Chill induction of the SigB-dependent general stress response in Bacillus subtilis and its contribution to low-temperature adaptation

Abstract

A variety of environmental and metabolic cues trigger the transient activation of the alternative transcription factor SigB of Bacillus subtilis, which subsequently leads to the induction of more than 150 general stress genes. This general stress regulon provides nongrowing and nonsporulated cells with a multiple, nonspecific, and preemptive stress resistance. By a proteome approach we have detected the expression of the SigB regulon during continuous growth at low temperature (15 degrees C). Using a combination of Western blot analysis and SigB-dependent reporter gene fusions, we provide evidence for high-level and persistent induction of the sigB operon and the SigB regulon, respectively, in cells continuously exposed to low temperatures. In contrast to all SigB-activating stimuli described thus far, induction by low temperatures does not depend on the positive regulatory protein RsbV or its regulatory phosphatases RsbU and RsbP, indicating the presence of an entirely new pathway for the activation of SigB by chill stress in B. subtilis. The physiological importance of the induction of the general stress response for the adaptation of B. subtilis to low temperatures is emphasized by the observation that growth of a sigB mutant is drastically impaired at 15 degrees C. Inclusion of the compatible solute glycine betaine in the growth medium not only improved the growth of the wild-type strain but rescued the growth defect of the sigB mutant, indicating that the induction of the general stress regulon and the accumulation of glycine betaine are independent means by which B. subtilis cells cope with chill stress.

FIG. 1.

Model for the regulation of SigB activity in B. subtilis. The environmental- and metabolic-stress-sensing branches of the signal transduction cascade conveying their output to RsbV are depicted (34, 53). Chill stress activates SigB via a new mechanism that operates independently of RsbV, RsbU, and RsbP.

FIG. 2.

Influence of growth at a low temperature on the protein profile of B. subtilis 168. (A and B) Cells were grown in SMM at either 37°C (A) or 15°C (B) to an OD at 578 nm of 1.0. Crude protein extracts were prepared and separated by 2-DE. After being stained with silver nitrate, the gels were scanned with an imaging system and analyzed with the Melanie 3.0 software package. Proteins induced or repressed by growth at the low temperature are marked with arrowheads or circles, respectively. (C) Enlarged sections of gels under both conditions. Representative members of the SigB-dependent general stress regulon are labeled.

FIG. 3.

Effect of low temperature and ethanol stress on the levels of products of the sigB operon. Crude protein extracts were prepared from cultures of B. subtilis strain 168 grown at either 37°C (lanes 1 to 4) or 15°C (lane 5). To activate SigB, cells were treated for either 30 min (lane 2) or 60 min (lane 3) with 4% ethanol during exponential growth (OD at 540 nm, 0.5). Cultures were propagated either in LB medium (lanes 1 to 3) or in SMM (lanes 4 and 5). Samples collected for lanes 1, 4, and 5 were harvested during exponential growth (OD at 540 nm, 0.5). After separation by sodium dodecyl sulfate-polyacrylamide protein gel electrophoresis and transfer of the proteins to a nitrocellulose membrane, the proteins were reacted with a set of monoclonal antibodies, and specific antibody binding was detected by an alkaline phosphatase-conjugated goat anti-mouse secondary antibody.

FIG. 4.

Temperature-dependent expression of SigB-controlled ctc-lacZ (A) and gsiB-gfp (B) fusions. (A) The B. subtilis ctc-lacZ fusion strain BSA46 was grown in SMM at the indicated temperatures either in the absence (filled bars) or in the presence (open bars) of 1 mM glycine betaine. Samples were removed for β-galactosidase assays during exponential growth (OD at 578 nm, 0.5). β-Galactosidase activities are expressed in Miller units (MU). (B) The B. subtilis gsiB-gfp fusion strain BSM269 was grown in SMM at the indicated temperatures, and samples were removed for phase-contrast and fluorescence microscopy during exponential growth (OD at 540 nm, 1.0). Left and right panels display phase-contrast and fluorescence images, respectively, of the same cells.

FIG. 5.

Time-resolved low-temperature induction of ctc-lacZ in different growth media. The B. subtilis ctc-lacZ fusion strains BSM151 (SigB⁺) (filled circles) and BSM156 (SigB⁻) (open circles) were precultured in SMM at 37°C and used to inoculate cultures in either SMM, LB medium, or DSM. The cultures were then propagated at 16°C. Samples were removed for β-galactosidase assays at the indicated time points. β-Galactosidase activities are expressed in Miller units (MU). Solid lines indicate the growth of the wild-type strain (BSM151); growth of the sigB mutant strain (BSM156) did not differ from that of the wild-type strain under these growth conditions.

FIG. 6.

Influence of sigB and glycine betaine on growth at a low temperature. The B. subtilis wild-type strain 168 (squares) and its isogenic sigB mutant BSM29 (circles) were precultured in SMM at 37°C and used to inoculate 25-ml cultures in 100-ml Erlenmeyer flasks with (filled symbols) or without (open symbols) 1 mM glycine betaine. The cultures were then propagated at 15°C.

FIG. 7.

An RsbV-independent signal transduction cascade controls chill induction of gsiB-gfp. The isogenic set of B. subtilis strains BSM269 (RsbV⁺ SigB⁺), BSM275 (RsbV⁻ SigB⁺), and BSM276 (RsbV⁺ SigB⁻) was precultured in SMM at 37°C and used to inoculate cultures in SMM that were subsequently either exposed to ethanol stress or grown at 16°C. Samples were removed for phase-contrast and fluorescence microscopy from cultures growing exponentially (exp.) at 37 or 16°C (OD at 540 nm, 1.0) or 60 min after cells grown at 37°C had been exposed to 4% ethanol.

FIG. 8.

Effects of a low growth temperature and ethanol stress on the levels of products of the sigB operon in a wild-type strain and in SigB regulatory mutants. Crude protein extracts were prepared from cultures of an isogenic set of B. subtilis strains grown in SMM at either 37 or 16°C or treated with 4% ethanol (Et) at 37°C for 60 min. After separation by sodium dodecyl sulfate-polyacrylamide protein gel electrophoresis and transfer of the proteins to a nitrocellulose membrane, the proteins were reacted with a set of monoclonal antibodies, and specific antibody binding was detected by an alkaline phosphatase-conjugated goat anti-mouse secondary antibody. The following strains were used: BSM151 (wild type), BSM152 (rsbP), BSM154 (rsbU), BSM153 (rsbU rsbP), and BSM155 (rsbV).

FIG. 9.

Time-resolved chill-stress-induced expression of ctc-lacZ in a wild-type strain and a set of isogenic SigB regulatory mutants. A set of B. subtilis ctc-lacZ fusion strains was precultured in SMM at 37°C and used to inoculate cultures that were then propagated at 16°C. Samples were removed for β-galactosidase assays at the indicated time points. β-Galactosidase activities are expressed in Miller units (MU). Solid lines indicate the growth of the various strains. Filled circles display β-galactosidase activity. In the panel showing the wild-type strain we additionally included the β-galactosidase activity of the sigB mutant, which is represented by open circles. The following strains were used: BSM151 (wild type), BSM156 (sigB), BSM154 (rsbU), BSM152 (rsbP), BSM153 (rsbU rsbP), BSM155 (rsbV), and BSM267 (rsbV rsbP rsbU).