Stress-Associated and Growth-Dependent Mutagenesis Are Divergently Regulated by c-di-AMP Levels in Bacillus subtilis.

A previous proteomic study uncovered a relationship between nutritional stress and fluctuations in levels of diadenylate cyclases (DACs) and other proteins that regulate DAC activity, degrade, or interact with c-di-AMP, suggesting a possible role of this second messenger in B. subtilis stress-associated mutagenesis (SAM). Here, we investigated a possible role of c-di-AMP in SAM and growth-associated mutagenesis (GAM). Our results showed that in growing cells of B. subtilis YB955 (hisC952, metB25 and leuC427), the DACs CdaA and DisA, which play crucial roles in cell wall homeostasis and chromosomal fidelity, respectively, counteracted spontaneous and Mitomycin-C-induced mutagenesis. However, experiments in which hydrogen peroxide was used to induce mutations showed that single deficiencies in DACs caused opposite effects compared to each other. In contrast, in the stationary-phase, DACs promoted mutations in conditions of nutritional stress. These results tracked with intracellular levels of c-di-AMP, which are significantly lower in cdaA- and disA-deficient strains. The restoration of DAC-deficient strains with single functional copies of the cdaA and/or disA returned SAM and GAM levels to those observed in the parental strain. Taken together, these results reveal a role for c-di-AMP in promoting genetic diversity in growth-limiting conditions in B. subtilis. Finally, we postulate that this novel function of c-di-AMP can be exerted through proteins that possess binding domains for this second messenger and play roles in DNA repair, ion transport, transcriptional regulation, as well as oxidative stress protection.

Role of Mfd and GreA in Bacillus subtilis Base Excision Repair-Dependent Stationary-Phase Mutagenesis.

We report that the absence of an oxidized guanine (GO) system or the apurinic/apyrimidinic (AP) endonucleases Nfo, ExoA, and Nth promoted stress-associated mutagenesis (SAM) in Bacillus subtilis YB955 (hisC952 metB5 leuC427). Moreover, MutY-promoted SAM was Mfd dependent, suggesting that transcriptional transactions over nonbulky DNA lesions promoted error-prone repair. Here, we inquired whether Mfd and GreA, which control transcription-coupled repair and transcription fidelity, influence the mutagenic events occurring in nutritionally stressed B. subtilis YB955 cells deficient in the GO or AP endonuclease repair proteins. To this end, mfd and greA were disabled in genetic backgrounds defective in the GO and AP endonuclease repair proteins, and the strains were tested for growth-associated and stress-associated mutagenesis. The results revealed that disruption of mfd or greA abrogated the production of stress-associated amino acid revertants in the GO and nfo exoA nth strains, respectively. These results suggest that in nutritionally stressed B. subtilis cells, spontaneous nonbulky DNA lesions are processed in an error-prone manner with the participation of Mfd and GreA. In support of this notion, stationary-phase ΔytkD ΔmutM ΔmutY (referred to here as ΔGO) and Δnfo ΔexoA Δnth (referred to here as ΔAP) cells accumulated 8-oxoguanine (8-OxoG) lesions, which increased significantly following Mfd disruption. In contrast, during exponential growth, disruption of mfd or greA increased the production of His+, Met+, or Leu+ prototrophs in both DNA repair-deficient strains. Thus, in addition to unveiling a role for GreA in mutagenesis, our results suggest that Mfd and GreA promote or prevent mutagenic events driven by spontaneous genetic lesions during the life cycle of B. subtilisIMPORTANCE In this paper, we report that spontaneous genetic lesions of an oxidative nature in growing and nutritionally stressed B. subtilis strain YB955 (hisC952 metB5 leuC427) cells drive Mfd- and GreA-dependent repair transactions. However, whereas Mfd and GreA elicit faithful repair events during growth to maintain genome fidelity, under starving conditions, both factors promote error-prone repair to produce genetic diversity, allowing B. subtilis to escape from growth-limiting conditions.

