A High-Frequency Mutation in Bacillus subtilis: Requirements for the Decryptification of the gudB Glutamate Dehydrogenase Gene

Abstract

Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10 ؊4 . Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes.A s the central amino group donor for nearly all biosynthetic pathways in any living cell, glutamate plays a key role in the biochemistry and physiology of all organisms (15). Investigations with Escherichia coli demonstrate that glutamate is by far the most abundant metabolite in these bacteria, accounting for ca. 40% of the internal metabolite pool (60). Moreover, glutamate is one of the most highly embedded metabolites. In the Gram-positive soil bacterium Bacillus subtilis, at least 37 reactions make use of this amino acid (42).In B. subtilis, glutamate is exclusively synthesized from 2-oxoglutarate and glutamine by the activity of glutamate synthase in the absence of exogenous glutamate or other sources of glutamate. 2-Oxoglutarate is replenished in the citric acid cycle, whereas glutamine can be synthesized with ammonium as the nitrogen source and one of the two molecules of glutamate that are generated by glutamate synthase as the acceptor. Glutamate does also serve as a precursor for proline biosynthesis and, under conditions of osmotic stress, molar concentrations of proline have to be produced (28). Thus, it is not surprising that glutamate synthesis has to be a highly efficient process and, indeed, interactions between enzymes of the branch of the citric acid cycle that generates 2-oxoglutarate and glutamate synthase have been reported (39). Glutamate can also serve as source of carbon and nitrogen. Its utilization is initiated by an oxidative deamination...

Experimental Evolution of Enhanced Growth by Bacillus subtilis at Low Atmospheric Pressure: Genomic Changes Revealed by Whole-Genome Sequencing

“…A well-studied example in B. subtilis is the activation of a second glutamate dehydrogenase (GDH) encoded by the cryptic gudB gene via an in-frame 9-bp deletion event; this deletion occurs at high frequency in response to glutamate starvation stress in mutants lacking the major GDH (27)(28)(29). A second example concerns in vivo development of daptomycin resistance in a clinical isolate of Enterococcus faecalis, which was shown to result from in-frame deletions in three genes involved in the response to antibiotics causing cell envelope stress (30).…”

Selection-Driven Accumulation of Suppressor Mutants in Bacillus subtilis: The Apparent High Mutation Frequency of the Cryptic gudB Gene and the Rapid Clonal Expansion of gudB+ Suppressors Are Due to Growth under Selection

“…However, only the rocG gene encodes a functional GDH, whereas the gudB CR gene is cryptic and encodes the enzymatically inactive GDH, GudB CR (formerly designated as GudB, [11], [25]). The GDH GudB CR is enzymatically inactive and extremely unstable because it contains a duplication of three amino acids in its active centre [11], [26], [27]. The duplication of these amino acids in GudB CR is due to a perfect 9 bp-long direct repeat (DR) that is present in the cryptic gudB CR gene.…”

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance

“…Pseudomonas aeruginosa and presumably other members of the genus Pseudomonas have a NADP + -specific and a NAD + -specific GDH, and it has been hypothesized than the latter acts specifically in arginine catabolism by converting glutamate, a product of the ammonia-producing arginine succinyl transferase (AST) pathway, into 2-OG, since it is allosterically modulated by arginine (positively) and citrate (negatively) [25]. Similarly, the only active GDH from Bacillus subtilis (RocG, NAD + -dependent) appears to be involved in arginine and proline catabolism [46]. On the other hand, despite the catabolic function assigned to NAD + -GDHs, the existence of an NAD + -specific GDH with an unusual biosynthetic role has been reported in the oral bacterium Capnocytophaga ochraea [ 3 5 ] .…”

“…While RocG is an enzymatically active GDH, GudB is inactive, due to a duplication of three amino acid residues at its active center. Decryptification of gudB in a rocG background is achieved by high-frequency acquisition of a suppressor mutation consisting of the precise deletion of part of the 9-bp direct repeat that prevents activity [46,47]. Both RocG and decryptified GudB are primarily catabolic dehydrogenases, and de novo glutamate synthesis in B. subtilis is performed exclusively by GOGAT.…”