranslated regions. (B) Partial sequence of the trpRS2 locus from S. coelicolor (2). An asterisk marks the transcriptional start site, and the probable translational start site is italicized. The bioinformatically proposed translational start site is underlined. (C) Partial sequence of the trpRS1 locus from S. coelicolor (2). The transcriptional start sites are marked by asterisks, and the predicted translational start site is underlined.

The conclusions of our 5′-RACE analysis of the trpRS1 transcript were validated by genetic complementation of the trpRS1 null mutant in trans (Fig. 5). The integrative plasmid pJS308 containing the trpRS1 open reading frame with 484 bp of upstream DNA restored antibiotic resistance when introduced into the trpRS1 null mutant. Consistent with the positions of the trpRS1 transcription start sites, the integrative plasmid pJS307, containing the trpRS1 open reading frame with only 49 bp upstream of the translation start site, failed to complement the trpRS1 null mutant.

FIG. 5.

Complementation of the trpRS1 null mutant. Shown is the region of the wild-type S. coelicolor chromosome spanned by the complementation constructs pJS307 and pJS308. The EcoNI restriction fragment in pJS307, which includes 49 bp upstream of the predicted trpRS1 translation start site (2), does not complement S. coelicolor B725 (ΔtrpRS1::apr). The EcoO109I restriction fragment in pJS308, which includes 484 bp upstream of the predicted trpRS1 translation start site (2), does complement S. coelicolor B725 (ΔtrpRS1::apr). See the supplemental material for complete information regarding the construction of these plasmids, strains, and growth conditions.

In summary, we have established that the trpRS1 gene in S. coelicolor confers resistance to two antibiotics that inhibit bacterial tryptophanyl-tRNA synthetases. The experiments reported here further show that indolmycin and chuangxinmycin are only indirectly responsible for the induction of trpRS1 transcription. The induction of trpRS1 by indolmycin, chuangxinmycin, serine hydroxamate, and hygromycin suggests that the inducing signal may be a perturbation of translation. Since the trpRS1 transcript was detected in the trpRS2 null mutant grown in medium without antibiotics, the absence of TrpRS2 activity must also perturb translation such that trpRS1 is induced. Serine hydroxamate and other aminoacyl-tRNA synthetase inhibitors are known to induce the stringent response (ppGpp production) in bacteria (25), including S. coelicolor (24). The induction of trpRS1 transcription by serine hydroxamate suggests that this phenomenon may be connected to the stringent response (14, 24). Efforts toward identifying the signal that activates trpRS1 transcription and the machinery that transduces this signal are in progress.