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.Since the mid-1950s, microbiologists have been aware of mutations occurring in nondividing populations of cells (22,29). The formation of these mutants was alternatively termed "starvation-associated mutagenesis" (29), "adaptive mutation" (8), or "stationary-phase mutagenesis" (13). Recently, variations of this phenomenon have been investigated with Escherichia coli (8,29,38), Pseudomonas (33), Bacillus subtilis (41), and the eukaryotic yeast Saccharomyces cerevisiae (15,40). These phenomena reveal that starving populations of cells can acquire mutations favoring growth after the application of selection.While the phenomenon of stationary-phase mutation is widespread, it is clear that the mechanism(s) by which it arises varies from organism to organism. To date, the most favored system for studying adaptive or stationary-phase mutagenesis is the RecA-dependent E. coli FC40 system investigated by, among others, the laboratories of Cairns, Foster, Rosenberg, and Roth (7,8,11,18). Recent results strongly suggest that this mutagenesis is the result of gene amplification followed by mutation in a transiently growing population of cells (18). In light of this, we chose to investigate the possibility that transiently growing cells may play a role in a B. subtilis system containing three chromosomal point mutations. The phenomenon of transcriptional mutagenesis, or retromutagenesis, whereby RNA polymerase bypasses an unrepaired DNA lesion or otherwise produces an altered mRNA, which is then translated into a protein of altered function, could provide a transient growth advantage for the cell. This mechanism has been proposed for other model systems, including eukaryotes (6, 9).We have previously shown the existence of one such mutagenic phenomenon occurring during stationary phase in B. subtilis cells starved for amino acids (41). This mutagenic process appears to enhance the survivability of cell populations undergoing nutritional stress. In brief, isogenic strains of B. subtilis carrying three amino acid auxotrophies conferred by hisC952 (amber), metB5 (ochre), and leuC427 (missense) are incubated on medium lacking one of the required amino acids. After several days of incubati...

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 stationaryphase-induced mutagenesis in this soil microorganism.Adaptive or stationary-phase mutagenesis can be defined as those mutations that permit organisms to grow and divide in response to natural or artificial selection (5) and that occur in nondividing cells during prolonged nonlethal selective pressure, e.g., starvation for an essential amino acid (32). Although this type of mutagenesis was first described in Escherichia coli (7), additional examples of adaptive mutagenesis in other prokaryotes (21, 41) and in eukaryotic organisms (14) have been published. In some cases, these mutations occurred in the absence of specific selection but in response to starvation (11). Regardless of the organisms utilized and the name used, these types of mutations and the processes that generate them are of real interest with respect to evolution and the generation of diversity across all domains of life.Studies with the FЈ lac frameshift reversion construct of E. coli (32) have demonstrated that the generation of Lac ϩ stationary-phase-associated revertants required functional recombination (15), as well as component(s) of the SOS system (24, 25). Further evidence suggests that the mutations generated by this lac system during stationary phase may also be the result of amplification of the plasmid-borne gene followed by SOS-induced mutagenesis and selection (18,38). Recent studies have demonstrated that DNA double-strand-break repair, in addition to the SOS DNA damage response and the error-prone DNA polymerase, are necessary for stress-induced reversion of the E. col...

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.The generation of mutations has been traditionally ascribed to spontaneous processes affecting actively growing, dividing cells. Nevertheless, by the mid-1950s, several reports describing mutagenesis in nondividing cells of bacteria, plants, flies, and fungi appeared in the scientific literature (reference 36 and references therein). Much of the initial characterization of this process in bacteria took place in the laboratory of Francis Ryan, who observed Escherichia coli mutants capable of synthesizing histidine arising from his mutant (auxotrophic) cells undergoing prolonged starvation (36) while cell turnover remained undetectable, and DNA replication slowed with increasing time (26). Renewed interest in adaptive mutation was generated when Cairns and coworkers published their work on the generation of Lac ϩ reversions in E. coli cells unable to use the lactose provided as the sole carbon source in a minimal medium (6). This work demonstrated that adaptive mutations can arise as a result of stress rather than from selection of preexisting mutations. The generation of stress-induced Lac ϩ reversions, assayed via a plasmid-borne system, has been studied intensively by several laboratories (reviewed in references 13, 15, and 34; 32) and is dependent on activation of the SOS and/or stress responses. Further studies have also suggested that a subpopulation within the Lac Ϫ stressed cells engage in an exquisitely regulated transient state of hypermutation limited in time and to DNA sites near double-stranded DNA breaks (reviewed in reference 15). Collectively, the results from studies on this system have provided interesting insights into the acquisition of beneficial mutations and demonstrated the role of several genetic factors in the adaptive mutation phenomenon.

AdnA is a transcription factor in Pseudomonas fluorescens that affects flagellar synthesis, biofilm formation, and sand adhesion. To identify the AdnA regulon, we used a promoterless Tn5-lacZ element to study the phenotypes of insertion mutants in the presence and absence of AdnA. Of 12,000 insertions, we identified seven different putative open reading frames (ORFs) activated by AdnA (named aba for activated by AdnA). aba120 and aba177 showed homology to flgC and flgI, components of the basal body of the flagella in Pseudomonas aeruginosa. Two other insertions, aba18 and aba51, disrupted genes affecting chemotaxis. The mutant loci aba160 (possibly affecting lipopolysaccharide synthesis) and aba175 (unknown function) led to loss of flagella. The mutant bearing aba203 became motile when complemented with adnA, but the mutated gene showed no similarity to known genes. Curiously, aba18, aba51, aba160, and aba203 mutants formed biofilms even in the absence of AdnA, suppressing the phenotype of the adnA deletion mutant. The combined findings suggest that flagella are nonessential for sand attachment or biofilm formation. Sequence and promoter analyses indicate that AdnA affects at least 23 ORFs either directly or by polar effects. These results support the concept that AdnA regulates cell processes other than those directly related to flagellar synthesis and define a broader cadre of genes in P. fluorescens than that described so far for its homolog, FleQ, in P. aeruginosa.

SummaryIn 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 nongrowing sporulating cells, Mfd played an antimutagenic role as its absence promoted UV-induced mutagenesis through a pathway involving YqjH/YqjWmediated 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.

Stress-promoted mutations that occur in nondividing cells (adaptive mutations)

The effect of trifolitoxin (TFX) production by Rhizobium etli on rhizosphere colonization and competition for nodulation in soil conditions was determined. TFX is a potent peptide antibiotic made by Rhizobium leguminosarum bv. trifolii T24 that inhibits many α-proteobacteria (E. W. Triplett, B. T. Breil, and G. A. Splitter, Appl. Environ. Microbiol. 60:4163–4166, 1994). Seeds of Phaseolus vulgaris were inoculated with a TFX-sensitive reference strain and either of two isogenic strains that differ only in their ability to produce TFX. The pair of strains were inoculated at different ratios in sterile and nonsterile soil. The representation of the strains in the rhizosphere and nodules was determined at 96 h after inoculation and 3 weeks after planting, respectively. The TFX-producing strain was significantly more competitive for both phenotypes versus the TFX-sensitive strain, compared with the TFX-nonproducing strain versus the TFX-sensitive strain. These results show that nodule occupancy by inoculant strains, often displaced from the nodules by indigenous strains, can be increased by addition of the TFX production phenotype to R. etli in plants grown in either sterile or nonsterile soil. Also, this work shows the efficacy of the TFX system for the first time on a legume host with determinant nodules.

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