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.

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.

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.

Transcriptional coupling of DNA repair in sporulating Bacillus subtilis cells.

In conditions of halted or limited genome replication, like those experienced in sporulating cells of Bacillus subtilis, a more immediate detriment caused by DNA damage is altering the transcriptional programme that drives this developmental process. Here, we report that mfd, which encodes a conserved bacterial protein that mediates transcription-coupled DNA repair (TCR), is expressed together with uvrA in both compartments of B. subtilis sporangia. The function of Mfd was found to be important for processing the genetic damage during B. subtilis sporulation. Disruption of mfd sensitized developing spores to mitomycin-C (M-C) treatment and UV-C irradiation. Interestingly, in non-growing sporulating cells, Mfd played an anti-mutagenic role as its absence promoted UV-induced mutagenesis through a pathway involving YqjH/YqjW-mediated translesion synthesis (TLS). Two observations supported the participation of Mfd-dependent TCR in spore morphogenesis: (i) disruption of mfd notoriously affected the efficiency of B. subtilis sporulation and (ii) in comparison with the wild-type strain, a significant proportion of Mfd-deficient sporangia that survived UV-C treatment developed an asporogenous phenotype. We propose that the Mfd-dependent repair pathway operates during B. subtilis sporulation and that its function is required to eliminate genetic damage from transcriptionally active genes.

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.

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.

Transcription-associated mutation in Bacillus subtilis cells under stress.

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.

Role of the Y-family DNA polymerases YqjH and YqjW in protecting sporulating Bacillus subtilis cells from DNA damage.

The role played by the Y-family DNA polymerases YqjH and YqjW in protecting sporulating cells of Bacillus subtilis from DNA damage was determined. The absence of either yqjH and/or yqjW not only reduced sporulation efficiency but also sensitized the sporulating cells to hydrogen peroxide, tert-butylhydroperoxide (t-BHP), mitomycin-C (M-C), and UV-C radiation. Moreover, these DNA-damaging agents increased the mutation frequency of wild-type sporulating cells to 4-azaleucine, but the production of mutants was YqjH- and YqjW-dependent. In conclusion, the results presented here indicate that YqjH/YqjW-dependent-translesion synthesis (TLS) operates in sporulating B. subtilis cells and contributes in processing spontaneous and artificially induced genetic damage, which is apparently required for an efficient sporulation process.

Defects in the error prevention oxidized guanine system potentiate stationary-phase mutagenesis in Bacillus subtilis.

Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationary-phase-induced mutagenesis in this soil microorganism.

Role of the Nfo and ExoA apurinic/apyrimidinic endonucleases in repair of DNA damage during outgrowth of Bacillus subtilis spores.

Germination and outgrowth are critical steps for returning Bacillus subtilis spores to life. However, oxidative stress due to full hydration of the spore core during germination and activation of metabolism in spore outgrowth may generate oxidative DNA damage that in many species is processed by apurinic/apyrimidinic (AP) endonucleases. B. subtilis spores possess two AP endonucleases, Nfo and ExoA; the outgrowth of spores lacking both of these enzymes was slowed, and the spores had an elevated mutation frequency, suggesting that these enzymes repair DNA lesions induced by oxidative stress during spore germination and outgrowth. Addition of H2O2 also slowed the outgrowth of nfo exoA spores and increased the mutation frequency, and nfo and exoA mutations slowed the outgrowth of spores deficient in either RecA, nucleotide excision repair (NER), or the DNA-protective alpha/beta-type small acid-soluble spore proteins (SASP). These results suggest that alpha/beta-type SASP protect DNA of germinating spores against damage that can be repaired by Nfo and ExoA, which is generated either spontaneously or promoted by addition of H2O2. The contribution of RecA and Nfo/ExoA was similar to but greater than that of NER in repair of DNA damage generated during spore germination and outgrowth. However, nfo and exoA mutations increased the spontaneous mutation frequencies of outgrown spores lacking uvrA or recA to about the same extent, suggesting that DNA lesions generated during spore germination and outgrowth are processed by Nfo/ExoA in combination with NER and/or RecA. These results suggest that Nfo/ExoA, RecA, the NER system, and alpha/beta-type SASP all contribute to the repair of and/or protection against oxidative damage of DNA in germinating and outgrowing spores.

