FIG. 5.

Ethanol treatment enhances RapA production. (A) β-Galactosidase activity of strain JH12981 (rapA-lacZ) grown in SSM in the absence or presence of ethanol (0.7 M). For the treated culture (open symbols) alcohol was added at early exponential phase (optical density at 525 nm [OD₅₂₅] of 0.25). Samples were removed at the indicated times and assayed for β-galactosidase activity. Data from a representative experiment are shown. (B) Levels of rapA expression in wild-type (JH12981) and two isogenic spo0A and comA mutants strains. Cultures were grown in SSM at 37°C until mid-early exponential phase (OD₅₂₅, 0.35), at which time ethanol (0.7 M) was added to one-half of each culture. Growth was resumed, and samples were taken every 30 min for measurement of β-galactosidase activity. The reported Miller units (M.U.) represent the sum of β-galactosidase activities accumulated from the moment of ethanol addition until 1 h after the end of the exponential phase. Data are the averages of three independent experiments done in triplicate. (C) Model for the RapA-dependent branch of inhibition of sporulation by alcohol treatment. In untreated exponentially growing cells (minus ethanol in the cartoon), expression of the rapA-phrA operon is driven from the σ^A-containing form of RNA polymerase under the temporal negative and positive control of Spo0A∼P and ComA∼P, respectively. Even though the presumed levels of both regulatory cell fate determinants (RapA and pro-PhrA) should be similar, the RapA phosphatase is constitutively active. This is because pro-PhrA is exported, in a SecA-dependent process, and accumulated in the extracellular medium as an inactive precursor. At the onset of stationary phase, pro-phrA is processed to an active pentapeptide and imported to the cellular cytosol (through the Opp oligopeptide permease) to inhibit RapA activity. As a result, more phosphate is transferred through the phosphorelay to Spo0A to reach the threshold of Spo0A∼P needed for the initiation of spore formation. In exponentially growing ethanol-treated cells (plus ethanol in the cartoon), the ComA-dependent enhanced rapA-phrA expression would result in higher levels of both regulatory proteins. However, since ethanol also induces the expression of the stress gene yjbG (coding for the pro-PhrA/PhrA oligopeptidase PepF), it is hypothesized that pro-PhrA/PhrA is proteolytically degraded under ethanol treatment (19). Hence, both during exponential and stationary phases of ethanol-treated cultures, only free and active RapA phosphatase would be present, producing a profound blockage of the ability of B. subtilis cells to initiate sporulation in the presence of alcohol.