Polyethylene glycol-assisted transfection of Streptomyces protoplasts

Publisher Website
Google Scholar
Abstract
In the presence of polyethylene glycol (concentration optimum 20%), protoplasts of appropriate Streptomyces strains could be transfected by DNA of 5 temperate phages (.vphi.C31, VP5, R4, .vphi.448 and S14) belonging to 4 different immunity groups. Quantitation of transfection was made possible by plating the transfection mixture with excess uninfected protoplasts in soft agar overlays on protoplast regeneration medium so that plaques were easily detected. Optimum frequencies of transfection in the ranges of 10-6/DNA molecule and 10-5/viable protoplast were invariably obtained. Apparently, single DNA molecules initiated transfection events and the conformation of the DNA (i.e., circular or linear) was not important. Inhibition of transfection by EDTA suggested that divalent cations were important in transfection. Effective competition by nontransfecting DNA was also observed. A minor subpopulation of protoplasts appeared to be particularly sensitive to transfection (i.e., competent) in some DNA-host combinations. In such cases the size of this subpopulation was the major limiting factor in obtaining high transfection frequencies. The same protoplasts appeared to be competent for DNA of 2 distinct phages (.vphi.C31 and R4).