Jill Stonesifer and Richard

Streptomyces fradiae JS6 (mcr-6) is defective in the repair of potentially lethal damage to DNA induced by mitomycin C (MC), hydroxylamine (NH2OH), methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (NQO), Nmethyl-N'-nitro-N-nitrosoguanidine (MNNG), and ultraviolet light (UV), but it exhibits nearly normal sensitivity to ethyl methanesulfonate (EMS)-induced lethality. JS6 is substantially less mutable by MNNG, MMS, NQO, UV, NH2OH, and also EMS than is the parental strain. A spontaneous revertant of JS6 showed wild-type levels of resistance to all of these agents and wild-type levels of induced mutagenesis, indicating that a single mutation caused the multiple traits displayed by JS6. The mcr-6 gene product thus appears to control an errorprone (mutagenic) DNA repair system. Mediation of EMS mutagenesis by an error-prone repair pathway in S. fradiae, rather than by direct mispairing as in Escherichia coli, suggests that the streptomycetes have evolved more efficient erroravoidance mechanisms than those commonly observed in the single-celled eubacteria. Streptomycetes are Gram-positive filamentous bacteria having remarkable capabilities to produce antibiotics of diverse chemical structure and biological activity (1). They have circular genomes (1, 2) approximately three times as large as the genome ofEscherichia coli (3), and they differentiate and form aerial spores as do many eukaryotic fungi. Recent reports indicate that streptomycetes can contain extensive amounts of reiterated DNA (4-8), a trait common to eukaryotic but not to prokaryotic organisms. Thus the streptomycetes may occupy an evolutionary position more advanced than the common unicellular eubacteria. We have begun studies on mechanisms of mutagenesis in Streptomycesfradiae, a commercially important species that produces the macrolide antibiotic tylosin (9, 10). We report here that most induced mutagenesis in S. fradiae is genetically controlled and appears to occur by error-prone DNA repair (or replication). The error-avoidance mechanisms in S. fradiae appear to be more highly evolved than those in E. coli, and closely resemble those observed in the eukaryote Saccharomyces cerevisiae. Thus, S. fradiae appears to occupy an evolutionary position somewhere between the simple eubacteria and the lower fungi in its responses to potentially mutagenic chemicals and radiations. MATERIALS AND METHODS Chemicals. Spectinomycin hydrochloride was a gift from Upjohn. Rifampin was purchased from Calbiochem. Streptomycin sulfate was purchased from Sigma. The chemical mutagens were obtained from the following sources: hydroxylamine (NH2OH) hydrochloride, methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS), Eastman Kodak; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4nitroquinoline 1-oxide (NQO), Aldrich; mitomycin C (MC), Calbiochem. Media and Growth Conditions. Streptomyces strains were grown in trypticase soy (TS) broth and fragmented by ultrasound as described (11). AS-1 agar medium was prepared as described (12). TS agar contained TS broth (Baltimore Biological Laboratory) and 20 g of agar (Difco) per liter. Czapek-Dox agar contained 35 g of Czapek-Dox broth (Difco) and 15 g of agar (Difco) per liter of distilled water. S. fradiae Strains. C4 (nar-J) is a mutant strain that produces high levels of the macrolide antibiotic tylosin (13-15). Ml (nar-J spo-J) is a spontaneous mutant of strain C4 (11) defective in sporulation. JS6 (nar-) spo-J mcr-6) is an MCsensitive (MCS) derivative of Ml induced by MNNG mutagenesis as described (13). (JS6 was selected by plating appropriate dilutions of a mnutagen-treated culture of Ml onto TS agar and replica plating cells onto TS agar plus MC at 0.5 gg/ml. Putative MCS colonies were retested by patching onto TS agar plus MC at 0.5 ,ug/ml.) JS96 (nar-J spo-J mcr+) is a spontaneous MCr revertant ofJS6 selected for growth on TS agar plus MC at 0.5 Mug/ml. Determination of Mutant Frequencies. Spontaneous and induced resistances to rifampin (Rifr) and spectinomycin (Spcr), and reversion of nitrate reductase deficiency (Nar-) to prototrophy (Nar') were determined, respectively, by overlaying cells in soft agar onto the following selective media: AS-1 plus Rif (50 ,ug/ml), AS-1 plus Spc (50 Akg/ml), or Czapek-Dox agar. Mutants (except Nar') were scored after incubation for 7 days at 34WC; Nar' revertants were scored after 7 days at 40'C, since the Narmutants were temperature sensitive. Viabilities before and after mutagenic treatments were determined by plating appropriate dilutions of cells (ultrasonic fragments) on AS-1 and counting colonies after 3-7 days at 34TC. Chemical mutagens, except MC, were inactivated or washed from the cells prior to plating or segregation. Viabilities on MC were determined by plating untreated cells directly onto TS agar containing various concentrations of MC. All mutagen-treated cells were diluted 1:10-1:20 and grown in TS broth for 18-24 hr at 34WC for segregation before mutant frequencies were determined. No significant selection for or against Spcr mutants is observed during segregation. Mutagenic Treatments. EMS. Late-exponential-phase cells suspended in 0.2-M potassium phosphate buffer, pH 7.0, were incubated for 10 min at 370C with 0.5-3.0% (vol/ Abbreviations: EMS, ethyl methanesulfonate; MC, mitomycin C; MMS, methyl methanesulfonate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; NQO, 4-nitroquinoline 1-oxide; Tes, 2-{ttris(hydroxymethyl)methyl]aminolethanesulfonic acid; Rif, rifampin; Spc, spectinomycin; r, resistant or resistance; ', sensitive or sensitivity; Nar, nitrate reductase. *To whom reprint requests should be addressed. tCommunication of this paper was initiated by George Streisinger and, after his death (August 11, 1984), was completed by Franklin W. Stahl. 1180 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. NatL Acad. Sci. USA 82 (1985) 1181 vol) EMS. The reactions were terminated by diluting the cells 1:10 in 0.16 M sodium thiosulfate (16). NH20H. Late-exponential-phase cells suspended in 0.1 M potassium phosphate buffer, pH 7.0, were incubated for 2 hr at 370C in 100-400 mM NH2OH. Reactions were terminated by diluting the cells 1:10 in chilled nutrient broth plus 10% (vol/vol) acetone. MMS. Late-exponential-phase cells suspended in 0.2 M potassium phosphate buffer, pH 7.0, were incubated for 30 min with 0.05-0.4% (vol/vol) MMS. Reactions were terminated as for EMS. MNNG. The MNNG treatment was as described (13). NQO. Late-exponential-phase cells suspended in 0.05 M potassium phosphate buffer, pH 7.0, were incubated for 2 hr at 37TC with freshly prepared NQO at 100-400 ,g/ml. Reactionis were terminated as for EMS. UV. Cells adjusted to an OD6N of 1.0 in 35 ml of 0.01 M 2{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (Tes) buffer, pH 7.2, were swirled gently in a Petri dish and irradiated at 20-160 J/m2 with a germicidal UV lamp. Manipulations were carried out at light levels sufficiently low to avoid photoreactivation.