FIG. 6.

Ethanol affects the SinR-SinI-dependent regulatory circuit controlling spore development in B. subtilis. (A to C) β-Galactosidase activities of the reporter fusions sinR-lacZ (RG438) (A) and sinI-lacZ (RG437) (B and C) in wild type (A and B) and hpr mutant (RG439) (C) strains. Cells were grown in SSM at 37°C until mid-exponential phase, at which time ethanol (0.7 M) was added to one-half of each culture (arrow). Growth was continued for several hours, and β-galactosidase activity was measured at the indicated times (closed symbols, without ethanol; open symbols, with ethanol). The results of a representative experiment are shown. M.U., Miller units. (D) Model for the SinR-dependent branch of inhibition of sporulation by alcohol treatment. In untreated ethanol cultures (minus ethanol in the cartoon), SinR synthesis blocks spo0A and early spoII gene expression during exponential phase (23, 24). Hence, the start of sporulation is prevented during vegetative growth. At the onset of stationary phase down-regulation of the Hpr repressor and the higher levels of Spo0A∼P activate transcription of sin. The increased levels of SinI overcome SinR levels, sequestering all the repressor protein in an inactive complex (SinI::SinR); hence sporulation begins. With alcohol treatment (plus ethanol in the figure), sinR expression is enhanced during both exponential and stationary phases. Simultaneously, down-regulation of spo0A (due to enhanced production SinR and RapA, see above) up-regulates abrB and hpr expression (data not shown). The decreased generation of Spo0A∼P and the increased levels of Hpr, activator and repressor of sinI expression, respectively, result in the production of low levels of SinI that cannot overcome the ethanol-dependent SinR overproduction; hence the beginning of sporulation is constitutively blocked. Also shown is the repressing effect of Hpr on opp, which contributes to the RapA-mediated inhibitory effect of alcohol on sporulation (see the text for details).