Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways

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.

Mechanistic insights into transcription coupled DNA repair

“…Therefore, it is important to understand the sequence of events during Mfd-mediated TCR. As fortuitous TCR is likely to be mutagenic [48][49][50] an intriguing question relates to how Mfd distinguishes RNAP stalling at a lesion from natural pauses at various DNA sequences during elongation.…”

Transcriptional coupling (Mfd) and DNA damage scanning (DisA) coordinate excision repair events for efficient Bacillus subtilis spore outgrowth

“…Together, these results strongly suggest that in spores returning to vegetative growth, spontaneous DNA lesions can be faithfully processed through NER or Mfd as well as in an error‐prone manner through coordination of Mfd with DisA. Previous evidence has associated Mfd with counteracting the noxious effects of bulky lesions during growth and sporulation (Ayora et al., ; Ramírez‐Guadiana et al., , ) and regulating transcription‐associated mutagenesis (Gómez‐Marroquín et al., ; Martin, Pedraza‐Reyes, Yasbin, & Robleto, ; Pybus et al., ). The evidence presented here also reveals a role for Mfd in coordinating with DisA to process ROS‐promoted lesions of DNA spontaneously generated by the metabolic conditions operating in germinating/outgrowing spores.…”

“…On the basis of these observations, we postulate that repair intermediaries of these oxidative lesions can stall RNA polymerase, the absence of Mfd precludes dislodging the stalled polymerase from DNA, and this eventually stalls the DNA replication machinery as well. In agreement with this notion, it has been proposed that a MutY‐AP site complex stalls RNA polymerase during transcription of the mutant leuC427 gene and activates an Mfd‐dependent event leading to leucine prototrophy (Gómez‐Marroquín et al., ). On the other hand, the DisA‐dependent error‐prone repair pathway could be activated by other DNA structures, including branched DNA as well as stalled replication forks (Gándara et al., ; Witte et al., ).…”

“…ROS directly or indirectly attack DNA generating a myriad of DNA lesions, including the oxidative products 8‐OxoG, 8‐OxoA, 2‐OxoA, 5‐OxoC, and thymine glycol (Tg), as well as products of guanine, adenine and cytosine deamination including xanthine, hypoxanthine, and uracil, respectively, among others (Chernikov, Gudkov, Shtarkman, & Bruskov, ; Cooke, Evans, Dizdaroglu, & Lunec, ). Although these lesions are mainly subjected to BER in an error‐free manner (Dalhus, Laerdahl, Backe, & Bjørås, ), alternative modes of error‐prone repair involving Mfd and excision repair proteins have been postulated in bacteria (Brégeon, Doddridge, You, Weiss, & Doetsch, ; Gómez‐Marroquín et al., ; Million‐Weaver et al., ; Wimberly, Chandan Shee, Thornton, Rosenberg, & Hastings, ). To further explore mechanistic aspects of repair events during spore outgrowth, we sequenced the rpoB gene from spontaneous Rif r colonies of outgrown spores proficient or deficient for disA , mfd, and disA/mfd .…”

The Bacillus Subtilis K-State Promotes Stationary-Phase Mutagenesis via Oxidative Damage

“…Interestingly, Bacillus subtilis also expresses stationary-phase mutagenesis. In these processes, the transcription repair coupling factor Mfd plays a central role, interacting with components of the nucleotide excision repair (NER) or base excision repair (BER) pathways and error-prone polymerases to produce mutations in stationary-phase cells [14]. This type of mutation occurs under endogenous levels of DNA damage or when cells are exposed to oxidants that inflict DNA lesions.…”