INTRODUCTION

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Members of the genus Streptomyces produce a wide variety of secondary metabolites that include about half of the known microbial antibiotics. Many of these compounds have been applied in both human medicine and agriculture. Streptomyces coelicolor A3(2), the most fully genetically characterized streptomycete, is an appropriate strain for studying the regulation of antibiotic production (11, 29, 30). It produces at least four antibiotics, including the blue-pigmented polyketide antibiotic actinorhodin that is normally produced in stationary-phase cultures (9, 17). Much progress has been made in elucidating the organization of antibiotic biosynthesis gene clusters in several Streptomyces species, and a number of pathway-specific regulatory genes have been identified that are required for the activation of their cognate biosynthetic genes (18, 21, 34, 46, 53). In the actinorhodin biosynthetic pathway, actII-ORF4 plays such a pathway-specific regulatory role. The production of actinorhodin is mediated at the transcriptional level through activation of the actII-ORF4 promoter (22). In addition to pathway-specific regulatory genes, S. coelicolor possesses several genes with pleiotropic effects on antibiotic production. These genes fall into two classes: those that affect only antibiotic production (absA, absB, afsB, afsR, and abaA) (1, 8, 19, 31, 32) and those that affect both antibiotic production and morphological differentiation (bldA, bldB, bldC, bldD, bldG, and bldH) (28). Many other factors influence directly or indirectly the production of actinorhodin in S. coelicolor A3(2). It has been stressed that ppGpp (guanosine 3'-diphosphate, 5'-diphosphate), which is responsible for the so-called stringent response, plays a role in triggering the onset of antibiotic production, including the production of actinorhodin in S. coelicolor (7, 37, 41, 48, 49, 51).

We reported previously that certain mutations conferring streptomycin resistance give rise to secondary metabolite production (by an unknown mechanism) without the requirement for ppGpp in Streptomyces lividans and S. coelicolor A3(2) (25, 51, 55). Later, we demonstrated that the introduction of a specific str mutation into other bacterial genera gave rise to a marked increase in antibiotic productivity, thus further elucidating the mechanism in S. coelicolor A3(2) (33). Recently, we found that acquisition of resistance to other aminoglycoside antibiotics, such as gentamicin and Geneticin, also confers the ability to produce actinorhodin in S. lividans 66, which normally does not produce actinorhodin (H. Hu and K. Ochi, unpublished). Furthermore, by inducing mutations conferring resistance to rifampin, an ansamycin antibiotic, we were able to restore the impaired actinorhodin production in the relA and relC mutants of S. coelicolor A3(2) (Y. Tozawa, J. Xu, and K. Ochi, unpublished data). These results offer some available strategies for improving the productivity of antibiotics. Since the development of methods to improve the production of antibiotics is of considerable industrial and economic importance, we attempted in the present study to develop novel approaches for improving antibiotic-producing strains, especially focusing on methods to induce combined drug-resistant mutations. Some physiological aspects of the mutant strains are also described.

	MATERIALS AND METHODS

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Bacterial strains and preparation of mutants. The wild-type strain (1147) of S. coelicolor A3(2) and its mutants used in this study are listed in Table 1. Spontaneous streptomycin-resistant (Str^r), gentamicin-resistant (Gen^r), Geneticin-resistant (Gne^r ), or rifampin-resistant (Rif^r) mutants were obtained from colonies that grew within 7 days after spores (or cells) were spread on GYM agar containing various concentrations of the above drugs (see Table 2).

Media and growth conditions. GYM, R3, and R4 media were described previously (48, 55). All strains were stored as spore suspensions at 20°C. For use in each experiment, spore suspensions were spread onto GYM agar plates and incubated for 7 to 10 days at 30°C to allow for sporulation. Sterile distilled water (5 ml) was added to each plate, and the surface was gently scraped to release the spores. Suspensions were collected by centrifugation and washed twice with distilled water. Before being used for inoculation, the spores were dispersed for 10 min in a sonic bath. The concentrations of spores were about 2 × 10⁹ spores per ml. A total of 0.5 ml of spore suspension was inoculated into 150 ml of media. Cultivation of cells was carried out with 500-ml flasks containing 150 ml of media and incubated on a rotary shaker (200 rpm) at 30°C. In some experiments, cultivation was performed by using 25-ml test tubes each containing 5 ml of media and incubated on a reciprocal shaker (350 rpm) at 30°C.

