Jeffrey Lynn Larson

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

SummaryA gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage λ Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes.

Uses of recombinant DNA for analyses of Streptomyces species.

The advent of recombinant DNA techniques has provided investigators with a new arsenal to attack problems of gene structure, genetic regulation, and genome organization. Already the intellectual rewards have been spectacular in providing solutions to problems that were intractable previously. However, the most important rewards are derived from applications to produce biological products that benefit mankind directly. The first-stage efforts have been mobilized for production of single proteins. September 20, 1982, is a historic date in stage one because Eli Lilly and Company marketed human insulin, which is the first clinically significant product from recombinant DNA that is available to the public. A plethora of additional protein products from recombinant DNA are in various stages of development or research. Undoubtedly, the results will have profound effects on the practice of medicine during the coming decades. Perhaps the next important milestone of pharmaceutical applications will be the production of metabolites such as antibiotics. Essential use of life-saving antibiotics is a cornerstone of modern medicine. Successful development of recombinant DNA methods will provide tools that will help increase the fermentation yields of antibiotics and generate new antibiotic structures that are not available through traditional routes of discovery.’ Full realization of these benefits necessitates developing recombinant DNA systems for Streptomyces (S.) species because two-thirds of all known antibiotics are produced by S. species.2 Researchers, led by David Hopwood and Keith Chater a t the John Innis Institute, have set the tempo for development of recombinant DNA systems in S. species: Hopwood working on plasmid vectors and Chater working on actinophage vectors. Their progress has been described recently and readers are referred to these excellent reviews.”’ Investigators with Stanley Cohen have reported progress in development of plasmid vectors,6.’ Isogai et aZ.* have potential actinophage vectors and Richardson et aL9 have published additional plasmid vectors. Multiple and parallel development of recombinant DNA systems provides the greatest likelihood for success because no one system is likely to work with all S. species or be applicable to all problems. This report describes progress in our program to apply recombinant DNA technology to S. species. Steps are described in the construction of the plasmids pJL197 and pJL198, which should be particularly useful as recombinant DNA cloning vectors. Discovery of amplified DNA in Streptomyces fradiae (S. fradiae) and application of recombinant DNA techniques in the characterization of amplified DNA are described in the latter portion of the report. The features of this high-level DNA amplification are unique to Streptomyces among bacterial species.