Novel Biochemical Properties and Physiological Role of the Flavin Mononucleotide Oxidoreductase YhdA from Bacillus subtilis.

Cr(VI) is mutagenic and teratogenic and considered an environmental pollutant of increasing concern. The use of microbial enzymes that convert this ion into its less toxic reduced insoluble form, Cr(III), represents a valuable bioremediation strategy. In this study, we examined the Bacillus subtilis YhdA enzyme, which belongs to the family of NADPH-dependent flavin mononucleotide oxide reductases and possesses azo-reductase activity as a factor that upon overexpression confers protection on B. subtilis from the cytotoxic effects promoted by Cr(VI) and counteracts the mutagenic effects of the reactive oxygen species (ROS)-promoted lesion 8-OxoG. Further, our in vitro assays unveiled catalytic and biochemical properties of biotechnological relevance in YhdA; a pure recombinant His10-YhdA protein efficiently catalyzed the reduction of Cr(VI) employing NADPH as a cofactor. The activity of the pure oxidoreductase YhdA was optimal at 30°C and at pH 7.5 and displayed Km and Vmax values of 7.26 mM and 26.8 µmol·min-1·mg-1 for Cr(VI), respectively. Therefore, YhdA can be used for efficient bioremediation of Cr(VI) and counteracts the cytotoxic and genotoxic effects of oxygen radicals induced by intracellular factors and those generated during reduction of hexavalent chromium.IMPORTANCE Here, we report that the bacterial flavin mononucleotide/NADPH-dependent oxidoreductase YhdA, widely distributed among Gram-positive bacilli, conferred protection to cells from the cytotoxic effects of Cr(VI) and prevented the hypermutagenesis exhibited by a MutT/MutM/MutY-deficient strain. Additionally, a purified recombinant His10-YhdA protein displayed a strong NADPH-dependent chromate reductase activity. Therefore, we postulate that in bacterial cells, YhdA counteracts the cytotoxic and genotoxic effects of intracellular and extracellular inducers of oxygen radicals, including those caused by hexavalent chromium.

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.

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-ß-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H2O2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. IMPORTANCE: Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions.

Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations.

Compelling evidence points to transcriptional processes as important factors contributing to stationary-phase associated mutagenesis. However, it has not been documented whether or not base excision repair mechanisms play a role in modulating mutagenesis under conditions of transcriptional derepression. Here, we report on a flow cytometry-based methodology that employs a fluorescent reporter system to measure at single-cell level, the occurrence of transcription-associated mutations in nutritionally stressed B. subtilis cultures. Using this approach, we demonstrate that (i) high levels of transcription correlates with augmented mutation frequency, and (ii) mutation frequency is enhanced in nongrowing population cells deficient for deaminated (Ung, YwqL) and oxidized guanine (GO) excision repair, strongly suggesting that accumulation of spontaneous DNA lesions enhance transcription-associated mutagenesis.

Role of Bacillus subtilis DNA Glycosylase MutM in Counteracting Oxidatively Induced DNA Damage and in Stationary-Phase-Associated Mutagenesis.

