SecA IS REQUIRED FOR THREE DISTINCT STAGES OF SPORULATION IN BACILLUS SUBTILIS

Bacillus subtilis secA341 mutant cells sporulate at 32°C but not at 39°C. Temperature shift-up and -down experiments indicated that the cells were sensitive to the high-temperature treatment from an early stage of sporulation to relatively late stages. Within this temperature-sensitive duration, three discontinuous periods were discriminated by means of pulse-treatment with the nonpermissive temperature. These periods were T-0.5-T0.5, T1-T2 and T3-T5; T0 indicates the end of exponential growth and subscript numbers represent hourly periods after T0. When the mutant cells harboring the spo0A-lacZ fusion with the sporulation specific promoter, Ps, were incubated at 39°C from T-0.5 to T0.5, the subsequent expression of lacZ gene at 32°C was almost completely abolished. Although the sporulation frequency of these temperature-treated cells was around 1/10 of the control culture, the remaining vegetative cells exhibited the so-called Spo0 phenotypes. Electron microscopic analysis with cells incubated at 39°C after developing to some stages of sporulation at 32°C, suggested that the second temperature-sensitive period, T1-T2, corresponded to a SecA-dependent stage for the initiation of asymmetric septation which occurred at around T3 in the control culture. The third period, T3-T5, dependent on the SecA function was shown to correspond to a stage of septum engulfment; the control culture at 32°C completed the engulfment process and forespore formation at T7.