Control of Sporulation in Fused Protoplasts of Bacillus subtilis 168

Abstract

Complementable spo mutants of Bacillus subtilis could be 'rescued' to form heat-resistant spores by fusing them with sporulating cells of the wild-type strain (Dancer & Mandelstam, 1981). The latter strain acted as a 'rescuer'. Rescuer protoplasts had to be derived from cells grown in a medium that allowed rapid and synchronous sporulation subsequently. Even protoplasts made from vegetative cells growing in such a medium (to protoplasts) acted as rescuers. Therefore, it was not necessary for the rescuer cells to have initiated sporulation before they were converted to protoplasts.The genetic requirement for to rescuer protoplasts was that they were spos for the gene to be complemented. Strains in the same complementation group did not rescue one another. Protoplasts of spo strains blocked at stages 0, IV and V of sporulation acted equally well as rescuers whether they were converted to protoplasts at to or t , (i.e. 3 h after transfer to sporulation medium). However, t, protoplasts of stage I1 and stage I11 mutants were much less effective rescuers than their to counterparts. This difference was not due to a dominance effect expressed only in t, protoplasts. I N T R O D U C T I O NProtoplasts of bacilli are able to complete sporulation if they are made from cells that have reached stage I11 of sporulation (completion of prespore engulfment) or beyond (Fitz-James, I9 64). Bacillus subtilis protoplasts containing forespores complete sporulation even when subjected to a fusion procedure involving treatment with 40 % (w/v) polyethyleneglycol 6000 (Dancer & Mandelstam, 198 1). This has been used to establish complementation relationships between asporogenous strains blocked at late stages of sporulation (stages 111, IV and V), i.e. the defective forespores of some strains can be 'rescued' to form heat-resistant spores by fusion with sporogenous strains or with asporogenous strains in other complementation groups.Having established that complementation by fusion was possible and that complementation groups could be distinguished, it was of interest to investigate the conditions for obtaining rescue of complementable strains and, in particular, the requirements for the 'rescuer' strain to act best in fusions. The experiments described in this paper show that t, protoplasts of complementable strains can be rescued by to protoplasts of strains in any other complementation group provided that growth took place in a sugar-free medium. Rescue was also effective when t , protoplasts of the spa+, sp00, spoW and spoV strains were used as rescuers but was poor when spoU or spolII strains were used.

Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis.

“…Another related method has been described by Dancer and Mandelstam (46,47). They showed that the defective sporulation of certain Spomutants could be rescued by converting them to protoplasts at about stage III and fusing them to protoplasts made from wild-type cells.…”