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.
Highlights
Short for Mutation frequency decline, Mfd is the transcription-coupling repair factor, which coalesces a stalled RNAP with the Nucleotide Excision Repair (NER) pathway to preferentially repair lesions in the template strand of actively transcribed genes before lesions in the coding strand or in non-actively transcribed genes (Hanawalt and Spivak, 2008)
Using RNA extracted from stationary-phase B. subtilis cultures exposed to either 0 or 1 mM of the protein oxidant diamide, we found that nearly half of the genes in the transcriptome and several biological functions were expressed differentially in the absence of Mfd
B. subtilis strains employed in this study (Table 1) were routinely isolated on tryptic blood agar base (TBAB) (Acumedia Manufacturers, Inc., Lansing, MI, United States), and liquid cultures were grown in Penassay broth (PAB) supplemented with 1X Ho-Le trace elements
Summary
Short for Mutation frequency decline, Mfd is the transcription-coupling repair factor, which coalesces a stalled RNAP with the Nucleotide Excision Repair (NER) pathway to preferentially repair lesions in the template strand of actively transcribed genes before lesions in the coding strand or in non-actively transcribed genes (Hanawalt and Spivak, 2008). As early as the 1990s, evidence suggested that Mfd influenced phenotypes unrelated to transcription-coupled repair. Reports showed that Mfd affected carbon catabolite repression of operons (Zalieckas et al, 1998b). Mfd Affects Bacillus Stress Biology that Mfd can facilitate repression of transcription by roadblock clearance at genes regulated by the global transcription regulator CodY (Belitsky and Sonenshein, 2011). Our lab demonstrated a role for Mfd in the expression of amino acid biosynthesis genes and protection from oxidative stress (Pybus et al, 2010; Martin et al, 2011, 2019). A recent report proposed that Mfd functions at hard-to-transcribe genes and affected gene expression and survival associated with toxin-antitoxin gene modules in B. subtilis (Ragheb et al, 2021)
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