Mutagenic DNA repair in Streptomyces.

Abstract

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 of Escherichia 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 4-nitroquinoline 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 bro...

recA gene of Escherichia coli complements defects in DNA repair and mutagenesis in Streptomyces fradiae JS6 (mcr-6)

“…A spontaneous MC-resistant mutant of JS6 expresses normal levels of resistance to all of the agents and shows wild-type levels of induced mutagenesis, indicating that a single mutation causes the multiple defects in JS6 (25). The mcr' gene product thus appears to control an SOS response in S. fradiae and to be functionally analogous to the RecA protein in Escherichia coli (28)(29)(30) and the RecE protein in Bacillus subtilis (15,16), One difference in the error-prone DNA repair systems expressed in S. fradiae and in E. coli or Salmonella typhimurium is that S. fradiae JS6 (mcr-6) is 10-to 100-fold less mutable by EMS than is its parent strain Ml (2,25), whereas E. coli and S. typhimurium recA mutants express normal levels of EMS-induced mutagenesis (8,12,24). The dependence of EMS mutagenesis on an error-prone repair system suggested that S. fradiae expresses error avoidance mechanisms more stringent than those observed in E. coli and similar to those expressed in the lower eucaryote Saccharomyces cerevisiae (25).…”

“…One mutant, JS6 (mcr-6), is defective in the repair of potentially lethal damage induced by a variety of agents, including MC, UV light, 4-nitroquinoline-1-oxide (NQO), methyl methanesulfonate (MMS), hydroxylamine (HA), and N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and is less mutable by MNNG, MMS, NQO, UV light, HA, and ethyl methanesulfonate (EMS) than is the wild-type strain (2,25). A spontaneous MC-resistant mutant of JS6 expresses normal levels of resistance to all of the agents and shows wild-type levels of induced mutagenesis, indicating that a single mutation causes the multiple defects in JS6 (25). The mcr' gene product thus appears to control an SOS response in S. fradiae and to be functionally analogous to the RecA protein in Escherichia coli (28)(29)(30) and the RecE protein in Bacillus subtilis (15,16), One difference in the error-prone DNA repair systems expressed in S. fradiae and in E. coli or Salmonella typhimurium is that S. fradiae JS6 (mcr-6) is 10-to 100-fold less mutable by EMS than is its parent strain Ml (2,25), whereas E. coli and S. typhimurium recA mutants express normal levels of EMS-induced mutagenesis (8,12,24).…”

“…To aid these studies, we have isolated mutants of S. fradiae Ml which were defective in repair of damage induced by mitomycin C (MC) and characterized their responses to treatment with a variety of mutagenic agents (2,3,6,25). One mutant, JS6 (mcr-6), is defective in the repair of potentially lethal damage induced by a variety of agents, including MC, UV light, 4-nitroquinoline-1-oxide (NQO), methyl methanesulfonate (MMS), hydroxylamine (HA), and N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and is less mutable by MNNG, MMS, NQO, UV light, HA, and ethyl methanesulfonate (EMS) than is the wild-type strain (2,25). A spontaneous MC-resistant mutant of JS6 expresses normal levels of resistance to all of the agents and shows wild-type levels of induced mutagenesis, indicating that a single mutation causes the multiple defects in JS6 (25).…”

“…The combined results indicate that the cloned recA gene from E. coli can overcome the defects in repair of 10- by UV light. Cells were grown in tryptic soy broth, homogenized and sonicated as previously described (1), treated with UV light, and plated on AS-1 agar as previously described (3,25). were grown in tryptic soy broth, homogenized and sonicated as previously described (1), and treated with EMS as previously described (3,25).…”

“…PM74 was completely resistant by MMS and MC. Cells were grown in tryptic soy broth, homogenized and sonicated to obtain single cells as previously described (1), treated with MMS or MC, and plated on AS-1 agar as previously described (3,25).…”