Assay for actinorhodin. Culture samples (1 ml) were taken at each time point and adjusted to pH 8.0. After centrifugation was carried out at 1,100 × g for 5 min, the amount of the blue-colored antibiotic actinorhodin was determined by measuring the optical density of supernatants at 633 nm. Measurements were always taken from duplicate or triplicate cultures, and the mean values are presented in Table 2 or legends to Fig. 2, 3, and 4.

Determination of MIC and resistance. The lowest concentration of an antibiotic that totally inhibited growth through a 48-h incubation period at 30°C on GYM agar was defined as the MIC. The resistance levels were determined similarly to the MIC.

Mutation analyses of the rpsL and rpoB genes. The rpsL gene fragment of the streptomycin-resistant mutant (S-1, S-2, or S-3) was obtained by PCR using the mutants' genomic DNA as a template and the synthetic oligonucleotide primers P1 (forward: 5'-ATTCGGCACACAGAAAC-3') and P2 (reverse: 5'-AGAGGAGAACCGTAGAC-3'), which were designed from the sequence for S. lividans (DDBJ accession no. D83746). ExTaq (Takara) was used to perform PCR according to the manufacturer's instructions. A Perkin-Elmer Cetus thermal cycler was used at the following conditions: 5 min of incubation at 96°C; 30 cycles of 96°C for 18 s, 55°C for 12 s, and 72°C for 30 s; and a final step at 72°C for 10 min. PCR products were directly sequenced by the dideoxy chain termination procedure (54) using the BigDye Terminator Cycle Sequencing kit (Perkin-Elmer Applied Biosystems, Foster City, Calif.). The sequence data were analyzed with the GENETIX program (Software Development Co., Tokyo, Japan). The partial rpoB gene fragment (nucleotides 600 to 1300) of the rifampin-resistant mutants (R-1, R-2, R-3, etc.) was obtained by PCR using the mutants' genomic DNA as a template and the synthetic oligonucleotide primers P3 (forward: 5'-GGCCGCTACAAGGTGAACAAGAAG-3') and P4 (reverse: 5'-CGATGACGAAGCGGTCCTCC-3'), which were designed from the sequence for S. coelicolor M145. PCR and DNA sequencing were performed under the same conditions as those for the rpsL gene.

Western blotting analysis. Cultures were grown on GYM agar plates covered with cellophane sheets at 30°C for 36 to 72 h. Cells were scraped off the cellophane sheet; suspended in 20 mM Tris-HCl (pH 7.0) containing 1 mM EDTA, 1 mM dithiothreitol, 10% (vol/vol) glycerol, and 0.5 mM phenylmethylsulfonyl fluoride; and disrupted by sonication as described previously by Gramajo et al. (23). Protein concentrations were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots were developed with the enhanced chemiluminescence Western blotting detection system for chemiluminescent detection as specified by the manufacturer. Polyclonal antiserum against the ActII-ORF4 protein was prepared in rabbits and used as a primary antibody at a dilution of 1:3,000.

	RESULTS

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Construction of combined resistant mutants. First, we introduced a single drug-resistant mutation into S. coelicolor wild-type strain 1147; the results are summarized in Table 2. The mutants with enhanced actinorhodin production (we tested 80 to 150 resistant isolates per antibiotic) were detected at a high frequency (5 to 10%) among streptomycin-resistant, gentamicin-resistant, or rifampin-resistant isolates. The highest productivity detected for each mutant strain ranged from 1.6 to 3 times the wild-type production level (Table 2). It is notable that the gen mutants with enhanced actinorhodin productivity all demonstrated low levels of resistance (a threefold-higher MIC) to gentamicin, whereas the str or rif mutants revealed either a low or high level of resistance (5- to 100-fold-higher MIC of streptomycin; 5- to 40-fold-higher MIC of rifampin). Several representative strains are listed in Tables 1 and 3.

