Genetic Control of Osmoadaptive Glycine Betaine Synthesis in Bacillus subtilis through the Choline-Sensing and Glycine Betaine-Responsive GbsR Repressor

Genetic control of gbsAB expression in response to the presence of external inducers, high salinity, and the GbsR regulator. Cells of the gbsA-treA reporter strains GNB45 (gbsR⁺) (A) or GNB48 (gbsR::neo) (B) were grown at 37°C either in SMM or in SMM with 0.4 M NaCl to an OD₅₇₈ of 0.25; 1 mM choline (Cho), glycine betaine aldehyde (GBA), or glycine betaine (GB), respectively, was then added to some of the cultures, and the activity of the TreA reporter enzyme was determined after 90 min of further cultivation.

DNA sequence of the intergenic gbsR-gbsAB regulatory region. (A) The −10 and −35 sequences of the SigA-type promoters for the gbsR gene and the gbsAB operon are indicated, and the mapped transcriptional start sites for these genes are shown by bent arrows. The transcriptional start site for the gbsAB operon was mapped by Boch et al. (5) and that of the gbsR gene was determined in this study (see Fig. S1 in the supplemental material). The putative GbsR binding site positioned downstream of the gbsAB transcription initiation site is indicated by a pair of inverted arrows. The beginning of the coding regions for the GbsR and GbsA proteins is marked. The two black triangles indicate the extent of the inferred minimal DNA fragment (Fig. 5) that permits GbsR-mediated induction of gbsAB transcription in response to choline. (B) Putative GbsR binding sites in the gbsAB and opuC regulatory regions. The −10 and −35 sequences of the SigA-type promoters for the opuB and opuC operons are indicated; the transcriptional start site for the opuB mRNA has been experimentally determined (36) (indicated by a bent arrow), and the promoter for the opuC operon was predicted by DNA sequence gazing. The putative GbsR binding sites are indicated by pairs of inverted arrows; the asterisks (*) mark differences in the DNA sequences of the putative GbsR binding site in the opuB and opuC regulatory regions.

Influence of GbsR on choline import via the OpuB and OpuC ABC transporters. Cells were cultivated at 37°C in SMM or in SMM containing 0.4 M NaCl until they had reached an OD₅₇₈ of about 0.4. Subsequently, uptake of [¹⁴C]choline was assayed at a final substrate concentration of 10 μM. (A) Uptake of [¹⁴C]choline in the wild-type strain JH642 (opuB⁺ opuC⁺ gbsR⁺) (□, ■) and strain JBB8 (opuB⁺ opuC⁺ gbsR::neo) (○, ●) grown in SMM (□, ○) or SMM with 0.4 M NaCl (■, ●). (B) Uptake of [¹⁴C]choline in strain RMKB25 [opuB⁺ opuC::Tn10 (spc) gbsR⁺] (♦), GNB25 [opuB⁺ opuC::Tn10 (spc) gbsR::neo] (♢), strain RMKB26 [opuB::Tn10 (spc) opuC⁺ gbsR⁺] (▲) and GNB26 [opuB::Tn10 (spc) opuC⁺ gbsR::neo] (▵); cells were grown at 37°C in SMM containing 0.4 M NaCl.

Choline-responsive and GbsR-dependent regulation of gbsA-treA expression. Strains with either an intact gbsR gene (+) or a gbsR::neo mutation (−) carrying chromosomal gbsA-treA fusions of various lengths were pregrown in SMM to early log phase (OD₅₇₈ of 0.25), and aliquots were assayed for TreA activity (noninduced condition). At this time point, 1 mM choline (final concentration) and 0.4 M NaCl (final concentration) were added to the cultures; after further growth for 90 min, the cells were harvested for assays of the TreA reporter enzyme (induced condition). NaCl was added to these cultures to elicit enhanced uptake of the inducer choline via the osmotically stimulated OpuB and OpuC transport systems (36). The symbol (Δ) indicates that the corresponding segment of the gbsR gene was deleted from the DNA fragment fused to the treA reporter gene. All fusion strains are derivatives of the B. subtilis strain JH642 (trp phe).

Binding of choline by the GbsR regulatory protein. (A) SDS-polyacrylamide gel electrophoresis of the purified GbsR protein. (B) Fluorescence spectrum of the purified GbsR protein (5 μM) in the absence or the presence of 1 mM choline. (C) Binding kinetics of choline to the purified GbsR protein (5 μM) as assessed by intrinsic fluorescence spectroscopy. (D) Intrinsic fluorescence spectrum of the purified GbsR protein (5 μM) in the absence or the presence of 1 mM glycine betaine.

Cells of the gbsA-treA fusion strain GNB46 (gbsAB::neo gbsR⁺) (A) and of strain GNB48 (gbsAB⁺ gbsR::neo) (B) were grown either in SMM without a compatible solute (−) or in SMM containing 1 mM choline (Cho) or 1 mM choline in combination with various compatible solutes: glycine betaine (GB), carnitine (Car), proline betaine (PB), dimethylsulfoniopropionate (DMSP), choline-O-sulfate (COS), l-proline (Pro), or ectoine (Ect). Cells were harvested for TreA reporter assays when they reached an OD₅₇₈ of 1 to 1.5.