UNLABELLED: Reactive oxygen species (ROS) promote the synthesis of the DNA lesion 8-oxo-G, whose mutagenic effects are counteracted in distinct organisms by the DNA glycosylase MutM. We report here that in Bacillus subtilis, mutM is expressed during the exponential and stationary phases of growth. In agreement with this expression pattern, results of a Western blot analysis confirmed the presence of MutM in both stages of growth. In comparison with cells of a wild-type strain, cells of B. subtilis lacking MutM increased their spontaneous mutation frequency to Rif(r) and were more sensitive to the ROS promoter agents hydrogen peroxide and 1,1'-dimethyl-4,4'-bipyridinium dichloride (Paraquat). However, despite MutM's proven participation in preventing ROS-induced-DNA damage, the expression of mutM was not induced by hydrogen peroxide, mitomycin C, or NaCl, suggesting that transcription of this gene is not under the control of the RecA, PerR, or σ(B) regulons. Finally, the role of MutM in stationary-phase-associated mutagenesis (SPM) was investigated in the strain B. subtilis YB955 (hisC952 metB5 leuC427). Results revealed that under limiting growth conditions, a mutM knockout strain significantly increased the amount of stationary-phase-associated his, met, and leu revertants produced. In summary, our results support the notion that the absence of MutM promotes mutagenesis that allows nutritionally stressed B. subtilis cells to escape from growth-limiting conditions. IMPORTANCE: The present study describes the role played by a DNA repair protein (MutM) in protecting the soil bacterium Bacillus subtilis from the genotoxic effects induced by reactive oxygen species (ROS) promoter agents. Moreover, it reveals that the genetic inactivation of mutM allows nutritionally stressed bacteria to escape from growth-limiting conditions, putatively by a mechanism that involves the accumulation and error-prone processing of oxidized DNA bases.

Error-prone processing of apurinic/apyrimidinic (AP) sites by PolX underlies a novel mechanism that promotes adaptive mutagenesis in Bacillus subtilis.

In growing cells, apurinic/apyrimidinic (AP) sites generated spontaneously or resulting from the enzymatic elimination of oxidized bases must be processed by AP endonucleases before they compromise cell integrity. Here, we investigated how AP sites and the processing of these noncoding lesions by the AP endonucleases Nfo, ExoA, and Nth contribute to the production of mutations (hisC952, metB5, and leuC427) in starved cells of the Bacillus subtilis YB955 strain. Interestingly, cells from this strain that were deficient for Nfo, ExoA, and Nth accumulated a greater amount of AP sites in the stationary phase than during exponential growth. Moreover, under growth-limiting conditions, the triple nfo exoA nth knockout strain significantly increased the amounts of adaptive his, met, and leu revertants produced by the B. subtilis YB955 parental strain. Of note, the number of stationary-phase-associated reversions in the his, met, and leu alleles produced by the nfo exoA nth strain was significantly decreased following disruption of polX. In contrast, during growth, the reversion rates in the three alleles tested were significantly increased in cells of the nfo exoA nth knockout strain deficient for polymerase X (PolX). Therefore, we postulate that adaptive mutations in B. subtilis can be generated through a novel mechanism mediated by error-prone processing of AP sites accumulated in the stationary phase by the PolX DNA polymerase.

Draft genome of Bacillus subtilis strain YB955, prophage-cured derivative of strain 168.

We report the genome sequence of Bacillus subtilis strain YB955, a prophage-cured strain used as a model in DNA repair, bacterial physiology, and mutagenesis studies. The assembled and annotated draft genome contains 4,031 coding genes, 5 rRNAs, and 73 tRNAs. Compared to 168, YB955 has a 134,402 bp deletion.

8-OxoG-Dependent Regulation of Global Protein Responses Leads to Mutagenesis and Stress Survival in Bacillus subtilis.

The guanine oxidized (GO) system of Bacillus subtilis, composed of the YtkD (MutT), MutM and MutY proteins, counteracts the cytotoxic and genotoxic effects of the oxidized nucleobase 8-OxoG. Here, we report that in growing B. subtilis cells, the genetic inactivation of GO system potentiated mutagenesis (HPM), and subsequent hyperresistance, contributes to the damaging effects of hydrogen peroxide (H2O2) (HPHR). The mechanism(s) that connect the accumulation of the mutagenic lesion 8-OxoG with the ability of B. subtilis to evolve and survive the noxious effects of oxidative stress were dissected. Genetic and biochemical evidence indicated that the synthesis of KatA was exacerbated, in a PerR-independent manner, and the transcriptional coupling repair factor, Mfd, contributed to HPHR and HPM of the ΔGO strain. Moreover, these phenotypes are associated with wider pleiotropic effects, as revealed by a global proteome analysis. The inactivation of the GO system results in the upregulated production of KatA, and it reprograms the synthesis of the proteins involved in distinct types of cellular stress; this has a direct impact on (i) cysteine catabolism, (ii) the synthesis of iron-sulfur clusters, (iii) the reorganization of cell wall architecture, (iv) the activation of AhpC/AhpF-independent organic peroxide resistance, and (v) increased resistance to transcription-acting antibiotics. Therefore, to contend with the cytotoxic and genotoxic effects derived from the accumulation of 8-OxoG, B. subtilis activates the synthesis of proteins belonging to transcriptional regulons that respond to a wide, diverse range of cell stressors.