View larger version (64K): [in this window] [in a new window]   FIG. 1.   Ability to produce aerial mycelia and actinorhodin in S. coelicolor wild-type strain 1147 and mutant strains. Spores were inoculated on R4 or R3 agar plates and incubated at 30°C for 6 days. A blue color represents the pigmented antibiotic actinorhodin.

Mutational analyses of the mutants. There is strong evidence that streptomycin resistance frequently results from a mutation in the rpsL gene, which encodes the ribosomal protein S12 (33, 55), while rifampin resistance results from a mutation in the rpoB gene, which encodes the -subunit of RNA polymerase (24, 35, 59). We therefore sequenced and compared the rpsL gene and rpoB gene from the mutants to the wild-type strain. As summarized in Table 3, the str mutant with high resistance to streptomycin (strain S-1) contained a mutation within the rpsL gene, where the altered nucleotide (from A to G) was found at position 262, resulting in an alteration of Lys-88 to Glu. str mutants with low levels of streptomycin resistance (S-2 and S-3) showed no mutation in the rpsL gene. Although certain mutations in the16S rRNAs or the rpsD gene, which encodes the ribosomal S4 protein, have been known to confer streptomycin resistance (4, 43), no mutation was found in either.

Physiological characterization of the mutants. Production of actinorhodin by wild-type and mutant strains was medium dependent. This was especially pronounced in the wild-type, single-mutant, and double-mutant strains (Fig. 2). In contrast, triple (str gen rif) mutants produced a high level of actinorhodin, irrespective of the medium used for cultivation, indicating the superior ability of these triple mutants to produce the antibiotic. Although triple mutants ultimately produced a high level of actinorhodin, production commenced at the same time as for the wild-type strain when examined with R4 and R3 media (Fig. 3). The triple mutants (i.e., SGR-1) (Fig. 3) revealed a somewhat reduced growth rate. It should be noted that the actinorhodin production by the triple mutant continued for a longer period of time (4 days) than by the single or double mutants (2 days).

View larger version (68K): [in this window] [in a new window]   FIG. 2.   Comparison of actinorhodin production between media R3 () and R4 (). Actinorhodin production was determined after 6 days of incubation.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Growth and actinorhodin production in media R4 and R3. Symbols: , 1147 (wild type); , S-1 (str); , SG-1 (str gen); , SGR-1 (str gen rif).

View larger version (13K): [in this window] [in a new window]   FIG. 4.   The effect of yeast extract, Casamino Acids, and KH2PO4 on the production of actinorhodin. Strains were grown in R4 medium supplemented with various concentrations of yeast extract, Casamino Acids, or KH2PO4 for 6 days. Symbols: , 1147 (wild type); , S-1 (str); , SG-1 (str gen) ; , SGR-1 (str gen rif).

Genetic characterization of the mutants. The ActII-ORF4 protein has been characterized as a DNA binding protein that positively regulates the transcription of the actinorhodin biosynthesis genes (2). Transcription of actII-ORF4 is growth-phase dependent in liquid culture, reaching a maximum output during the transition from exponential to stationary phase (22). We therefore analyzed the expression level of the ActII-ORF4 protein by Western analysis in the wild-type and mutant strains (Fig. 5). Cells were grown on GYM agar covered with a cellophane sheet, scraped off, disrupted by sonication, and then subjected to Western analysis (see Materials and Methods). Wild-type strain 1147 produced a basal level of ActII-ORF4 during the middle (36 h) and late (60 h) growth phases that increased when cells entered into the stationary phase (72 h). It should be stressed that the amount of ActII-ORF4 increased (especially at transition phase) in the following hierarchical order: single (S-1), double (SG-1), and triple (SGR-1) mutants (Fig. 5). The increase in ActII-ORF4 was especially pronounced in the triple mutant (SGR-1) at 60 h, reflecting the superior ability (see above) of this strain. The dramatically increased expression of ActII-ORF4 is transient, as the expression of the triple mutant decreased later (at 72 h) to the same level as that of the wild-type strain (Fig. 5).

