Previously, using a chromosomal reversion assay system, we established that an adaptive mutagenic process occurs in nongrowing Bacillus subtilis cells under stress, and we demonstrated that multiple mechanisms are involved in generating these mutations (41, 43). In an attempt to delineate how these mutations are generated, we began an investigation into whether or not transcription and transcription-associated proteins influence adaptive mutagenesis. In B. subtilis, the Mfd protein (transcription repair coupling factor) facilitates removal of RNA polymerase stalled at transcriptional blockages and recruitment of repair proteins to DNA lesions on the transcribed strand. Here we demonstrate that the loss of Mfd has a depressive effect on stationary-phase mutagenesis. An association between Mfd mutagenesis and aspects of transcription is discussed.

Adaptive (stationary phase) mutagenesis is a phenomenon by which nondividing cells acquire beneficial mutations as a response to stress. Although the generation of adaptive mutations is essentially stochastic, genetic factors are involved in this phenomenon. We examined how defects in a transcriptional factor, previously reported to alter the acquisition of adaptive mutations, affected mutation levels in a gene under selection. The acquisition of mutations was directly correlated to the level of transcription of a defective leuC allele placed under selection. To further examine the correlation between transcription and adaptive mutation, we placed a point-mutated allele, leuC427 , under the control of an inducible promoter and assayed the level of reversion to leucine prototrophy under conditions of leucine starvation. Our results demonstrate that the level of Leu ⁺ reversions increased significantly in parallel with the induced increase in transcription levels. This mutagenic response was not observed under conditions of exponential growth. Since transcription is a ubiquitous biological process, transcription-associated mutagenesis may influence evolutionary processes in all organisms.

One of the experimental platforms to study programs increasing genetic diversity in cells under stressful or nondividing conditions is adaptive mutagenesis, also called stationary phase mutagenesis or stress-induced mutagenesis. In some model systems, there is evidence that mutagenesis occurs in genes that are actively transcribed. Some of those genes may be actively transcribed as a result of environmental stress giving the appearance of directed mutation. That is, cells under conditions of starvation or other stresses accumulate mutations in transcribed genes, including those transcribed because of the selective pressure. An important question concerns how, within the context of stochastic processes, a cell biases mutation to genes under selection pressure? Because the mechanisms underlying DNA transactions in prokaryotic cells are well conserved among the three domains of life, these studies are likely to apply to the examination of genetic programs in eukaryotes. In eukaryotes, increasing genetic diversity in differentiated cells has been implicated in neoplasia and cell aging. Historically, Escherichia coli has been the paradigm used to discern the cellular processes driving the generation of adaptive mutations; however, examining adaptive mutation in Bacillus subtilis has contributed new insights. One noteworthy contribution is that the B. subtilis' ability to accumulate chromosomal mutations under conditions of starvation is influenced by cell differentiation and transcriptional derepression, as well as by proteins homologous to transcription and repair factors. Here we revise and discuss concepts pertaining to genetic programs that increase diversity in B. subtilis cells under nutritional stress.

In replication-limited cells of Bacillus subtilis, Mfd is mutagenic at highly transcribed regions, even in the absence of bulky DNA lesions. However, the mechanism leading to increased mutagenesis through Mfd remains currently unknown. Here, we report that Mfd may promote mutagenesis in nutritionally stressed B. subtilis cells by coordinating error-prone repair events mediated by UvrA, MutY and PolI. Using a point-mutated gene conferring leucine auxotrophy as a genetic marker, it was found that the absence of UvrA reduced the Leu⁺ revertants and that a second mutation in mfd reduced mutagenesis further. Moreover, the mfd and polA mutants presented low but similar reversion frequencies compared to the parental strain. These results suggest that Mfd promotes mutagenic events that required the participation of NER pathway and PolI. Remarkably, this Mfd-dependent mutagenic pathway was found to be epistatic onto MutY; however, whereas the MutY-dependent Leu⁺ reversions required Mfd, a direct interaction between these proteins was not apparent. In summary, our results support the concept that Mfd promotes mutagenesis in starved B. subtilis cells by coordinating both known and previously unknown Mfd-associated repair pathways. These mutagenic processes bias the production of genetic diversity towards highly transcribed regions in the genome.

In recent years, it has been proposed that conflicts between transcription and active chromosomal replication engender genome instability events. Furthermore, transcription elongation factors have been reported to prevent conflicts between transcription and replication and avoid genome instability. Here, we examined transcriptional de-repression as a genetic diversity-producing agent and showed, through the use of physiological and genetic means, that transcriptional de-represssion of a leuC defective allele leads to accumulation of Leu⁺ mutations. We also showed, by using riboswitches that activate transcription in conditions of tyrosine or methionine starvation, that the effect of transcriptional de-repression of the leuC construct on the accumulation of Leu⁺ mutations was independent of selection. We examined the role of Mfd, a transcription elongation factor involved in DNA repair, in this process and showed that proficiency of this factor promotes mutagenic events. These results are in stark contrast to previous reports in Escherichia coli, which showed that Mfd prevents replication fork collapses. Because our assays place cells under non-growing conditions, by starving them for two amino acids, we surmised that the Mfd mutagenic process associated with transcriptional de-repression does not result from conflicts with chromosomal replication. These results raise the interesting concept that transcription elongation factors may serve two functions in cells. In growing conditions, these factors prevent the generation of mutations, while in stress or non-growing conditions they mediate the production of genetic diversity.