Novel Role of mfd : Effects on Stationary-Phase Mutagenesis in Bacillus subtilis

Abstract

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...

Transcription-Associated Mutation in Bacillus subtilis Cells under Stress

“…Thus, one can speculate that the combination of transcriptional derepression and DNA repair inadequacies under conditions of nongrowth biases mutations to transcribed regions. Interestingly, results from stationary-phase mutagenesis assays in B. subtilis have alluded to the role of transcription in adaptation (33). As with the E. coli paradigm, adaptive mutation decreases in the absence of RelA (35).…”

“…Surprisingly, this also occurs in the absence of the transcription repair coupling factor Mfd (33), which is counterintuitive because Mfd is a repair factor, and its absence would be expected to increase mutations. It should also be noted that this novel property of mfd is not observed under conditions of exponential growth (33), and it is expressed in bacterial species other than B. subtilis (18). This factor mediates transcription coupling repair and is known to dissociate RNA polymerase stalled at a lesion in DNA (1,38), which would consequently result in repeated attempts at transcription.…”

“…on May 10, 2018 by guest http://jb.asm.org/ strated that the bacterial concentrations of the auxotrophic cells on the plates do not impede the growth of prototrophic revertant colonies (33,41,42). Furthermore, the viabilities of the nonrevertant colonies are similar in both strains (33); therefore, differential cell survival cannot account for the difference in mutation frequencies.…”

“…First, a Bacillus subtilis 168 strain derivative lacking the Mfd transcription factor, previously shown to be decreased in the accumulation of stationary-phase mutants (33), showed a dramatic decrease in levels of mRNA of a gene under selection. This correlation between transcription and mutagenesis was further investigated by placing a defective leu allele under the control of an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible promoter and comparing the accumulations of Leu ϩ revertants under conditions of transcriptional induction and repression.…”

DNA Repair and Genome Maintenance in Bacillus subtilis

“…Deletion of mfd causes an increase in mutagenesis during vegetative growth, and interestingly, an mfd deficiency substantially reduces "adaptive" or "stationary-phase" mutagenesis in B. subtilis (330). Stationary-phase mutagenesis refers to the formation of mutations under nonlethal selection when cells are not growing, including conditions of nutrient limitation (402,403).…”