View larger version (40K): [in this window] [in a new window]   FIG. 5.   Western blotting analysis of the ActII-ORF4 protein, a pathway-specific positive regulator in the actinorhodin biosynthesis pathway. Cultures were grown at 30°C for the denoted time on a GYM agar plate covered with a cellophane sheet (see Materials and Methods). Each lane contained 20 µg of total proteins.

	DISCUSSION

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In the present study, we successfully developed a new approach for improving antibiotic producers by inducing combined drug-resistant mutations. This method not only results in a remarkable increase in antibiotic productivity (48-fold higher in GYM medium) but also makes it possible to generate positive mutations at a high frequency (5 to 15%). Although much progress has been made in improving antibiotic producers (10, 39, 40), our method is characterized by the host cell's amenability (generation of spontaneous drug-resistant mutation) and the method's applicability to a number of microorganisms (if not all) as demonstrated previously with other bacteria (33, 55). It should also be emphasized that combined resistant mutations (triple mutations) demonstrated no significant impairment in growth or sporulation under the conditions tested. However, since the triple mutants (e.g., SGR-1) had a reduced growth rate (Fig. 3), it is highly likely that the effect of the mutations is actually to alter growth rates and the timing of entrance into secondary metabolism.

A high level of resistance to streptomycin has been previously shown to result from a point mutation in the rpsL gene, which encodes the ribosomal protein S12 (20, 25, 27, 33, 42, 55). In agreement with this, our str mutant (S-1), which showed a high level of resistance, contained a mutation (Lys-88 to Glu) in the rpsL gene. However, those strains with low levels of resistance (S-2 and S-3) showed no mutation in the rpsL gene. It has been reported that mutations in the 16S rRNAs (530 loop region) or the rpsD gene, which encodes the ribosomal S4 protein, can confer streptomycin resistance (4, 43). Gentamicin resistance is known to result from a mutation in the rplF gene, which codes for the ribosomal L6 protein (5, 38). Mutations occur within their putative RNA-binding sites (14). Nevertheless, we could not detect this mutation in our mutant strains, which had a low level of resistance to streptomycin or gentamicin, although we analyzed every 16S rRNA, rpsD, and rplF sequence. However, it is possible that our mutants have a mutation in certain ribosomal components other than the 16S rRNAs, S4, and L6 proteins.

Aminoglycosides are the best characterized as a class of antibiotics that bind directly to rRNA, cause a decrease in translational accuracy, and inhibit translocation of the ribosome (15, 16). These antibiotics bind to a conserved sequence in the rRNA that is near the site of codon-anticodon recognition in the aminoacyl-tRNA site (A site) of the 30S subunit. Aminoglycoside binding stabilizes the tRNA-mRNA interaction in the A site by decreasing tRNA dissociation rates; this decrease interferes with proofreading steps that ensure translational fidelity (36). The action of streptomycin on bacterial ribosomes has been studied in great detail (reviewed by Wallace et al. [58] and Cundliffe [13]), and among the numerous effects attributed to this drug, the misreading of the mRNA codons is the best known. Gentamicin (and also Geneticin) belongs to the kanamycin class of aminoglycosides but is structurally different from streptomycin when classified on the basis of structural characteristics. The L6 mutations are drastic because they result in large deletions of an RNA-binding region; thus, they may indirectly affect proofreading by locally distorting the EF-Tu-GTP-aminoacyl tRNA-binding site on the large subunit (14, 60). It is well known that S12 mutations, which can confer streptomycin resistance, can in general increase the accuracy of protein synthesis. More recently, defined regions of the 16S rRNA have also come to be associated with the ribosome's accuracy function (43, 45, 52). It is notable that translational accuracy can also be affected by two components of the 50S subunit, the ribosomal protein L6 and the 2660 loop region of the 23S rRNA. Mutations have been identified in these components that result in a decreased rate of translation, greater accuracy in protein synthesis, and increased resistance to many of the misreading-inducing aminoglycoside antibiotics, in particular gentamicin (5, 38, 44, 57). Failure in identifying these mutations in our mutant strains may be due to the fact that, unlike the previously identified mutations as described above, we are dealing with mutations which confer only a low level of (or even slight) resistance to streptomycin or gentamicin.