Implementation of a loss-of-function system to determine growth and stress-associated mutagenesis in Bacillus subtilis.

A forward mutagenesis system based on the acquisition of mutations that inactivate the thymidylate synthase gene (TMS) and confer a trimethoprim resistant (Tmpr) phenotype was developed and utilized to study transcription-mediated mutagenesis (TMM). In addition to thyA, Bacillus subtilis possesses thyB, whose expression occurs under conditions of cell stress; therefore, we generated a thyB- thyA+ mutant strain. Tmpr colonies of this strain were produced with a spontaneous mutation frequency of ~1.4 × 10-9. Genetic disruption of the canonical mismatch (MMR) and guanine oxidized (GO) repair pathways increased the Tmpr frequency of mutation by ~2-3 orders of magnitude. A wide spectrum of base substitutions as well as insertion and deletions in the ORF of thyA were found to confer a Tmpr phenotype. Stationary-phase-associated mutagenesis (SPM) assays revealed that colonies with a Tmpr phenotype, accumulated over a period of ten days with a frequency of ~ 60 ×10-7. The Tmpr system was further modified to study TMM by constructing a ΔthyA ΔthyB strain carrying an IPTG-inducible Pspac-thyA cassette. In conditions of transcriptional induction of thyA, the generation of Tmpr colonies increased ~3-fold compared to conditions of transcriptional repression. Further, the Mfd and GreA factors were necessary for the generation of Tmpr colonies in the presence of IPTG in B. subtilis. Because GreA and Mfd facilitate transcription-coupled repair, our results suggest that TMM is a mechanim to produce genetic diversity in highly transcribed regions in growth-limited B. subtilis cells.

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.

Transcriptional de-repression and Mfd are mutagenic in stressed Bacillus subtilis cells.

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.

Novel role of mfd: effects on stationary-phase mutagenesis in Bacillus subtilis.

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.

YtkD and MutT protect vegetative cells but not spores of Bacillus subtilis from oxidative stress.

ytkD and mutT of Bacillus subtilis encode potential 8-oxo-dGTPases that can prevent the mutagenic effects of 8-oxo-dGTP. Loss of YtkD but not of MutT increased the spontaneous mutation frequency of growing cells. However, cells lacking both YtkD and MutT had a higher spontaneous mutation frequency than cells lacking YtkD. Loss of either YtkD or MutT sensitized growing cells to hydrogen peroxide (H2O2) and t-butylhydroperoxide (t-BHP), and the lack of both proteins sensitized growing cells to these agents even more. In contrast, B. subtilis spores lacking YtkD and MutT were not sensitized to H2O2, t-BHP, or heat. These results suggest (i) that YtkD and MutT play an antimutator role and protect growing cells of B. subtilis against oxidizing agents, and (ii) that neither YtkD nor MutT protects spores against potential DNA damage induced by oxidative stress or heat.

Contribution of the mismatch DNA repair system to the generation of stationary-phase-induced mutants of Bacillus subtilis.

A reversion assay system previously implemented to demonstrate the existence of adaptive or stationary-phase-induced mutagenesis in Bacillus subtilis was utilized in this report to study the influence of the mismatch DNA repair (MMR) system on this type of mutagenesis. Results revealed that a strain deficient in MutSL showed a significant propensity to generate increased numbers of stationary-phase-induced revertants. These results suggest that absence or depression of MMR is an important factor in the mutagenesis of nongrowing B. subtilis cells because of the role of MMR in repairing DNA damage. In agreement with this suggestion, a significant decrease in the number of adaptive revertant colonies, for the three markers tested, occurred in B. subtilis cells which overexpressed a component of the MMR system. Interestingly, the single overexpression of mutS, but not of mutL, was sufficient to decrease the level of adaptive mutants in the reversion assay system of B. subtilis. The results presented in this work, as well as in our previous studies, appear to suggest that an MMR deficiency, putatively attributable to inactivation or saturation with DNA damage of MutS, may occur in a subset of B. subtilis cells that differentiate into the hypermutable state.