Bacillus subtilis stress-associated mutagenesis and developmental DNA repair.

SUMMARYThe metabolic conditions that prevail during bacterial growth have evolved with the faithful operation of repair systems that recognize and eliminate DNA lesions caused by intracellular and exogenous agents. This idea is supported by the low rate of spontaneous mutations (10-9) that occur in replicating cells, maintaining genome integrity. In contrast, when growth and/or replication cease, bacteria frequently process DNA lesions in an error-prone manner. DNA repairs provide cells with the tools needed for maintaining homeostasis during stressful conditions and depend on the developmental context in which repair events occur. Thus, different physiological scenarios can be anticipated. In nutritionally stressed bacteria, different components of the base excision repair pathway may process damaged DNA in an error-prone approach, promoting genetic variability. Interestingly, suppressing the mismatch repair machinery and activating specific DNA glycosylases promote stationary-phase mutations. Current evidence also suggests that in resting cells, coupling repair processes to actively transcribed genes may promote multiple genetic transactions that are advantageous for stressed cells. DNA repair during sporulation is of interest as a model to understand how transcriptional processes influence the formation of mutations in conditions where replication is halted. Current reports indicate that transcriptional coupling repair-dependent and -independent processes operate in differentiating cells to process spontaneous and induced DNA damage and that error-prone synthesis of DNA is involved in these events. These and other noncanonical ways of DNA repair that contribute to mutagenesis, survival, and evolution are reviewed in this manuscript.

Colonization strategies of Pseudomonas fluorescens Pf0-1: activation of soil-specific genes important for diverse and specific environments.

BACKGROUND: Pseudomonas fluorescens is a common inhabitant of soil and the rhizosphere environment. In addition to potential applications in biocontrol and bioremediation, P. fluorescens is of interest as a model for studying bacterial survival and fitness in soil. A previous study using in vivo expression technology (IVET) identified 22 genes in P. fluorescens Pf0-1 which are up-regulated during growth in Massachusetts loam soil, a subset of which are important for fitness in soil. Despite this and other information on adaptation to soil, downstream applications such as biocontrol or bioremediation in diverse soils remain underdeveloped. We undertook an IVET screen to identify Pf0-1 genes induced during growth in arid Nevada desert soil, to expand our understanding of growth in soil environments, and examine whether Pf0-1 uses general or soil type-specific mechanisms for success in soil environments. RESULTS: Twenty six genes were identified. Consistent with previous studies, these genes cluster in metabolism, information storage/processing, regulation, and 'hypothetical', but there was no overlap with Pf0-1 genes induced during growth in loam soil. Mutation of both a putative glutamine synthetase gene (Pfl01_2143) and a gene predicted to specify a component of a type VI secretion system (Pfl01_5595) resulted in a decline in arid soil persistence. When examined in sterile loam soil, mutation of Pfl01_5595 had no discernible impact. In contrast, the Pfl01_2143 mutant was not impaired in persistence in sterile soil, but showed a significant reduction in competitive fitness. CONCLUSIONS: These data support the conclusion that numerous genes are specifically important for survival and fitness in natural environments, and will only be identified using in vivo approaches. Furthermore, we suggest that a subset of soil-induced genes is generally important in different soils, while others may contribute to success in specific types of soil. The importance of glutamine synthetase highlights a critical role for nitrogen metabolism in soil fitness. The implication of Type 6 secretion underscores the importance of microbial interactions in natural environments. Understanding the general and soil-specific genes will greatly improve the persistence of designed biocontrol and bioremediation strains within the target environment.