Previous studies dealing with various bacteria have indicated that mutations in the rpoB gene, which codes for the -subunit of RNA polymerase, are responsible for the acquisition of resistance to rifampin (24, 35, 56, 59). Almost all the mutations found are located on a specified conserved region of the rpoB gene that can be divided into three clusters: I, II, and III. In the present study, most of the rif mutants exhibited point mutations in either cluster I or II, resulting in an amino acid alteration in one of eight sites. All of these sites correspond to previously known positions conferring rifampin resistance, although some new substitution types have been found. These results are apparently related to previous findings that show that the guanine nucleotide ppGpp is a pivotal signal molecule for the onset of antibiotic production (7, 41, 47, 51), since ppGpp has been proven to bind to the -subunit of E. coli RNA polymerase (12). ppGpp [and (p)ppGpp] is believed to be responsible for the stringent response, which causes an immediate cessation of RNA synthesis and other cellular reactions (for reviews, see Cashel et al. [6]). Strains with mutated relA (which codes for ppGpp synthetase) or relC = rplK (which codes for the ribosomal L11 protein) fail to synthesize normal levels of ppGpp. Although the relA and relC mutants of various Streptomyces spp. exhibit a severely impaired ability to produce antibiotics due to the failure to synthesize ppGpp (7, 37, 48, 50, 51), we have found that the acquisition of certain rif mutations by S. coelicolor relA and relC mutants restores the antibiotic productivity lost in these mutants (Y. Tozawa, J. Xu, and K. Ochi, unpublished data). These rif mutants, like the rif mutants used in the present study, have mutations in the -subunit of RNA polymerase. The dependence of S. coelicolor on ppGpp to initiate antibiotic production is therefore apparently bypassed by certain mutations in RNA polymerase. It is possible that the remarkable enhancement of ActII-ORF4 expression which accompanies the overproduction of actinorhodin (Fig. 5) is based on the independence of cells on ppGpp in initiating the secondary metabolism. The mutant RNA polymerase may function by mimicking the ppGpp bound form.

Despite the lack of detail on the molecular level for the effects of these mutations, overexpression of the ActII-ORF4 protein by introducing str, gen, and rif mutations accounts well for the observed hierarchical increase in actinorhodin productivity, since ActII-ORF4 plays a crucial role in activating the genes necessary for actinorhodin biosynthesis (2). Antibiotic production is in general subjected to the suppressive effects caused by an excess of nutrients such as carbon, nitrogen, and phosphate sources (26). In particular, ammonium and phosphate both appear to be major regulators of antibiotic production in S. coelicolor A3(2) and their control systems may be interrelated in some way (26). Consistent with this notion, our results revealed that actinorhodin production in wild-type and mutant strains is more or less medium dependent, displaying more production in R4 medium (containing less yeast extract and phosphate) than in R3 medium (Fig. 3). It is important to point out that the triple (str gen rif) mutants constructed in the present study all revealed less sensitivity to such suppressive effects (Fig. 2 through 4). Our new breeding approach could be effective especially for initially improving the production of antibiotics from wild-type strains and may be effective not only for antibiotic production but also for certain enzyme production linked with secondary metabolism.