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.

The major cause of death in people with cystic fibrosis (CF), a human autosomal recessive genetic disease, is respiratory failure due to chronic lung infection. Pseudomonas aeruginosa is a prevalent CF respiratory pathogen (Cystic Fibrosis Foundation Patient Registry, 2019). The CF lung environment selects for mucoid P. aeruginosa mutants, which overproduce the exopolysaccharide alginate and are associated with poor disease prognosis (

Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the accumulation of mutations in stationary phase in B. subtilis. Since recombination is not involved in this process even though ComK is, we investigated if the development of a subpopulation (K-cells) could be involved in stationary-phase mutagenesis. Using genetic knockout strains and a point-mutation reversion system, we investigated the effects of ComK, ComEA (a protein involved in DNA transport during transformation), and oxidative damage on stationary-phase mutagenesis. We found that stationary-phase revertants were more likely to have undergone the development of competence than the background of non-revertant cells, mutations accumulated independently of DNA uptake, and the presence of exogenous oxidants potentiated mutagenesis in K-cells. Therefore, the development of the K-state creates conditions favorable to an increase in the genetic diversity of the population not only through exogenous DNA uptake but also through stationary-phase mutagenesis.Genes 2020, 11, 190 2 of 15 surrounding DNA double-stranded breaks [4], which occur at a 10 −3 frequency spontaneously [5]. Repair of double-stranded breaks, processed by recombination, can generate point mutations produced via error-prone synthesis or genetic amplifications that confer fitness to cells experiencing limited replication [6][7][8]. Interestingly, genetic regions undergoing transcription in stressed cells can precipitate the formation of double-stranded breaks via the formation of R-loops [9]. Also, factors affecting transcription termination influence stress-induced mutagenesis [10]. Therefore, the observations above support a model in which stressed E. coli manage increases in genetic diversity by activating mechanisms that operate on a subpopulation of cells and transcribed DNA undergoing repair of double-stranded breaks [1].In the context of increasing genetic diversity in times of nutritional stress, B. subtilis halts replication and differentiates subpopulations [11,12]. One subpopulation of cells become naturally competent, a strategy to acquire new genes from the environment by the process of recombination [13]. Interestingly, Bacillus subtilis also expresses stationary-phase mutagenesis. In these processes, the transcription repair coupling factor Mfd plays a central role, interacting with components of the nucleotide excision repair (NER) or base exc...

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF), and this bacterium undergoes selection in the CF lung environment over the course of these life-long infections. One genetic adaptation frequently observed in CF P. aeruginosa isolates is mutation of mucA. MucA inhibits the sigma factor AlgU. Clinical mucA mutations lead to misregulation of AlgU, resulting in a mucoid bacterial phenotype that is associated with poor CF disease outcomes. Here we show that paradoxically a portion of the mucA gene is essential for P. aeruginosa viability. We demonstrate that mucA is no longer essential in a strain lacking algU, and mucA alleles that encode for proteins that do not bind to AlgU are insufficient for bacterial viability. Furthermore, we found that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity, suggesting that reducing AlgU activity can suppress the requirement for mucA. Finally, we found that overexpression of algU from an inducible promoter prevents cell growth in the absence of MucA, and that this phenotype can be rescued by overproduction of RpoD, the housekeeping sigma factor. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity leads to sigma factor competition, preventing the expression of essential housekeeping genes and resulting in the loss of bacterial viability.

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