Abstract
Soil bacteria like Bacillus subtilis can cope with many growth conditions by adjusting gene expression and metabolic pathways. Alternatively, bacteria can spontaneously accumulate beneficial mutations or shape their genomes in response to stress. Recently, it has been observed that a B. subtilis mutant lacking the catabolically active glutamate dehydrogenase (GDH), RocG, mutates the cryptic gudBCR gene at a high frequency. The suppressor mutants express the active GDH GudB, which can fully replace the function of RocG. Interestingly, the cryptic gudBCR allele is stably inherited as long as the bacteria synthesize the functional GDH RocG. Competition experiments revealed that the presence of the cryptic gudBCR allele provides the bacteria with a selective growth advantage when glutamate is scarce. Moreover, the lack of exogenous glutamate is the driving force for the selection of mutants that have inactivated the active gudB gene. In contrast, two functional GDHs are beneficial for the cells when glutamate was available. Thus, the amount of GDH activity strongly affects fitness of the bacteria depending on the availability of exogenous glutamate. At a first glance the high mutation frequency of the cryptic gudBCR allele might be attributed to stress-induced adaptive mutagenesis. However, other loci on the chromosome that could be potentially mutated during growth under the selective pressure that is exerted on a GDH-deficient mutant remained unaffected. Moreover, we show that a GDH-proficient B. subtilis strain has a strong selective growth advantage in a glutamate-dependent manner. Thus, the emergence and rapid clonal expansion of the active gudB allele can be in fact explained by spontaneous mutation and growth under selection without an increase of the mutation rate. Moreover, this study shows that the selective pressure that is exerted on a maladapted bacterium strongly affects the apparent mutation frequency of mutational hot spots.
Highlights
The high abundance of glutamate in many living organisms suggests that this metabolite fulfils fundamental tasks in the cell [1,2,3,4]
We have previously shown that only mutants of the laboratory strain 168, devoid of any glutamate-degrading glutamate dehydrogenase (GDH) activity can grow in CS medium containing succinate and ammonium as poor sources of carbon and nitrogen, respectively, even though this strain possesses the genetic equipment for glutamate biosynthesis under these conditions [20]
The observation that the cryptic gudBCR gene is rapidly mutated with a high frequency of 1024 in a B. subtilis rocG– mutant raised the question whether other loci on the B. subtilis chromosome that could be potentially mutated are modified by the same factor(s) during growth under strong selective pressure that is exerted on the rocG– mutant
Summary
The high abundance of glutamate in many living organisms suggests that this metabolite fulfils fundamental tasks in the cell [1,2,3,4]. The Grampositive model organism Bacillus subtilis, needs glutamate in high amounts to synthesize proline, which serves as a compatible solute to protect cells growing under high external osmotic pressure [9]. In B. subtilis glutamate is exclusively synthesized by the combined action of the glutamine synthetase (GS) and the glutamate synthase (GOGAT) that are encoded by the glnA and gltAB genes, respectively (for a recent review [10]). The inability of RocG to synthesize glutamate in the background of a B. subtilis cell is caused by the very low affinity of the enzyme for ammonium [12,13]
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