Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity

Bacillus subtilis is known to accumulate large amounts of the compatible solute proline viade novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on theproJ -encoded γ-glutamyl kinase, theproA -encoded γ-glutamyl phosphate reductase, and theproH -encoded Δ1 -pyrroline-5-caboxylate reductase. Disruption of theproHJ operon abolished osmoadaptive proline production and strongly impaired the ability ofB. subtilis to cope with high-osmolarity growth conditions. Disruption of theproA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption ofproHJ , proline auxotrophy. Northern blot analysis demonstrated that the transcription of theproHJ operon is osmotically inducible, whereas that of theproBA operon is not. Reporter gene fusion studies showed thatproHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlledproHJ promoter, a promoter that resembles typical SigA-type promoters ofB. subtilis . Deletion analysis of theproHJ promoter region identified a 126-bp DNA segment carrying all sequences required incis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes inB. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.