Mfd Affects Global Transcription and the Physiology of Stressed Bacillus subtilis Cells

Abstract

For several decades, Mfd has been studied as the bacterial transcription-coupled repair factor. However, recent observations indicate that this factor influences cell functions beyond DNA repair. Our lab recently described a role for Mfd in disulfide stress that was independent of its function in nucleotide excision repair and base excision repair. Because reports showed that Mfd influenced transcription of single genes, we investigated the global differences in transcription in wild-type and mfd mutant growth-limited cells in the presence and absence of diamide. Surprisingly, we found 1,997 genes differentially expressed in Mfd– cells in the absence of diamide. Using gene knockouts, we investigated the effect of genetic interactions between Mfd and the genes in its regulon on the response to disulfide stress. Interestingly, we found that Mfd interactions were complex and identified additive, epistatic, and suppressor effects in the response to disulfide stress. Pathway enrichment analysis of our RNASeq assay indicated that major biological functions, including translation, endospore formation, pyrimidine metabolism, and motility, were affected by the loss of Mfd. Further, our RNASeq findings correlated with phenotypic changes in growth in minimal media, motility, and sensitivity to antibiotics that target the cell envelope, transcription, and DNA replication. Our results suggest that Mfd has profound effects on the modulation of the transcriptome and on bacterial physiology, particularly in cells experiencing nutritional and oxidative stress.

Molecular Mechanisms of Transcription-Coupled Repair

“…In these and other cases (91), Mfd was found to enhance suppression, as "without Mfd to release the polymerase, the RNA polymerase will in some instances eventually overcome the roadblock as the repressor protein dissociates" (90, p. 7). Regarding overall transcription, genome-wide gene expression analysis has shown that in mfd − cells, ∼1,000 genes are upregulated, and a comparable number of genes are downregulated (92). Significantly, in E. coli, chromosome immunoprecipitation (ChIP) sequencing analysis has shown a close correspondence between RNAP pause sites and Mfd-binding sites in the genome, and these sites in general are known or suspected sites of DNA secondary structures, including hairpins, high GC content, or G-quadruplexes (81).…”

8-OxoG-Dependent Regulation of Global Protein Responses Leads to Mutagenesis and Stress Survival in Bacillus subtilis

“…As shown in this work, these processes can be subjected to regulation with Mfd ( Figure 2 ), a transcription factor commonly associated with the repair of bulky DNA lesions in bacteria [ 81 ]. However, recent evidence has revealed that Mfd functions as a global regulator of gene transcription and cell differentiation in B. subtilis [ 34 , 82 ]. Therefore, elucidating the Mfd-dependent mechanisms that cells employ to directly or indirectly regulate the expression of the factors associated with the HPM and HPHR phenotypes will contribute to understanding the novel aspects of bacterial physiology in stressed bacterial cells.…”