The Bacillus subtilis SinR and RapA developmental regulators are responsible for inhibition of spore development by alcohol

Abstract

Even though there is a large body of information concerning the harmful effects of alcohol on different organisms, the mechanism(s) that affects developmental programs, at a single-cell level, has not been clearly identified. In this respect, the spore-forming bacterium Bacillus subtilis constitutes an excellent model to study universal questions of cell fate, cell differentiation, and morphogenesis. Here, we demonstrate that treatment with subinhibitory concentrations of alcohol that did not affect vegetative growth inhibited the initiation of spore development through a selective blockage of key developmental genes under the control of the master transcription factor Spo0A approximately P. Isopropyl-beta-D-thiogalactopyranoside-directed expression of a phosphorylation-independent form of Spo0A (Sad67) and the use of an in vivo mini-Tn10 insertional library permitted the identification of the developmental SinR repressor and RapA phosphatase as the effectors that mediated the inhibitory effect of alcohol on spore morphogenesis. A double rapA sinR mutant strain was completely resistant to the inhibitory effects of different-C-length alcohols on sporulation, indicating that the two cell fate determinants were the main or unique regulators responsible for the spo0 phenotype of wild-type cells in the presence of alcohol. Furthermore, treatment with alcohol produced a significant induction of rapA and sinR, while the stationary-phase induction of sinI, which codes for a SinR inhibitor, was completely turned off by alcohol. As a result, a dramatic repression of spo0A and the genes under its control occurred soon after alcohol addition, inhibiting the onset of sporulation and permitting the evaluation of alternative pathways required for cellular survival.

FIG. 1.

Ethanol blocks expression of early Spo0A∼P-dependent developmental genes. (A to J). Cells were grown in liquid SSM, and at the time indicated by the arrow ethanol (0.7 M; 4%) was added to one-half of each culture. Samples of untreated (filled symbols) and ethanol-treated (open symbols) cultures were collected at the indicated times and assayed for β-galactosidase activity, expressed in Miller units (M.U.) (1). Time zero represents the transition from vegetative to stationary phase. The wild-type B. subtilis strains utilized for the experiments, RG1679 (A), RG2051 (B), JH16480 (C), JH16182 (D), JH16124 (E), JH19000 (F), JH16102 (G), JH19003 (H), JH19004 (I), and JH19005 (J), harbored transcriptional β-galactosidase fusions to the sporulation promoters indicated in each panel. Each panel shows the data from a representative experiment done in triplicate. (K) Cartoon interpreting the results shown in panels A to J, indicating that ethanol treatment blocks the onset of spore morphogenesis (stage zero).

FIG. 2.

Ethanol does not affect the stability of Spo0A-Sad67. Western blot experiment showing the in vivo stability of the Spo0A∼P equivalent form Spo0A-Sad67 in ethanol-treated and untreated cultures. A 100-ml culture of B. subtilis strain Sik31 (Δspo0A::Erm^r P_spac-sad67) was grown in SSM at 37°C until mid-exponential phase. At this point IPTG (1 mM) was added, and growth was continued for another 2 h. After this induction period (production of Spo0A-Sad67), the 100-ml culture was washed three times and growth was resumed in fresh prewarmed SSM without IPTG. Five minutes later, ethanol (0.7 M) was added to one-half of the washed culture, and both halves (with and without ethanol) were further incubated at 37°C. At the indicated times, samples were removed to prepare the cell extracts. Lanes 1 and 2, levels of Spo0A-Sad67 after the 2-h incubation period with IPTG before and immediately after its removal (IPTG washing), respectively. Lanes 3 to 10, levels of Spo0A-Sad67 at the indicated times after resumption of growth in fresh IPTG-free SSM with or without ethanol supplementation. Protein extracts, electrophoresis conditions, and reaction with anti-Spo0A antibodies were as indicated in Materials and Methods.

FIG. 3.

Identification of cell fate determinants involved in alcohol-produced inhibition of cellular morphogenesis in B. subtilis. (A) Cartoon summarizing a hypothetical scenario in which alcohol treatment triggers the expression of more than one negative regulator of spore development that would interfere with the activation of Spo0A by the phosphorelay (possibility 1) and its activity as a transcription factor (possibility 2). These induced antisporulation regulatory factors should be responsible for the inhibition of the activity of the phosphorelay signaling system (phosphorylation of Spo0A) and the inhibition of the activity of Spo0A∼P itself. (B) Identification of alcohol-resistant transposants after the generation of the insertional mini-Tn10 library, enrichment, isolation, and DNA sequencing (40). The DNA sequence information permitted the determination of the precise location of the original transpositions of mini-Tn10 in clones 1 and 2 of B. subtilis that generated the alcohol-resistant phenotype observed on the sporulation plates.

FIG. 4.

The cell fate determinants SinR and RapA are responsible for the inhibition of spore morphogenesis under alcohol stress. Levels of Spo0A∼P-dependent β-galactosidase activity from different JH16304-derived isogenic strains harboring rapA and/or sinR mutations in the presence or absence of ethanol (black and white bars, respectively). Cultures of JH16304 (wild type) and the isogenic derivatives RG1400 (ΔrapA::ery), RG1401 (ΔsinR::cat), and RG1402 (ΔrapA::ery ΔsinR::cat) were grown in SSM until the late exponential phase (T_−1.5), when ethanol (0.7 M) was added to one-half of each culture. Growth of the eight cultures was continued for several hours, and samples were collected and assayed for β-galactosidase activity. The reported Miller units (M.U.) represent the sum of β-galactosidase activities determined every 30 min from T₀ to T_2.5 for each culture. Data are the averages from three independent experiments done in triplicate. The activity of the Spo0A reporter lacZ fusion and the efficiency of spore formation (+/− ethanol) were essentially the same for strains RG19 (rapA::mini-Tn10-Spc^r)/RG1401 (ΔrapA::Ery^r) and RG23 (ΔsinR::mini-Tn10-Spc^r)/RG1400 (ΔsinR::Cat^r), respectively (data not shown).

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.

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).

FIG. 7.

The inhibitory effect of alcohol on sporulation constitutes a developmental checkpoint assuring cellular survival in the absence of lipid synthesis. (A) Autoradiographic pattern of lipids synthesized by the reference strain JH642 in the presence of the specific inhibitor of fatty acid synthesis cerulenin or ethanol in sporulation medium. A culture of strain JH642 was grown until T₀ in SSM at 37°C. At this time several 1-ml samples were taken and treated with ethanol (0.7 M) or cerulenin (5 μg ml⁻¹) and exposed to 10 μCi of [¹⁴C]isoleucine for 3 h at 37°C. A duplicate 1-ml sample was incubated for the same period with the radioactivity but without cerulenin or ethanol supplementation (positive control). After these incubation periods lipids were extracted and chromatographed as described in Material and Methods. The radioactive compounds were located by autoradiography, eluted from the silica gel, and then quantified by scintillation counting. The sample in lane 1 (positive control) contained 10,000 and 13,000 cpm of radioactivity in the phospholipid (PL) and diacylglycerol (DG) fractions, respectively. Lanes 2 (cerulenin treated) and 3 (ethanol treated) contained 390 and 430 cpm in the PL fractions, and 400 and 350 cpm in DG fractions, respectively. Essentially, the same results were observed in several independent experiments done with [¹⁴C]acetate or [¹⁴C]isoleucine as precursors of radioactive lipids. (B) Schematic cartoon representing the effects of and the key regulators triggered by alcohol treatment and their roles in spore formation and cellular survival.