Fusion of bacterial protoplasts.

Abstract

Fig. 1. Bacteria were first grown in nutrient broth (17), and protoplasted in SMM, the sucrose-magnesium-maleate buffer of Wyrick and Rogers (13), to which 5 ,ug/ml of DNase I (Worthington Biochem. Corp.) had been added (SMMD). Protoplasts were made to revert to bacillary forms by plating on RDR, a rich regeneration agar medium of high tonicity (13), to which 5 /g/ml each of DNase I and rifamycin (Lepetit Labs, Milano) were added. Prototrophic clones within the film of growth that appeared on RDR plates after incubation were selected out by replica plating onto variously supplemented SDR medium. This is a nonhypertonic minimal medium (14), to which 20 ,M MnCl2, 5 gg/ml of DNase, 1 Ag/ml of rifamycin, and 15 g/liter of (Difco) agar have been added. It was used as a selection medium, both unsupplemented (SDR) and supplemented (see Table 2).Procedure Adopted for Fusion Experiments. Overnight precultures of both parental strains in nutrient broth at 300 were inoculated, before growth ceased, into 20 ml of broth, to give an initial optical density (OD570) = 0.05. These cultures were incubated with shaking at 370 until an OD of 0.4 was reached. From each culture, 15 ml were centrifuged, the pellets were taken up in 3 ml of SMMD (OD570 = 2, or about 4 X 108 colony forming units/ml), and lysozyme was added to a concentration of 200,ug/ml. Complete protoplast formation was usually seen after 10 min of gentle shaking at 420, but exposure to lysozyme was continued for 20 more minutes. Samples (0.1 ml) of each suspension were then plated on ordinary nutrient agar. The plates usually remained sterile, and indicated that the frequency of osmotic shock resistant forms was below 2.5 X 10-8.One milliliter samples from each of the two suspensions were mixed in a third tube, the three tubes were centrifuged, and each pellet was resuspended in 0.2 ml of SMMD. To one tube, 1.8 ml of a 40% (wt/vol) solution of polyethylene glycol (PEG)t in SMM was added and the suspension immediately homogenized by shaking. After a 1 min exposure to PEG, either at 200 or at 00, several 0.05 ml samples were spread on the surface of duplicate RDR plates and used to make 10-1 and 10-2 dilutions in SMMD, from which, in turn, further reversion plates and also tThe molecular weight is not critical; PEG 6000 from Merck was usually used.

Spontaneous zygogenesis in Escherichia coli, a form of true sexuality in prokaryotes

“…Diploidy in Escherichia coli, first proposed by Lederberg in 1949 to account for various observations on prototrophs obtained in conjugation experiments with E. coli K-12, was confirmed by analyses of clonal pedigrees from isolated single cells (Zelle & Lederberg, 1951). More recently, diploidy has been achieved artificially, by polyethylene-glycol-induced protoplast fusion in various bacteria: Bacillus megatherium (Fodor & Alfoldi, 1976), Bacillus subtilis (Schaeffer et al, 1976), Providencia alcalifaciens (Coetzee et al, 1979) and even Streptomyces spp. (Hopwood et al, 1977).…”

Ploidy of Bacillus subtilis exfusants: the haploid nature of cells forming colonies with biparental or prototrophic phenotypes

“…NB is the complex medium of Schaeffer et al (1965). RD is the cell wall regeneration medium of Wyrick & Rogers (1973) The haploid nature of B. subtilis exfusants 1079 supplemented with 5 pg DNAase I ml-l (Schaeffer et al, 1976). mR2 is the minimal cell wall regeneration medium of Sanchez-Rivas (1982).…”

“…mR2 is the minimal cell wall regeneration medium of Sanchez-Rivas (1982). SMMD is the protoplasting medium of Wyrick & Rogers (1973) supplemented with 5pg DNAase I ml-l (Schaeffer et al, 1976). SMMAD is SMMD supplemented with 1% (w/v) BSA (Gabor & Hotchkiss, 1979).…”

“…Hauser and D . Karamata generally complementary multiple auxotrophs, are converted into protoplasts by lysozyme treatment, fused by addition of polyethylene glycol (PEG) and plated for single colonies on a rich regeneration medium on which cell wall synthesis can proceed (Schaeffer et al, 1976). Exfusants are transferred, by toothpicks, onto three solid minimal media : minimal medium alone, that containing the growth requirements of parent 1 and that containing those of parent 2.…”

Induction of L‐Phase Variant from Protoplast of Staphylococcus aureus

“…The induction of protoplast fusion by polyethylene glycol, which was first demonstrated in plant protoplasts by Kao and Michayluk (5), was extended to various microbial protoplasts (3,7) including Staphylococcus aureus (4,6). The cell fusion method will provide an excellent opportunity for studying genetic recombination and chromosomal mapping in Staphylococcus aureus, concerning which there has been little work done.…”