Adaptive, or stationary-phase, mutagenesis, a component of bacterial differentiation in Bacillus subtilis.

Adaptive (stationary-phase) mutagenesis occurs in the gram-positive bacterium Bacillus subtilis. Furthermore, taking advantage of B. subtilis as a paradigm for the study of prokaryotic differentiation and development, we have shown that this type of mutagenesis is subject to regulation involving at least two of the genes that are involved in the regulation of post-exponential phase prokaryotic differentiation, i.e., comA and comK. On the other hand, a functional RecA protein was not required for this type of mutagenesis. The results seem to suggest that a small subpopulation(s) of the culture is involved in adaptive mutagenesis and that this subpopulation(s) is hypermutable. The existence of such a hypermutable subpopulation(s) raises important considerations with respect to evolution, the development of specific mutations, the nature of bacterial populations, and the level of communication among bacteria in an ecological niche.

Roles of YqjH and YqjW, homologs of the Escherichia coli UmuC/DinB or Y superfamily of DNA polymerases, in stationary-phase mutagenesis and UV-induced mutagenesis of Bacillus subtilis.

YqjH and YqjW are Bacillus subtilis homologs of the UmuC/DinB or Y superfamily of DNA polymerases that are involved in SOS-induced mutagenesis in Escherichia coli. While the functions of YqjH and YqjW in B. subtilis are still unclear, the comparisons of protein structures demonstrate that YqjH has 36% identity to E. coli DNA polymerase IV (DinB protein), and YqjW has 26% identity to E. coli DNA polymerase V (UmuC protein). In this report, we demonstrate that both YqjH and the products of the yqjW operon are involved in UV-induced mutagenesis in this bacterium. Furthermore, resistance to UV-induced damage is significantly reduced in cells lacking a functional YqjH protein. Analysis of stationary-phase mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the ability of B. subtilis to generate revertants at the hisC952 allele via this system. These data suggest a role for YqjH in the generation of at least some types of stationary-phase-induced mutagenesis.

The ytkD (mutTA) gene of Bacillus subtilis encodes a functional antimutator 8-Oxo-(dGTP/GTP)ase and is under dual control of sigma A and sigma F RNA polymerases.

The regulation of expression of ytkD, a gene that encodes the first functional antimutator 8-oxo-dGTPase activity of B. subtilis, was studied here. A ytkD-lacZ fusion integrated into the ytkD locus of wild-type B. subtilis 168 revealed that this gene is expressed during both vegetative growth and early stages of sporulation. In agreement with this result, ytkD mRNAs were detected by both Northern blotting and reverse transcription-PCR during both developmental stages. These results suggested that ytkD is transcribed by the sequential action of RNA polymerases containing the sigma factors sigma(A) and sigma(F), respectively. In agreement with this suggestion, the spore-associated expression was almost completely abolished in a sigF genetic background but not in a B. subtilis strain lacking a functional sigG gene. Primer extension analysis mapped transcriptional start sites on mRNA samples isolated from vegetative and early sporulating cells of B. subtilis. Inspection of the sequences lying upstream of the transcription start sites revealed the existence of typical sigma(A)- and sigma(F)-type promoters. These results support the conclusion that ytkD expression is subjected to dual regulation and suggest that the antimutator activity of YtkD is required not only during vegetative growth but also during the early sporulation stages and/or germination of B. subtilis. While ytkD expression obeyed a dual pattern of temporal expression, specific stress induction of the transcription of this gene does not appear to occur, since neither oxidative damage (following either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment or sigma(B) general stress inducers (sodium chloride, ethanol, or heat) affected the levels of the gene product produced.