Roles of endonuclease V, uracil-DNA glycosylase, and mismatch repair in Bacillus subtilis DNA base-deamination-induced mutagenesis.

The disruption of ung, the unique uracil-DNA-glycosylase-encoding gene in Bacillus subtilis, slightly increased the spontaneous mutation frequency to rifampin resistance (Rif(r)), suggesting that additional repair pathways counteract the mutagenic effects of uracil in this microorganism. An alternative excision repair pathway is involved in this process, as the loss of YwqL, a putative endonuclease V homolog, significantly increased the mutation frequency of the ung null mutant, suggesting that Ung and YwqL both reduce the mutagenic effects of base deamination. Consistent with this notion, sodium bisulfite (SB) increased the Rif(r) mutation frequency of the single ung and double ung ywqL strains, and the absence of Ung and/or YwqL decreased the ability of B. subtilis to eliminate uracil from DNA. Interestingly, the Rif(r) mutation frequency of single ung and mutSL (mismatch repair [MMR] system) mutants was dramatically increased in a ung knockout strain that was also deficient in MutSL, suggesting that the MMR pathway also counteracts the mutagenic effects of uracil. Since the mutation frequency of the ung mutSL strain was significantly increased by SB, in addition to Ung, the mutagenic effects promoted by base deamination in growing B. subtilis cells are prevented not only by YwqL but also by MMR. Importantly, in nondividing cells of B. subtilis, the accumulations of mutations in three chromosomal alleles were significantly diminished following the disruption of ung and ywqL. Thus, under conditions of nutritional stress, the processing of deaminated bases in B. subtilis may normally occur in an error-prone manner to promote adaptive mutagenesis.

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.

Mismatch repair modulation of MutY activity drives Bacillus subtilis stationary-phase mutagenesis.

Stress-promoted mutations that occur in nondividing cells (adaptive mutations) have been implicated strongly in causing genetic variability as well as in species survival and evolutionary processes. Oxidative stress-induced DNA damage has been associated with generation of adaptive His(+) and Met(+) but not Leu(+) revertants in strain Bacillus subtilis YB955 (hisC952 metB5 leuC427). Here we report that an interplay between MutY and MutSL (mismatch repair system [MMR]) plays a pivotal role in the production of adaptive Leu(+) revertants. Essentially, the genetic disruption of MutY dramatically reduced the reversion frequency to the leu allele in this model system. Moreover, the increased rate of adaptive Leu(+) revertants produced by a MutSL knockout strain was significantly diminished following mutY disruption. Interestingly, although the expression of mutY took place during growth and stationary phase and was not under the control of RecA, PerR, or σ(B), a null mutation in the mutSL operon increased the expression of mutY several times. Thus, in starved cells, saturation of the MMR system may induce the expression of mutY, disturbing the balance between MutY and MMR proteins and aiding in the production of types of mutations detected by reversion to leucine prototrophy. In conclusion, our results support the idea that MMR regulation of the mutagenic/antimutagenic properties of MutY promotes stationary-phase mutagenesis in B. subtilis cells.

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

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.

Transcriptional coupling and repair of 8-OxoG activate a RecA-dependent checkpoint that controls the onset of sporulation in Bacillus subtilis.

