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The only compound which fully replaced protamine sulfate in facilitating transfection ofEscherichia coli spheroplasts by phage DNAs was spermine; poly-l -lysine, poly-l -arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor fromStreptococci . By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10−3 and 3 × 10−4 for lambda DNA; between 10−6 and 3 × 10−8 for T7 DNA; and between 3 × 10−6 and 10−7 for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.

Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers