title: Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.
abstract: Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations.
authors.affiliation: Department of General Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Dormeyer
authors.affiliation: Department of General Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
authors.forename: Anastasia L
authors.initials: AL
authors.lastname: Lübke
authors.affiliation: Department of Genetics, Bacterial Genetics, Friedrich Schiller University Jena, Philosophenweg 12, Jena, 07743, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Müller
authors.affiliation: Department of General Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Lentes
authors.affiliation: Department of General Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Reuß
authors.affiliation: Department of Genomic and Applied Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Thürmer
authors.affiliation: Department of General Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Stülke
authors.affiliation: Department of Genomic and Applied Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Daniel
authors.affiliation: Department of Genetics, Bacterial Genetics, Friedrich Schiller University Jena, Philosophenweg 12, Jena, 07743, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Brantl
authors.affiliation: Department of General Microbiology, Georg August University Göttingen, Grisebachstr. 8, Göttingen, 37077, Germany.
authors.forename: Fabian M
authors.initials: FM
authors.lastname: Commichau
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D005784:Gene Amplification / Q000235:genetics; D018628:Gene Dosage / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005970:Glutamate Synthase / Q000235:genetics*; D018698:Glutamic Acid / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C095882:GltC protein, Bacillus subtilis; D012097:Repressor Proteins; D015534:Trans-Activators; D018698:Glutamic Acid; D005970:Glutamate Synthase