Mutational activation of the RocR activator and of a cryptic rocDEF promoter bypass loss of the initial steps of proline biosynthesis in Bacillus subtilis.

The gamma-glutamyl-phosphate reductase (ProA) interlinks both the anabolic and osmostress adaptive proline biosynthetic routes of Bacillus subtilis. Because no paralogous protein to ProA exists in this microorganism, proA mutants should exhibit a tight proline auxotrophic growth phenotype. Contrary to expectations, proA mutants formed microcolonies on agar plates lacking proline and faster growing Pro(+) suppressor mutants arose. These mutants carried alterations in the rocR-rocDEF region encoding enzymes of the arginine degradation pathway and its transcriptional activator RocR. They were of two types: (i) mutants carrying single amino acid substitutions in RocR resulting in partial inducer-independent variants and (ii) mutants carrying single base-pair changes in the vicinity of the SigL/Sig-54-dependent -12/-24 class rocDEF promoter that activate a cryptic SigA-type promoter. Consequently, enhanced rocDEF transcription should lead to increased cellular amounts of the RocD ornithine aminotransferase, an enzyme that synthesizes the same reaction product as ProA, gamma-glutamic-semialdehyde/delta-1-pyrroline-5-carboxylate. This compound can be enzymatically converted into proline. The Pro(+) suppressors also exhibited a new regulatory pattern by allowing enhanced rocDEF transcription in response to proline availability when ammonium is present. Our work provides an example how flexibly bacteria can genetically develop routes to bypass constraints imposed on their biosynthetic networks and evolve new regulatory mechanisms.