The Impact of RNA-DNA Hybrids on Genome Integrity in Bacteria.

During the essential processes of DNA replication and transcription, RNA-DNA hybrid intermediates are formed that pose significant risks to genome integrity when left unresolved. To manage RNA-DNA hybrids, all cells rely on RNase H family enzymes that specifically cleave the RNA portion of the many different types of hybrids that form in vivo. Recent experimental advances have provided new insight into how RNA-DNA hybrids form and the consequences to genome integrity that ensue when persistent hybrids remain unresolved. Here we review the types of RNA-DNA hybrids, including R-loops, RNA primers, and ribonucleotide misincorporations, that form during DNA replication and transcription and discuss how each type of hybrid can contribute to genome instability in bacteria. Further, we discuss how bacterial RNase HI, HII, and HIII and bacterial FEN enzymes contribute to genome maintenance through the resolution of hybrids.

Two distinct regulatory systems control pulcherrimin biosynthesis in Bacillus subtilis.

Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MarR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important role for pulcherrimin in B. subtilis physiology.

Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria.

There is increasing evidence that prokaryotes maintain chromosome structure, which in turn impacts gene expression. We recently characterized densely occupied, multi-kilobase regions in the E. coli genome that are transcriptionally silent, similar to eukaryotic heterochromatin. These extended protein occupancy domains (EPODs) span genomic regions containing genes encoding metabolic pathways as well as parasitic elements such as prophages. Here, we investigate the contributions of nucleoid-associated proteins (NAPs) to the structuring of these domains, by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and B. subtilis. We identify key NAPs contributing to the silencing of specific EPODs, whose deletion opens a chromosomal region for RNA polymerase binding at genes contained within that region. We show that changes in E. coli EPODs facilitate an extra layer of transcriptional regulation, which prepares cells for exposure to exotic carbon sources. Furthermore, we distinguish novel xenogeneic silencing roles for the NAPs Fis and Hfq, with the presence of at least one being essential for cell viability in the presence of domesticated prophages. Our findings reveal previously unrecognized mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements.

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB.

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids.

The current model for Okazaki fragment maturation in bacteria invokes RNA cleavage by RNase H, followed by strand displacement synthesis and 5' RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5'-3' flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Bacillus subtilis Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on nicked double-flap, 5' single flap, and nicked RNA-DNA hybrid substrates. We show that the 5' nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5' flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that ΔpolA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5' nuclease domain fails to complement ΔpolA. Cells lacking FEN (ΔfenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model resembles Okazaki fragment processing in eukaryotes, where Pol δ catalyzes strand displacement synthesis followed by 5' flap cleavage using FEN-1. Together our work highlights the conservation of ordered steps for Okazaki fragment processing in cells ranging from bacteria to human.

Structure and kinase activity of bacterial cell cycle regulator CcrZ.

CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over-initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 Å resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation.

Diverse Mechanisms of Helicase Loading during DNA Replication Initiation in Bacteria.

Initiation of DNA replication is required for cell viability and passage of genetic information to the next generation. Studies in Escherichia coli and Bacillus subtilis have established ATPases associated with diverse cellular activities (AAA+) as essential proteins required for loading of the replicative helicase at replication origins. AAA+ ATPases DnaC in E. coli and DnaI in B. subtilis have long been considered the paradigm for helicase loading during replication in bacteria. Recently, it has become increasingly clear that most bacteria lack DnaC/DnaI homologs. Instead, most bacteria express a protein homologous to the newly described DciA (dnaC/dnaI antecedent) protein. DciA is not an ATPase, and yet it serves as a helicase operator, providing a function analogous to that of DnaC and DnaI across diverse bacterial species. The recent discovery of DciA and of other alternative mechanisms of helicase loading in bacteria has changed our understanding of DNA replication initiation. In this review, we highlight recent discoveries, detailing what is currently known about the replicative helicase loading process across bacterial species, and we discuss the critical questions that remain to be investigated.

The roles of replication-transcription conflict in mutagenesis and evolution of genome organization.