During sporulation Bacillus subtilis Mfd couples transcription to nucleotide excision repair (NER) to eliminate DNA distorting lesions. Here, we report a significant decline in sporulation following Mfd disruption, which was manifested in the absence of external DNA-damage suggesting that spontaneous lesions activate the function of Mfd for an efficient sporogenesis. Accordingly, a dramatic decline in sporulation efficiency took place in a B. subtilis strain lacking Mfd and the repair/prevention guanine oxidized (GO) system (hereafter, the ∆GO system), composed by YtkD, MutM and MutY. Furthermore, the simultaneous absence of Mfd and the GO system, (i) sensitized sporulating cells to H2O2, and (ii) elicited spontaneous and oxygen radical-induced rifampin-resistance (Rifr) mutagenesis. Epifluorescence (EF), confocal and transmission electron (TEM) microscopy analyses, showed a decreased ability of ∆GO ∆mfd strain to sporulate and to develop the typical morphologies of sporulating cells. Remarkably, disruption of sda, sirA and disA partially, restored the sporulation efficiency of the strain deficient for Mfd and the ∆GO system; complete restoration occurred in the RecA- background. Overall, our results unveil a novel Mfd mechanism of transcription-coupled-repair (TCR) elicited by 8-OxoG which converges in the activation of a RecA-dependent checkpoint event that control the onset of sporulation in B. subtilis.

Microbially mediated aerobic and anaerobic degradation of acrylamide in a western United States irrigation canal.

Acrylamide (AMD), a neurotoxin and suspected carcinogen, is present at concentrations of up to 0.05% in linear anionic polyacrylamide, which is under evaluation as a temporary sealant in unlined irrigation canal systems across the United States. We examined the microbially mediated degradation of AMD and diversity of AMD-degrading microbial physiotypes in the Rocky Ford Highline Canal, Colorado to better constrain the potential fate ofAMD in a canal environment. Microorganisms able to use AMD (500 mg L(-1)) as a sole nitrogen source were relatively abundant (2.3 x 10(3) to 9.4 x 10(3) cells mL(-1) in water and 4.2 x 10(3) to 2.3 x 10(5) cells g(-1) in sediment). Only sediment samples contained microorganisms able to use AMD as a sole carbon source. Acrylamide (up to 100 mg L(-1)) was efficiently removed from amended canal water and sediment slurries under aerobic conditions, but no AMD degradation was observed in abiotic controls. Anaerobic degradation of AMD by nitrate-, sulfate-, and iron-reducing microorganisms was also tested, with nitrate reducers affecting the highest amounts of AMD removal (70.3-85%) after 60 d. All representatives (n=15) from a collection of 256 AMD-degrading microbial isolates from Rocky Ford Highline Canal were closely related to well characterized environmental bacteria capable of facultative nitrate respiration. Our results demonstrate that natural microbial populations within this canal are capable of AMD degradation under aerobic and anaerobic conditions and that this degradation is performed by naturally abundant bacteria likely to be present in other freshwater irrigation canals or similar lotic habitats.

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.

Stationary-phase Mutagenesis Soft-agar Overlay Assays in Bacillus subtilis.

Elucidating how a population of non-growing bacteria generates mutations improves our understanding of phenomena like antibiotic resistance, bacterial pathogenesis, genetic diversity and evolution. To evaluate mutations that occur in nutritionally stressed non-growing bacteria, we have employed the strain B. subtilis YB955, which measures the reversions rates to the chromosomal auxotrophies hisC952, metB5 and leuC427 (Sung and Yasbin, 2002). This gain-of-function system has successfully allowed establishing the role played by repair systems and transcriptional factors in stress-associated mutagenesis (SPM) (Barajas- Ornelas et al., 2014 ; Gómez- Marroquín et al., 2016 ). In a recent study (Castro- Cerritos et al., 2017 ), it was found that Ribonucleotide Reductase (RNR) was necessary for SPM; this enzyme is essential in this bacterium. We engineered a conditional mutant of strain B. subtilis YB955 in which expression of the nrdEF operon was modulated by isopropyl-ß-D-thiogalactopyranoside (IPTG) (Castro- Cerritos et al., 2017 ). The conditions to determine mutation frequencies conferring amino acid prototrophy in three genes (hisC952, metB5, leuC427) under nutritional stress in this conditional mutant are detailed here. This technique could be used to evaluate the participation of essential genes in the mutagenic processes occurring in stressed B. subtilis cells.