The Influence of G4 DNA Structures on Stationary Phase Mutagenesis in Bacillus subtilis

Until the late 1980’s it was widely understood that bacterial variance emerges randomly during growth. Research that followed has convincingly shown evidence for mutations arising in non-growing conditions, a phenomenon known as stationary-phase mutagenesis. In Bacillus subtilis, an elegant mutagenic mechanism has been elucidated in non-growing cells that biases mutations to transcribed regions of a subpopulation. One interesting possibility is that mutations can be further biased to hotspots within genes through alternate DNA structures known as non-B DNA. Non-B DNA have been linked to genomic instability and disease in humans, lesser is known about its role in bacteria. Here we investigate if G4 DNA, a type of non-B DNA, are mutagenic hotspots in non-growing B. subtilis cells. We hypothesize that G4 DNA can block RNA polymerase and trigger gratuitous transcription coupled repair. Gratuitous repair, or repair occurring in the absence of DNA damage, can lead to mutagenesis via repetitive re-synthesis of DNA, which can introduce mutations. In order to test this hypothesis, we constructed strains differing in their ability to form G4 DNA in a gene of interest and measured the effect on mutagenesis. We found that a strain having the potential to form G4 DNA in the coding strand had the highest levels of mutagenesis and this effect was dependent on a transcription coupled repair factor Mfd. Our data adds to the evidence of how B. subtilis avoids genetic load by having an elegant mechanism that biases mutations to distinct regions of genes under selection. Further, elucidating how alternate DNA structures promote genetic instability can lead to a better understanding of bacterial evolution and genetic diseases in humans

Mfd Protects Against Oxidative Stress in Bacillus Subtilis Independently of its Canonical Function in DNA Repair

Background: Previous reports showed that mutagenesis in nutrient-limiting conditions is dependent on Mfd in Bacillus subtilis. Mfd initiates one type of transcription-coupled repair (TCR); this type of repair is known to target bulky lesions, like those associated with UV exposure. Interestingly, the roles of Mfd in repair of oxidative-promoted DNA damage and regulation of transcription differ. Here, we used a genetic approach to test whether Mfd protected B. subtilis from exposure to two different oxidants. Results: Wild-type cells survived tert-butyl hydroperoxide (t-BHP) exposure significantly better than Mfd-deficient cells. This protective effect was independent of UvrA, a component of the canonical TCR/nucleotide excision repair (NER) pathway. Further, our results suggest that Mfd and MutY, a DNA glycosylase that processes 8-oxoG DNA mismatches, work together to protect cells from lesions generated by oxidative damage. We also tested the role of Mfd in mutagenesis in starved cells exposed to t-BHP. In conditions of oxidative stress, Mfd and MutY may work together in the formation of mutations. Unexpectedly, Mfd increased survival when cells were exposed to the protein oxidant diamide. Under this type of oxidative stress, cells survival was not affected by MutY or UvrA. Conclusions: These results are significant because they show that Mfd mediates error-prone repair of DNA and protects cells against oxidation of proteins by affecting gene expression; Mfd deficiency resulted in increased gene expression of the OhrR repressor which controls the cellular response to organic peroxide exposure. These observations point to Mfd functioning beyond a DNA repair factor in cells experiencing oxidative stress

Mfd Affects Global Transcription and the Physiology of Stressed Bacillus subtilis Cells

© Copyright © 2021 Martin, Sundararajan, Ermi, Heron, Gonzales, Lee, Anguiano-Mendez, Schilkey, Pedraza-Reyes and Robleto. For several decades, Mfd has been studied as the bacterial transcription-coupled repair factor. However, recent observations indicate that this factor influences cell functions beyond DNA repair. Our lab recently described a role for Mfd in disulfide stress that was independent of its function in nucleotide excision repair and base excision repair. Because reports showed that Mfd influenced transcription of single genes, we investigated the global differences in transcription in wild-type and mfd mutant growth-limited cells in the presence and absence of diamide. Surprisingly, we found 1,997 genes differentially expressed in Mfd– cells in the absence of diamide. Using gene knockouts, we investigated the effect of genetic interactions between Mfd and the genes in its regulon on the response to disulfide stress. Interestingly, we found that Mfd interactions were complex and identified additive, epistatic, and suppressor effects in the response to disulfide stress. Pathway enrichment analysis of our RNASeq assay indicated that major biological functions, including translation, endospore formation, pyrimidine metabolism, and motility, were affected by the loss of Mfd. Further, our RNASeq findings correlated with phenotypic changes in growth in minimal media, motility, and sensitivity to antibiotics that target the cell envelope, transcription, and DNA replication. Our results suggest that Mfd has profound effects on the modulation of the transcriptome and on bacterial physiology, particularly in cells experiencing nutritional and oxidative stress

Non-B DNA-Forming Motifs Promote Mfd-Dependent Stationary-Phase Mutagenesis in Bacillus subtilis

Transcription-induced mutagenic mechanisms limit genetic changes to times when expression happens and to coding DNA. It has been hypothesized that intrinsic sequences that have the potential to form alternate DNA structures, such as non-B DNA structures, influence these mechanisms. Non-B DNA structures are promoted by transcription and induce genome instability in eukaryotic cells, but their impact in bacterial genomes is less known. Here, we investigated if G4 DNA- and hairpin-forming motifs influence stationary-phase mutagenesis in Bacillus subtilis. We developed a system to measure the influence of non-B DNA on B. subtilis stationary-phase mutagenesis by deleting the wild-type argF at its chromosomal position and introducing IPTG-inducible argF alleles differing in their ability to form hairpin and G4 DNA structures into an ectopic locus. Using this system, we found that sequences predicted to form non-B DNA structures promoted mutagenesis in B. subtilis stationary-phase cells; such a response did not occur in growing conditions. We also found that the transcription-coupled repair factor Mfd promoted mutagenesis at these predicted structures. In summary, we showed that non-B DNA-forming motifs promote genetic instability, particularly in coding regions in stressed cells; therefore, non-B DNA structures may have a spatial and temporal mutagenic effect in bacteria. This study provides insights into mechanisms that prevent or promote mutagenesis and advances our understanding of processes underlying bacterial evolution