Replication-transcription conflicts promote mutagenesis and give rise to evolutionary signatures, with fundamental importance to genome stability ranging from bacteria to metastatic cancer cells. This review focuses on the interplay between replication-transcription conflicts and the evolution of gene directionality. In most bacteria, the majority of genes are encoded on the leading strand of replication such that their transcription is co-directional with the direction of DNA replication fork movement. This gene strand bias arises primarily due to negative selection against deleterious consequences of head-on replication-transcription conflict. However, many genes remain head-on. Can head-on orientation provide some benefit? We combine insights from both mechanistic and evolutionary studies, review published work, and analyze gene expression data to evaluate an emerging model that head-on genes are temporal targets for adaptive mutagenesis during stress. We highlight the alternative explanation that genes in the head-on orientation may simply be the result of genomic inversions and relaxed selection acting on nonessential genes. We seek to clarify how the mechanisms of replication-transcription conflict, in concert with other mutagenic mechanisms, balanced by natural selection, have shaped bacterial genome evolution.

A positive perspective on DNA methylation: regulatory functions of DNA methylation outside of host defense in Gram-positive bacteria.

The presence of post-replicative DNA methylation is pervasive among both prokaryotic and eukaryotic organisms. In bacteria, the study of DNA methylation has largely been in the context of restriction-modification systems, where DNA methylation serves to safeguard the chromosome against restriction endonuclease cleavage intended for invading DNA. There has been a growing recognition that the methyltransferase component of restriction-modification systems can also regulate gene expression, with important contributions to virulence factor gene expression in bacterial pathogens. Outside of restriction-modification systems, DNA methylation from orphan methyltransferases, which lack cognate restriction endonucleases, has been shown to regulate important processes, including DNA replication, DNA mismatch repair, and the regulation of gene expression. The majority of research and review articles have been focused on DNA methylation in the context of Gram-negative bacteria, with emphasis toward Escherichia coli, Caulobacter crescentus, and related Proteobacteria. Here we summarize the epigenetic functions of DNA methylation outside of host defense in Gram-positive bacteria, with a focus on the regulatory effects of both phase variable methyltransferases and DNA methyltransferases from traditional restriction-modification systems.

The Bacillus subtilis PriA Winged Helix Domain Is Critical for Surviving DNA Damage.

DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis. We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The important in vivo function of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restart in vivo. Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis. Furthermore, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms. IMPORTANCE PriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain. The E. coli PriA WH domain is dispensable and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.

DNA damage checkpoint activation affects peptidoglycan synthesis and late divisome components in Bacillus subtilis.

During normal DNA replication, all cells encounter damage to their genetic material. As a result, organisms have developed response pathways that provide time for the cell to complete DNA repair before cell division occurs. In Bacillus subtilis, it is well established that the SOS-induced cell division inhibitor YneA blocks cell division after genotoxic stress; however, it remains unclear how YneA enforces the checkpoint. Here, we identify mutations that disrupt YneA activity and mutations that are refractory to the YneA-induced checkpoint. We find that YneA C-terminal truncation mutants and point mutants in or near the LysM peptidoglycan binding domain render YneA incapable of checkpoint enforcement. In addition, we develop a genetic method which isolated mutations in the ftsW gene that completely bypassed checkpoint enforcement while also finding that YneA interacts with late divisome components FtsL, Pbp2b, and Pbp1. Characterization of an FtsW variant resulted in considerably shorter cells during the DNA damage response indicative of hyperactive initiation of cell division and bypass of the YneA-enforced DNA damage checkpoint. With our results, we present a model where YneA inhibits septal cell wall synthesis by binding peptidoglycan and interfering with interaction between late arriving divisome components causing DNA damage checkpoint activation.

RnhP is a plasmid-borne RNase HI that contributes to genome maintenance in the ancestral strain Bacillus subtilis NCIB 3610.

RNA-DNA hybrids form throughout the chromosome during normal growth and under stress conditions. When left unresolved, RNA-DNA hybrids can slow replication fork progression, cause DNA breaks, and increase mutagenesis. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA-DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper-replication, RnhP compensates for the loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore, resulting in SOS induction and inhibition of cell division. We conclude that all three functional RNase H enzymes are present in B. subtilis NCIB 3610 and that the plasmid-encoded RNase HI contributes to chromosome stability, while the chromosomally encoded RNase HIII is important for chromosome stability and plasmid hyper-replication.

Methyltransferase DnmA is responsible for genome-wide N6-methyladenosine modifications at non-palindromic recognition sites in Bacillus subtilis.

The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.

Hydroxyurea Induces a Stress Response That Alters DNA Replication and Nucleotide Metabolism in Bacillus subtilis.

