Complementation of Sporulation Mutations in Fused Protoplasts of Bacillus subtilis

Abstract

Asporogenous and oligosporogenous mutants of Bacillus subtilis blocked at stages III, IV and V and carrying mutations at 18 different genetic loci have been tested for complementation. This was done by preparing protoplasts 3 h after induction of sporulation (t 3) and fusing them with wild-type protoplasts prepared in parallel. After incubation to t 10 the suspension was assayed for heat-resistant (80 °C, 40 min) colony-forming units. Mutations in 9 of the 18 loci were complemented, i.e. the spore count was increased > 1000-fold. All of the complemented mutants, except for one stage V mutant, were blocked at stage IV. Spo− mutants that were complementable by wild-type were also complementable by other Spo− mutants provided that they did not carry the same spo mutation. Complementation analysis was applied to three alleles in the spoIVC locus. The complementation pattern suggests that there are at least two cistrons in the locus and this agrees with the mapping data.