Peter E. Burby

Summary: All organisms possess several DNA repair pathways to maintain the integrity of their genetic material. Although there are several DNA repair pathways that are well understood, we recently identified several genes in Bacillus subtilis that are important for surviving treatment with drugs that damage DNA. Here, we report a drug specific DNA repair pathway in B. subtilis . We identified genes coding for a previously uncharacterized helicase and exonuclease, mrfA and mrfB , respectively. Deletion of mrfA and mrfB resulted in sensitivity to the DNA damaging agent mitomycin C, but not other types of DNA damage. We found that MrfAB operate independently of canonical nucleotide excision repair, forming a novel excision repair pathway in bacteria. A phylogenetic analysis demonstrates that MrfAB homologs are present in diverse bacterial phyla, and a cross-complementation assay shows that MrfAB function is conserved in closely related species. Mitomycin C is a natural antibiotic that is produced by the soil dwelling bacterium Streptomyces lavendulae , and B. subtilis is also a soil dwelling organism. The specificity of the Δ mrfAB phenotype suggests that MrfAB have been adapted as a countermeasure to mitomycin producing bacteria. Abbreviated Summary: Bacteria possess DNA repair pathways to maintain the integrity of their genetic material. The helicase MrfA and the exonuclease MrfB are part of a mitomycin C specific DNA repair pathway in Bacillus subtilis . Despite being present in many bacterial species, MrfAB activity in repairing MMC damaged DNA appears to be restricted to closely related species, suggesting that these proteins have likely been adapted to the specific needs of each bacterium.

Bacteria coordinate DNA replication and cell division, ensuring that a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA damage or impediments to DNA replication, a cell cycle checkpoint is activated to delay cell division by expressing a cell division inhibitor. The prevailing model for bacterial DNA damage checkpoints is that activation of the DNA damage response and protease mediated degradation of the cell division inhibitor is sufficient to regulate the checkpoint process. Our recent genome-wide screens identified the gene ddcA as critical for surviving exposure to a broad spectrum of DNA damage. The ddcA deletion phenotypes are dependent on the checkpoint enforcement protein YneA. We found that expression of the checkpoint recovery proteases could not compensate for ddcA deletion. Similarly, expression if ddcA could not compensate for the absence of the checkpoint recovery proteases, indicating that DdcA function is distinct from the checkpoint recovery step. Deletion of ddcA resulted in sensitivity to yneA overexpression independent of YneA protein levels or stability, further supporting the conclusion that DdcA regulates YneA through a proteolysis independent mechanism. Using a functional GFP-YneA we found that DdcA inhibits YneA activity independent of YneA localization, suggesting that DdcA may regulate YneA access to its target. These results uncover a regulatory step that is important for controlling the DNA damage checkpoint in bacteria, and suggests that the typical mechanism of degrading the checkpoint enforcement protein is insufficient to control the rate of cell division in response to DNA damage. Author Summary: All cells coordinate DNA replication and cell division. When cells encounter DNA damage, the process of DNA replication is slowed and the cell must also delay cell division. In bacteria, the process has long been thought to occur using two principle modes of regulation. The first, is RecA coated ssDNA transmits the signal of DNA damage through inactivation of the repressor of the DNA damage (SOS) response regulon, which results in expression of a cell division inhibitor establishing the checkpoint. The second principle step is protease mediated degradation of the cell division inhibitor relieving the checkpoint. Recent work by our lab and others has suggested that this process may be more complex than originally thought. Here, we investigated a gene of unknown function that we previously identified as important for survival when the bacterium Bacillus subtilis is exposed to DNA damage. We found that this gene negatively regulates the cell division inhibitor, but is functionally distinct from the checkpoint recovery process. We provide evidence that this gene functions as an antagonist to establishing the DNA damage checkpoint. Our study uncovers a novel layer of regulation in the bacterial DNA damage checkpoint process challenging the longstanding models established in the bacterial DNA damage response field.

The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells sense successful DNA repair and promote checkpoint recovery is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis . Deletion of both proteases leads to accumulation of the checkpoint protein YneA. DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA . Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that one of the two proteases, CtpA, directly cleaves YneA in vitro . With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.

Curli amyloid fibers are the major protein component of the extracellular matrix produced by Enterobacteriaceae during biofilm formation. Curli are required for proper biofilm development and environmental persistence by Escherichia coli. Here, we present a complete and vetted genetic analysis of functional amyloid fiber biogenesis. The Keio collection of single gene deletions was screened on Congo red indicator plates to identify E. coli mutants that had defective amyloid production. We discovered that more than three hundred gene products modulated curli production. These genes were involved in fundamental cellular processes such as regulation, environmental sensing, respiration, metabolism, cell envelope biogenesis, transport, and protein turnover. The alternative sigma factors, σ^S and σ^E, had opposing roles in curli production. Mutations that induced the σ^E or Cpx stress response systems had reduced curli production, while mutant strains with increased σ^S levels had increased curli production. Mutations in metabolic pathways, including gluconeogenesis and the biosynthesis of lipopolysaccharide (LPS), produced less curli. Regulation of the master biofilm regulator, CsgD, was diverse, and the screen revealed several proteins and small RNAs (sRNA) that regulate csgD messenger RNA (mRNA) levels. Using previously published studies, we found minimal overlap between the genes affecting curli biogenesis and genes known to impact swimming or swarming motility, underlying the distinction between motile and sessile lifestyles. Collectively, the diversity and number of elements required suggest curli production is part of a highly regulated and complex developmental pathway in E. coli.