The concentration of ribonucleoside triphosphates (rNTPs) in cells is far greater than the concentration of deoxyribonucleoside triphosphates (dNTPs), and this pool imbalance presents a challenge for DNA polymerases (Pols) to select their proper substrate. This report examines the effect of nucleotide pool imbalance on the rate and fidelity of the Escherichia coli replisome. We find that rNTPs decrease replication fork rate by competing with dNTPs at the active site of the C-family Pol III replicase at a step that does not require correct base-pairing. The effect of rNTPs on Pol rate generalizes to B-family eukaryotic replicases, Pols δ and e. Imbalance of the dNTP pool also slows the replisome and thus is not specific to rNTPs. We observe a measurable frequency of rNMP incorporation that predicts one rNTP incorporated every 2.3 kb during chromosome replication. Given the frequency of rNMP incorporation, the repair of rNMPs is likely rapid. RNase HII nicks DNA at single rNMP residues to initiate replacement with dNMP. Considering that rNMPs will mark the new strand, RNase HII may direct strand-specificity for mismatch repair (MMR). How the newly synthesized strand is recognized for MMR is uncertain in eukaryotes and most bacteria, which lack a methyl-directed nicking system. Here we demonstrate that Bacillus subtilis incorporates rNMPs in vivo, that RNase HII plays a role in their removal, and the RNase HII gene deletion enhances mutagenesis, suggesting a possible role of incorporated rNMPs in MMR.T he structures of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) differ by a single atom, yet each must be distinguished by DNA and RNA polymerases (Pols). This is more challenging for DNA Pols because the intracellular concentration of rNTPs is 10-100-fold higher than that of dNTPs (1-3). DNA is more stabile than RNA, and rNMP residues in DNA could lead to spontaneous strand breaks. Hence, it is important to genomic integrity that DNA Pols exclude rNMPs from incorporation into the genome (1, 4).Structural studies reveal that DNA Pols distinguish ribo and deoxyribo sugars via a "steric gate," in which a bulky residue or main chain atom sterically occludes binding of the ribo 2′OH (5, 6). However, the single-atom difference between rNTPs and dNTPs imposes an upper limit to sugar recognition, and thus DNA Pols incorporate rNMPs at a low frequency. For example, studies in yeast demonstrate that rNMPs are incorporated in vivo, and studies in vitro demonstrate a frequency of rNMP incorporation predicting that 10,000 rNMPs or more may be incorporated each replication cycle (2,7,8). Given their abundant incorporation, there are probably multiple pathways to remove rNMPs, as their persistence is associated with genomic instability in yeast (7, 9).The current study reconstitutes the Escherichia coli replisome and examines the cost of rNTP/dNTP nucleotide pool imbalance on the frequency of rNMP incorporation and the rate of fork progression. We find that a nucleotide pool imbalance slows the ...

The cardiotonic steroid marinobufagenin (MBG) has been implicated in the pathogenesis of experimental uremic cardiomyopathy, which is characterized by progressive cardiac fibrosis. We examined whether the transcription factor Friend leukemia integration-1 (Fli-1) might be involved in this process. Fli-1-knockdown mice demonstrated greater cardiac collagen-1 expression and fibrosis compared with wild-type mice; both developed increased cardiac collagen expression and fibrosis after 5/6 nephrectomy. There was a strong inverse relationship between the expressions of Fli-1 and procollagen in primary culture of rat cardiac and human dermal fibroblasts as well as a cell line derived from renal fibroblasts and MBG-induced decreases in nuclear Fli-1 as well as increases in procollagen-1 expression in these cells. Transfection of a Fli-1 expression vector prevented increased procollagen-1 expression from MBG. MBG exposure induced a rapid translocation of the δ-isoform of protein kinase C (PKCδ) to the nucleus. This translocation was prevented by pharmacological inhibition of phospholipase C, and MBG-induced increases in procollagen-1 expression were prevented with a PKCδ- but not a PKCα-specific inhibitor. Finally, immunoprecipitation studies strongly suggest that MBG induced phosphorylation of Fli-1. We feel these data support a causal relationship with MBG-induced translocation of PKCδ, which results in phosphorylation of as well as decreases in nuclear Fli-1 expression, which, in turn, leads to increases in collagen production. Should these findings be confirmed, we speculate that this pathway may represent a therapeutic target for uremic cardiomyopathy as well as other conditions associated with excessive fibrosis.