Hydroxyurea (HU) is classified as a ribonucleotide reductase (RNR) inhibitor and has been widely used to stall DNA replication by depleting deoxyribonucleoside triphosphate (dNTP) pools. Recent evidence in Escherichia coli shows that HU readily forms breakdown products that damage DNA directly, indicating that toxicity is a result of secondary effects. Because HU is so widely used in the laboratory and as a clinical therapeutic, it is important to understand its biological effects. To determine how Bacillus subtilis responds to HU-induced stress, we performed saturating transposon insertion mutagenesis followed by deep sequencing (Tn-seq), transcriptome sequencing (RNA-seq) analysis, and measurement of replication fork progression. Our data show that B. subtilis cells elongate, and replication fork progression is slowed, following HU challenge. The transcriptomic data show that B. subtilis cells initially mount a metabolic response likely caused by dNTP pool depletion before inducing the DNA damage response (SOS) after prolonged exposure. To compensate for reduced nucleotide pools, B. subtilis upregulates the purine and pyrimidine biosynthetic machinery and downregulates the enzymes producing ribose 5-phosphate. We show that overexpression of the RNR genes nrdEF suppresses the growth interference caused by HU, suggesting that RNR is an important target of HU in B. subtilis. Although genes involved in nucleotide and carbon metabolism showed considerable differential expression, we also find that genes of unknown function (y-genes) represent the largest class of differentially expressed genes. Deletion of individual y-genes caused moderate growth interference in the presence of HU, suggesting that cells have several ways of coping with HU-induced metabolic stress. IMPORTANCE Hydroxyurea (HU) has been widely used as a clinical therapeutic and an inhibitor of DNA replication. Some evidence suggests that HU inhibits ribonucleotide reductase, depleting dNTP pools, while other evidence shows that toxic HU breakdown products are responsible for growth inhibition and genotoxic stress. Here, we use multiple, complementary approaches to characterize the response of Bacillus subtilis to HU. B. subtilis responds by upregulating the expression of purine and pyrimidine biosynthesis. We show that HU challenge reduced DNA replication and that overexpression of the ribonucleotide reductase operon suppressed growth interference by HU. Our results demonstrate that HU targets RNR and several other metabolic enzymes contributing to toxicity in bacteria.

RecA Is Required for the Assembly of RecN into DNA Repair Complexes on the Nucleoid.

Homologous recombination requires the coordinated effort of several proteins to complete break resection, homologous pairing, and resolution of DNA crossover structures. RecN is a conserved bacterial protein important for double-strand break repair and is a member of the structural maintenance of chromosomes (SMC) protein family. Current models in Bacillus subtilis propose that RecN responds to double-stranded breaks prior to RecA and end processing, suggesting that RecN is among the very first proteins responsible for break detection. Here, we investigate the contribution of RecA and end processing by AddAB to RecN recruitment into repair foci in vivo. Using this approach, we found that recA is required for RecN-green fluorescent protein (GFP) focus formation on the nucleoid during normal growth and in response to DNA damage. In the absence of recA function, RecN foci form in a low percentage of cells, RecN localizes away from the nucleoid, and RecN fails to assemble in response to DNA damage. In contrast, we show that the response of RecA-GFP foci to DNA damage is unchanged in the presence or absence of recN. In further support of RecA activity preceding RecN, we show that ablation of the double-strand break end-processing enzyme addAB results in a failure of RecN to form foci in response to DNA damage. With these results, we conclude that RecA and end processing function prior to RecN, establishing a critical step for the recruitment and participation of RecN during DNA break repair in Bacillus subtilis. IMPORTANCE Homologous recombination is important for the repair of DNA double-strand breaks. RecN is a highly conserved protein that has been shown to be important for sister chromatid cohesion and for surviving break-inducing clastogens. Here, we show that the assembly of RecN into repair foci on the bacterial nucleoid requires the end-processing enzyme AddAB and the recombinase RecA. In the absence of either recA or end-processing RecN-GFP, foci are no longer DNA damage inducible, and foci form in a subset of cells as large complexes in regions away from the nucleoid. Our results establish the stepwise order of action, where double-strand break end processing and RecA association precede the participation of RecN in break repair in Bacillus subtilis.

DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes.

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (ΔRSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the ΔRSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the ΔRSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the ΔRSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.

