A modified regeneration method for streptomycete protoplasts.

Abstract

In 1974, we1} reported on the culture media and conditions for preparing stable protoplasts of streptomycetes and the procedures for reverting them at high frequency to a filamentous state by plating the surface of the synthetic agar media (Rl and R2). This method, sometimes with minor modifications, has been employed by many researchers on protoplast fusion2~7) or genetic transformation,8~n) including recombinant DNAexperiments, in streptomycetes.However, it has been found that the regeneration frequency varies according to the species used and that high regeneration frequency is limited to a narrow range of streptomycete species. Therefore, we have worked out a new regeneration method which is applicable to some regeneration-negative species or mutants, and this is the subject of the present communication. R. H. Baltz, independently, has reported on the conditions for effective regeneration of streptomycete protoplasts at the International Fermentation Symposium held in London, Canada, in July 1980. The effective conditions he reported were quite similar to the those we had found at that time, viz., a) to overlay soft agar together with protoplasts over the basal agar layer of medium R2; b) to dry the surface of the underlayer of the agar plates before plating protoplasts; and c) to incubate the protoplasts at a lower temperature for regeneration. The emphasis in the present paper is on a newly established medium and the procedure for, regeneration which differs from that reported by Baltz, as well as on the effect of temperature on the stability of protoplasts.Streptomyces kasugaensis MB 273-18a (Atp~Amy" Arg~) were used in most of the experiments. To prepare the protoplasts, cells from a slant culture were shakecultured at 28°C for 2 days in mediumS.1} The culture was transferred with 2% inoculum size to fresh medium S supplemented with glycine (0.4%, this value differed depending on the species used1}), and shake-cultured for 26hr. Cells from 8ml culture were harvested by centrifugation, and washed with 0.35m sucrose solution. The washed cells were suspended in 8ml of medium P3 consisting of70 mM NaCl, 5 mM MgCl2, 5 him CaCl^, 0.5 m sucrose and 0.025 m TES buffer (TV-tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid, pH 7.2). Then lysozyme was added to a concentration of 0.3~1.0mg/ml, followed by incubation at 28°C for 30~60min. Since the cells of S. kasugaensis grow as short hyphae resembling bacilli in liquid medium, the filtration procedure1> was inadequate to remove the intact cells. The lysozymetreated cell suspension was therefore centrifuged at 200x0 for lOmin to remove the intact cells, and the turbid supernatant containing the protoplasts was centrifuged again at 500 x g for lOmin. The protoplasts precipitated were gently suspended in 8ml of medium PWP consisting of 70mM NaCl, 10mM MgCl2, 20mM CaCl2, 0.5m sucrose and 0.025m TES buffer (pH 7.2). After treatment with lysozyme, centrifugation and resuspension of protoplasts were carried out as soon as possible, because the protoplasts were unstab...

Isolation and characterization of a strong promoter element from the Streptomyces ghanaensis phage I19 using the gentamicin resistance gene (aacC1) of Tn1696 as reporter

“…For S. lividans, MICs for Gm were determined using solid medium supplemented with different amounts of the antibiotic. MICs for Km were determined using microtitre plates with each well containing 200 pl R3 medium (Shirahama et al, 1981) supplemented with different amounts of the antibiotic. After spore inoculation, the plates were incubated at 28 "C for 3 d and the MIC defined as the concentration of antibiotic that reduced colony number by Plasmid copy number determinations, DNA manipulations and Southern blotting.…”

Application of Protoplast Fusion to the Development ofl-Threonine andl-Lysine Producers

“…Sakaguchi et al 7 ) showed the possibility of protoplast fusion in B.flavum. Their procedure was modified as follows: 0.1 M CaCI 2 10 ) was added to the solutions used in the fusion and regeneration processes, and low melting point agarose 8 ) was used as the upper layer for plating instead of the usual agar (Fig. 1).…”

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes

“…Regeneration of mycelium from protoplasts was performed using the overlay technique suggested by Shirahama et al 23 Plates were seeded by pouring 0.2 ml of protoplast suspension on hypertonic media (M3 or M103 or MP3), and the upper layer was medium VMS0.1. 12 To assess residual hyphal contamination of protoplast suspensions, control plates, with medium V0.1 as the under layer and medium VM0.1 as the upper layer, were seeded.…”