SUMMARY From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis.

MutS is responsible for initiating the correction of DNA replication errors. To understand how MutS searches for and identifies rare base-pair mismatches, we characterized the dynamic movement of MutS and the replisome in real time using superresolution microscopy and single-molecule tracking in living cells. We report that MutS dynamics are heterogeneous in cells, with one MutS population exploring the nucleoid rapidly, while another MutS population moves to and transiently dwells at the replisome region, even in the absence of appreciable mismatch formation. Analysis of MutS motion shows that the speed of MutS is correlated with its separation distance from the replisome and that MutS motion slows when it enters the replisome region. We also show that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition. Using variants of MutS and the replication processivity clamp to impair mismatch repair, we find that MutS dynamically moves to and from the replisome before mismatch binding to scan for errors. Furthermore, a block to DNA synthesis shows that MutS is only capable of binding mismatches near the replisome. It is well-established that MutS engages in an ATPase cycle, which is necessary for signaling downstream events. We show that a variant of MutS with a nucleotide binding defect is no longer capable of dynamic movement to and from the replisome, showing that proper nucleotide binding is critical for MutS to localize to the replisome in vivo. Our results provide mechanistic insight into the trafficking and movement of MutS in live cells as it searches for mismatches.super-resolution microscopy | DNA replication | bacterial cell biology | Bacillus subtilis | single-cell analysis D NA mismatch repair (MMR) is the highly conserved process responsible for correcting DNA replication errors (1). Although replication errors occur infrequently in bacteria (∼1 error per 31 million bp) (2), the consequences of MMR failure on human health are severe (3). MutS is the first protein involved in the MMR pathway, and it is responsible for detecting rare base-pairing errors. In Bacillus subtilis, MutS then recruits MutL, an endonuclease in most bacteria and eukaryotic organisms, to incise the DNA (4, 5). After MutL incision, the error-containing strand is removed, and the DNA is resynthesized to complete error correction (6).The mechanism by which MutS homologs locate a single mismatch among millions of correctly paired nucleotides has been studied extensively by bulk biochemistry, in vitro atomic force microscopy and single-molecule imaging, and visualizing MutS using in vivo cell biology approaches (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In vitro single-molecule studies largely indicate that MutS operates as a searching clamp diffusing along DNA in a 1D search process (8,10,13). In this model, after mismatch recognition, MutS can dwell at the mismatch before exchanging ADP for ATP, converting into a stable ATP-bound sliding clamp, and subseque...

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.

Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology. IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

SUMMARY The most abundant oxidants controlling bacterial colonization on mucosal barrier epithelia are hypochlorous acid (HOCl), hypobromous acid (HOBr), and hypothiocyanous acid (HOSCN). All three oxidants are highly antimicrobial but little is known about their relative efficacies, their respective cellular targets, or what specific responses they elicit in bacteria. To address these important questions, we directly tested the individual oxidants on the virulent Pseudomonas aeruginosa strain PA14. We discovered that HOCl and HOBr work almost interchangeably, impacting non-growing bacterial cultures more significantly than actively growing bacteria, and eliciting similar stress responses, including the heat shock response. HOSCN treatment is distinctly different, affecting primarily actively growing PA14, and evoking stress responses suggestive of membrane damage. What all three oxidants have in common, however, is their ability to cause substantial protein aggregation. This effect became particularly obvious in strains lacking polyphosphate, a newly recognized chemical chaperone. Treatment of PA14 with the FDA-approved anti-inflammatory drug mesalamine, which has recently been shown to attenuate polyP production in a wide range of bacteria, effectively decreased the resistance of PA14 towards all three oxidants, suggesting that we have discovered a novel, targetable defense system in P. aeruginosa.

Dedicated to Professor Siegjiried Petersen on the occasion of his 65th birthdayOptical brighteners are used in detergents, textiles, paper, plastics, and paints. Most optical brighteners contain heterocycles such as coumarins, 2-pyrazolines, naphthalimides, pyrazoles, triazoles, oxadiazoles, and triazines. In this article the syntheses, properties, and applications of new optical brighteners are described with references to patent literature.