Binding of the regulatory domain of MutL to the sliding ß-clamp is species specific.

The ß-clamp is a protein hub central to DNA replication and fork management. Proteins interacting with the ß-clamp harbor a conserved clamp-binding motif that is often found in extended regions. Therefore, clamp interactions have -almost exclusively- been studied using short peptides recapitulating the binding motif. This approach has revealed the molecular determinants that mediate the binding but cannot describe how proteins with clamp-binding motifs embedded in structured domains are recognized. The mismatch repair protein MutL has an internal clamp-binding motif, but its interaction with the ß-clamp has different roles depending on the organism. In Bacillus subtilis, the interaction stimulates the endonuclease activity of MutL and it is critical for DNA mismatch repair. Conversely, disrupting the interaction between Escherichia coli MutL and the ß-clamp only causes a mild mutator phenotype. Here, we determined the structures of the regulatory domains of E. coli and B. subtilis MutL bound to their respective ß-clamps. The structures reveal different binding modes consistent with the binding to the ß-clamp being a two-step process. Functional characterization indicates that, within the regulatory domain, only the clamp binding motif is required for the interaction between the two proteins. However, additional motifs beyond the regulatory domain may stabilize the interaction. We propose a model for the activation of the endonuclease activity of MutL in organisms lacking methyl-directed mismatch repair.

Discovery of a dual protease mechanism that promotes DNA damage checkpoint recovery.

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 promote checkpoint recovery after sensing successful repair 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. We show that 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 both proteases interact with YneA and 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.

Sources of spontaneous mutagenesis in bacteria.

Mutations in an organism's genome can arise spontaneously, that is, in the absence of exogenous stress and prior to selection. Mutations are often neutral or deleterious to individual fitness but can also provide genetic diversity driving evolution. Mutagenesis in bacteria contributes to the already serious and growing problem of antibiotic resistance. However, the negative impacts of spontaneous mutagenesis on human health are not limited to bacterial antibiotic resistance. Spontaneous mutations also underlie tumorigenesis and evolution of drug resistance. To better understand the causes of genetic change and how they may be manipulated in order to curb antibiotic resistance or the development of cancer, we must acquire a mechanistic understanding of the major sources of mutagenesis. Bacterial systems are particularly well-suited to studying mutagenesis because of their fast growth rate and the panoply of available experimental tools, but efforts to understand mutagenic mechanisms can be complicated by the experimental system employed. Here, we review our current understanding of mutagenic mechanisms in bacteria and describe the methods used to study mutagenesis in bacterial systems.

Regulation of DNA Binding and High-Order Oligomerization of the DnaB Helicase Loader.

DnaB is an essential primosomal protein that coloads the replicative helicase in many Gram-positive bacteria, including several human pathogens. Although DnaB is tetrameric in solution, it is from a protein family whose members can oligomerize into large complexes when exposed to DNA. It is currently unknown how DNA binding by DnaB is regulated or how these interactions induce changes in its oligomeric state. Here, we investigated DNA binding by DnaB from Bacillus subtilis and the critical human pathogen Staphylococcus aureus We found that B. subtilis DnaB binds double-stranded DNA as a tetramer; however, M13mp18 single-stranded DNA induces high-order oligomerization. Mutating a conserved motif at the C-terminal end of DnaB stimulates single-stranded DNA binding, suggesting that conformational changes in this region regulate DNA substrate preferences. S. aureus DnaB could also be induced to form high-order oligomers with either M13mp18 or PhiX174 single-stranded DNA. Therefore, oligomeric shifts in DnaB are tightly controlled and this activity is conserved between B. subtilis and a pathogenic species.IMPORTANCE DnaB is a replicative helicase loader involved in initiating DNA replication in many bacterial species. We investigated the binding preferences of DnaB for its DNA substrate and determined that the C-terminal end of the protein plays a critical role in controlling DNA interactions. Furthermore, we found that DNA binding in general did not trigger changes to the oligomeric state of DnaB, but rather, certain types of single-stranded DNA substrates specifically induced DnaB to self-assemble into a large complex. This indicates that the structure of DNA itself is an important regulatory element that influences the behavior of DnaB. Importantly, these observations held for both Bacillus subtilis and the pathogenic species Staphylococcus aureus, demonstrating conserved biochemical functions of DnaB in these species.