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

Abstract

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia 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 polyamineor protamine-like, natural competence factor from Streptococci. 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 x 10-4 for lambda DNA; between 10-6 and 3 x 10-8 for T7 DNA; and between 3 x 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 Enterobacteriaceae and its applications.

“…There are also several disadvantages to the calcium shock technique. (i) The efficiencies of transfection are generally 100-fold lower than those obtained with lysozyme-EDTA spheroplasts (154). (ii) Transfection experments with larger DNAs require a fresh preparation of cell every day since competence is stable for only a few hours (P. Nevers, personal communication; M. Oishi, personal communication); however, cells can be stored for long time periods in 0.01 M MgSO4 at 5 x 108 cells/ml before calcium shock (34).…”

“…In summary, bacterial transformation studies have focused on the mechanism of uptake and genetic recombination of heterogeneous transforming DNAs, whereas transfection studies have been used to measure the infectivity of homogeneous viral nucleic acids after in vivo and in vitro alteration. The recent development of efficient transfection (29, 154,167,217,221) and transformation systems for Escherichia coli (70,72,429) justifies the conclusion that these systems will yield a variety offruitful results not obtainable in other systems because the genetics and biochemistry of E. coli and its phages have been elucidated in far more detail than in any other system. Therefore, this review will focus on results obtained with transfection of E. coli.…”

“…The reproducible preparation of competent cells of D. pneumoniae was discovered by Fox and Hotchkiss (119). Until recent improvements (154), 100-fold variations in day-to-day competence levels were common with E. coli (29).…”

“…Such stable preparations have been available for B. subtilis, H. influenzae, and D. pneunwniae (119,263,471). However, only recent improvements (154)…”

“…The lysozyme-EDTA spheroplast technique was first described in detail by Guthrie and Sin-sheimer (145). It has subsequently been modified to yield higher and more reproducible levels of competence (29,154). Thus the following description is adapted from these more recent papers (29, 154).…”