Conjugal Transferring of Resistance Gene ptr for Improvement of Pristinamycin-Producing Streptomyces pristinaespiralis

Abstract

Improving pristinamycin production from Streptomyces pristinaespiralis was performed by introducing the resistance gene ptr followed by selection for enhanced tolerance to pristinamycin and fermentation test. To transfer ptr into S. pristinaespiralis, an effective method was established for the first time by using the intergeneric conjugation of DNA from Escherichia coli to S. pristinaespiralis. The procedure was optimized with heat treatment, spore concentration, optimum medium used in conjugation, concentration of MgCl(2), etc. With the optimized conditions, the conjugation frequency was up to 1.36 x 10(-3) exconjugants per recipient. The procedure was used to transfer the ptr gene into S. pristinaespiralis, resulting in 146 exconjugants. These exconjugants were screened on the pristinamycin-resistant plates, and then the fermentation test subsequently. Finally, two strains (SPR1 and SPR2) were obtained with a high yield of 0.11 and 0.15 g/l, respectively, which is about six to eight times more than that of wild-strain ATCC25486. The subculture experiments indicated that the hereditary character of the high-producing S. pristinaespiralis SPR1 and SPR2 was stable. Our work suggests that introducing resistance gene ptr into S. pristinaespiralis could be the way to improve the production of pristinamycin through the enhancement of antibiotic tolerance.

Methods for the genetic manipulation of Nonomuraea sp. ATCC 39727

“…The frequency of 10 -3 exconjugants per recipient Nonomuraea sp. ATCC 39727 CFU is comparable with data reported for the same vector in other actinomycetes, such as Actinoplanes teichomyceticus (*10 -3 ) [10] and Streptomyces pristinaespiralis (*10 -3 ) [22]. Use of the UC31 att/int system in Streptomyces lividans is reported to give a higher frequency (8 9 10 -2 ) [3,10].…”

“…ATCC 39727 CFU determined from the biomass used for conjugation) varies widely depending on the agar medium used. With MS (the most commonly used media for streptomycetes [3,22] and for Actinoplanes teichomyceticus [10]), we did not obtain any exconjugants, whereas in media specifically developed for Nonomuraea sp. ATCC 39727 [8], we had the highest number of resistant clones.…”

“…ATCC 39727 mycelium before conjugation enhanced dispersion. The agar medium used to select exconjugants can greatly influence the efficiency of conjugation in actinomycetes [10,22]. Therefore, six representative media were tested.…”

An Efficient Intergeneric Conjugation of DNA from Escherichia coli to Mycelia of the Lincomycin-Producer Streptomyces lincolnensis

“…When 20 mM of MgCl 2 was added to the MS medium, the conjugation frequency of S. lincolnensis mycelia increased 9-fold compared without MgCl 2 (Figure 3). However, the conjugation frequency decreased significantly as the concentration of MgCl 2 increased, considering the spore formation and mycelia growth were severely inhibited at concentrations of 40 mM or higher for S. pristinaespiralis [28]. Thus, MS medium supplemented with 20 mM of MgCl 2 proved to be optimal for the conjugation of E. coli - S. lincolnensis mycelia.…”

Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

“…To select the appropriate medium for intergeneric conjugation, five agar media (MS, ISP4, 2CMY, YMS and TSA) containing 10 mmol/L MgCl 2 were assessed as conjugative medium. Among them, MS, ISP4 and 2CMY has been used as conjugal medium for several Streptomyces [1,21,25,26,27,28]; TSA is usually applied in the growth of Streptomyces species [1,22]; and YMS is the suitable medium for the growth and spore formation of S. noursei xinao-4. S. noursei xinao-4 grow and sporulate fast within 7 days on YMS agar at 28 °C.…”