Fusion of Bacterial Protoplasts

Prototrophic Bacillus subtilis cells can be formed in the presence of DNase as a result of cell fusion occurring in mixed populations of protoplasts derived from two parental strains which are both nutritionally-complementing and polyauxotrophic. No prototrophs ever appear from mixed nonprotoplasted bacteria, or from the auxotrophic parental protoplasts plated separately. The frequency of prototroph formation, which is appreciable only when the mixed protoplasts are exposed to polyethylene glycol treatment, may exceed 1 × 10-4of the total protoplast population initially present, which is 1 to 4 × 10-3of those protoplasts which reverted to the bacillary form. It is strongly dependent on the number and chromosomal location of the markers used in the selection of the prototrophs, and it is unaffected when either one of the parental strains bears the phage φ 105 in the inducible prophage state. No auxotrophic bacteria, parental or otherwise, were found as segregants from repeatedly isolated prototrophic clones growing in a nonselective medium. Unselected markers segregate among the selected recombinants. It is concluded that the observed formation of prototrophic bacteria is due to protoplast fusion, a process which does not induce prophage development, and that the only stable products of the resulting diploid state are haploid recombinants.