Dynamics of Mismatch and Alternative Excision-Dependent Repair in Replicating Bacillus subtilis DNA Examined Under Conditions of Neutral Selection.

Spontaneous DNA deamination is a potential source of transition mutations. In Bacillus subtilis, EndoV, a component of the alternative excision repair pathway (AER), counteracts the mutagenicity of base deamination-induced mispairs. Here, we report that the mismatch repair (MMR) system, MutSL, prevents the harmful effects of HNO2, a deaminating agent of Cytosine (C), Adenine (A), and Guanine (G). Using Maximum Depth Sequencing (MDS), which measures mutagenesis under conditions of neutral selection, in B. subtilis strains proficient or deficient in MutSL and/or EndoV, revealed asymmetric and heterogeneous patterns of mutations in both DNA template strands. While the lagging template strand showed a higher frequency of C → T substitutions; G → A mutations, occurred more frequently in the leading template strand in different genetic backgrounds. In summary, our results unveiled a role for MutSL in preventing the deleterious effects of base deamination and uncovered differential patterns of base deamination processing by the AER and MMR systems that are influenced by the sequence context and the replicating DNA strand.

Analysing the fitness cost of antibiotic resistance to identify targets for combination antimicrobials.

Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) confer antibiotic resistance and often have global effects on transcription that compromise fitness and stress tolerance of resistant mutants. We suggested that the non-essential genome, through its impact on the bacterial transcription cycle, may represent an untapped source of targets for combination antimicrobial therapies. Using transposon sequencing, we carried out a genome-wide analysis of fitness cost in a clinically common rpoB H526Y mutant. We find that genes whose products enable increased transcription elongation rates compound the fitness costs of resistance whereas genes whose products function in cell wall synthesis and division mitigate it. We validate our findings by showing that the cell wall synthesis and division defects of rpoB H526Y result from an increased transcription elongation rate that is further exacerbated by the activity of the uracil salvage pathway and unresponsiveness of the mutant RNAP to the alarmone ppGpp. We applied our findings to identify drugs that inhibit more readily rpoB H526Y and other RifR alleles from the same phenotypic class. Thus, genome-wide analysis of fitness cost of antibiotic-resistant mutants should expedite the discovery of new combination therapies and delineate cellular pathways that underlie the molecular mechanisms of cost.

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.

The Bacillus Subtilis K-State Promotes Stationary-Phase Mutagenesis via Oxidative Damage.

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.

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.

Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways.

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.