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...
Genes encoding enzymes for tylosin biosynthesis, genes involved in the expression of resistance to tylosin (Tyl), hygromycin B (Hm), chloramphenicol (Cm), and mitomycin C (MC), and a single copy of an amplifiable unit of DNA (AUD) were jointly transferred at very high frequencies by conjugation from several different Streptomyces fradiae strains to S. fradiae JS85, a mutant defective in many or possibly all tylosin biosynthetic reactions and containing a multiple tandem reiteration of the AUD. No recombination was observed between nar, rif and spc genes in conjugal matings, but recombination was observed between these genes after protoplast fusion. Tylosin biosynthetic genes were transferred at a much lower frequency to S. fradiae JS87, another mutant defective in many or all tylosin biosynthetic reactions, but deleted for the AUD and other DNA sequences. These findings suggest that tylosin structural genes, several genes encoding antibiotic resistance determinants, and amplifiable DNA are present on a self-transmissible element that does not mobilize chromosomal genes, and that JS85 and JS87 contain deletions, and JS85 an amplification, of overlapping portions of this element.
Two mutants of the tylosin-producing Streptomyces fradiae defective in the biosynthesis of the macrolide antibiotic tylosin were isolated from colonies derived from regenerated protoplasts.Both strains were unable to carry out any of at least seven tylosin biosynthetic steps and were sensitive to tylosin. One strain, JS82, was also more sensitive to chloramphenicol (Cm), mitomycin C (Mc), hygromycin B (Hm) and kanamycin (Km) than its parent strain. The other strain, JS87, was also more sensitive to Cm than wild type but expressed normal levels of resistance to Me and I-Im. Both strains expressed genetic instabilities associated with auxotrophy or expression of antibiotic resistance. Since the genetic instabilities were not due to defective error-free or error-prone DNA repair, they appear to be due to genetic rearrangements associated with the deletion or amplification of sequences linked to and perhaps encompassing tylosin biosynthesis genes. ptomycesfradiae produces tylosin1,2) an economically important macrolide antibiotic. Recent Stre studies with mutants blocked in different steps of tylosin biosynthesis have elucidated the biosynthetic pathway from tylactone to tylosin1,2,3) and have identified a rate limiting step in the biosynthetic pathway suitable for applied genetic manipulation4,5).We have been interested in defining the genetic locations and possible physical linkage of tylosin biosynthesis genes in S. fradiae. Since in conjugal coatings we were not able to demonstrate recombination between mutants of S. fradiae carrying auxotrophic mutations or antibiotic resistance mutations (which were presumably chromosomal)6), we developed a protoplast fusion procedure to carry out genetic recombination and mapping7,8).In preliminary experiments to map the tylosin biosynthesis genes, it was observed that production of tylosin or tylosin-like intermediates or branch products of specific mutants was lost at high frequencies after genetic recombination mediated by protoplast fusion6). Since protoplast formation and regeneration can cause loss of plasmids in Streptomyces9,10.11) and in other Gram-positive bacteria12,13) we initiated studies to determine if the tylosin biosynthesis genes were located on a plasmid or plasmid-like element.To approach this question, we first attempted to obtain strains of S. fradiae cured of plasmid by protoplast regeneration.Two strains, JS82 and JS87, which were obtained by protoplast regeneration of wild type and high tylosin producing strains, respectively, were unable to produce tylosin and were sensitive to tylosin.In conjugation experiments with various strains proficient in the production of tylosin or blocked in specific steps of tylosin biosynthesis used as donors, JS82 was shown to be a high frequency recipient for expression of tylosin resistance and tylosin structural genes, but not for other (presumably chromosomal) genes14) . Attempts to isolate plasmid DNA from JS82, JS87 and the parental strains, however, were unsuccessful15) (MATSUSHIMA and BALTZ, unpublished). In ...
Two mutants of Streptomyces fradiae defective in DNA repair have been characterized for their responses to the mutagenic and lethal effects of several chemical mutagens and ultraviolet (UV) light. S. fradiae JS2 (mcr-2) was more sensitive than wild type to agents which produce bulky lesions resulting in large distortions of the double helix [i.e. UV-light, 4-nitroquinoline-1-oxide (NQO), and mitomycin C (MC)] but not to agents which produce small lesions [i.e. hydroxylamine (HA), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)]. JS2 expressed a much higher frequency of mutagenesis induced by UV-light at low doses and thus appeared to be defective in an error-free excision repair pathway for bulky lesions analogous to the uvr ABC pathway of Escherichia coli. S. fradiae JS4 (mcr-4) was defective in repair of damage by most agents which produce small or bulky lesions (i.e., HA, NQO, MMS, MNNG, MC, and UV, but not EMS). JS4 was slightly hypermutable by EMS and MMS but showed reduced mutagenesis by NQO and HA. This unusual phenotype suggests that the mcr-4+ protein plays some role in error-prone repair in S. fradiae.
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