title: Mutational activation of the RocR activator and of a cryptic rocDEF promoter bypass loss of the initial steps of proline biosynthesis in Bacillus subtilis.
abstract: 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.
authors.affiliation: Department of Biology, Laboratory for Microbiology, Philipps-University Marburg, Karl-von-Frisch Str. 8, Marburg, D-35032, Germany.
authors.forename: Adrienne
authors.initials: A
authors.lastname: Zaprasis
authors.forename: Tamara
authors.initials: T
authors.lastname: Hoffmann
authors.forename: Guido
authors.initials: G
authors.lastname: Wünsche
authors.forename: Lope A
authors.initials: LA
authors.lastname: Flórez
authors.forename: Jörg
authors.initials: J
authors.lastname: Stülke
authors.forename: Erhard
authors.initials: E
authors.lastname: Bremer
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000096:biosynthesis / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000096:biosynthesis* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011758:Pyrroles; D016415:Sequence Alignment; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011758:Pyrroles; D015534:Trans-Activators; C086032:rocR protein, Bacillus subtilis; C015485:delta-1-pyrroline-5-carboxylate; D001120:Arginine; D011392:Proline