Abstract
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.
Highlights
Stationary-phase mutagenesis (SPM), referred to as stress-induced mutagenesis, is the collection of cellular processes that produces genetic alterations in non-growing cells
B. subtilis strains employed in this study (Table 1) were routinely isolated on tryptic blood agar base (TBAB) (Acumedia Manufacturers, Inc., Lansing, MI, USA), and liquid cultures were grown in Penassay broth (PAB)
Cells were harvested by centrifugation (10,000 ˆ g, 10 min), resuspended in 10 mL of Spizizen minimal salts (SMS) [36] and plated in quintuplicate onto solid Spizizen minimal medium (SMM; 1ˆ Spizizen salts supplemented with 0.5% glucose, 50 μgmL1 of each methionine, histidine, isoleucine and glutamic acid, and 200 ngmL1 of leucine)
Summary
Stationary-phase mutagenesis (SPM), referred to as stress-induced mutagenesis, is the collection of cellular processes that produces genetic alterations in non-growing cells. In Bacillus subtilis, a recent report showed that Mfd, the NER system and PolY1, an error-prone DNA polymerase, are all part of a mutagenic pathway that prevents conflicts between transcription and replication [21]. NusA, proposed to take part in UvrD-mediated TCR [28], and error-prone DNA synthesis, have been associated with stress-induced mutagenesis in E. coli [29,30] Another possibility addressed here was the association of Mfd with BER proteins, the DNA glycosylase MutY, which has been implicated in the generation of stationary-phase mutations in. We present evidence indicating that Mfd combines with UvrA, MutY and PolI in the formation of mutations at leuC427 mutant allele and expanding the transcription-mediated mutagenic functions of Mfd beyond NER in replication-limited cells
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