scholarly journals A Roadblock-and-Kill Mechanism of Action Model for the DNA-Targeting Antibiotic Ciprofloxacin

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Nikola Ojkic ◽  
Elin Lilja ◽  
Susana Direito ◽  
Angela Dawson ◽  
Rosalind J. Allen ◽  
...  
Keyword(s):  
Growth Rate ◽  
Dna Damage ◽  
Production Rate ◽  
Mechanism Of Action ◽  
Sos Response ◽  
Dynamical Response ◽  
Bacterial Cells ◽  
Content Type ◽  
Dna Production

ABSTRACT Fluoroquinolones, antibiotics that cause DNA damage by inhibiting DNA topoisomerases, are clinically important, but their mechanism of action is not yet fully understood. In particular, the dynamical response of bacterial cells to fluoroquinolone exposure has hardly been investigated, although the SOS response, triggered by DNA damage, is often thought to play a key role. Here, we investigated the growth inhibition of the bacterium Escherichia coli by the fluoroquinolone ciprofloxacin at low concentrations. We measured the long-term and short-term dynamical response of the growth rate and DNA production rate to ciprofloxacin at both the population and single-cell levels. We show that, despite the molecular complexity of DNA metabolism, a simple roadblock-and-kill model focusing on replication fork blockage and DNA damage by ciprofloxacin-poisoned DNA topoisomerase II (gyrase) quantitatively reproduces long-term growth rates in the presence of ciprofloxacin. The model also predicts dynamical changes in the DNA production rate in wild-type E. coli and in a recombination-deficient mutant following a step-up of ciprofloxacin. Our work highlights that bacterial cells show a delayed growth rate response following fluoroquinolone exposure. Most importantly, our model explains why the response is delayed: it takes many doubling times to fragment the DNA sufficiently to inhibit gene expression. We also show that the dynamical response is controlled by the timescale of DNA replication and gyrase binding/unbinding to the DNA rather than by the SOS response, challenging the accepted view. Our work highlights the importance of including detailed biophysical processes in biochemical-systems models to quantitatively predict the bacterial response to antibiotics.

scholarly journals A roadblock-and-kill model explains the dynamical response to the DNA-targeting antibiotic ciprofloxacin

2019 ◽  
Author(s):  
Nikola Ojkic ◽  
Elin Lilja ◽  
Susana Direito ◽  
Angela Dawson ◽  
Rosalind J. Allen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Production Rate ◽  
Topoisomerase Ii ◽  
Sos Response ◽  
Dynamical Response ◽  
Bacterial Cells ◽  
Resistance Evolution ◽  
Dna Production ◽  
Biochemical Systems

AbstractFluoroquinolones - antibiotics that cause DNA damage by inhibiting DNA topoisomerases - are clinically important, but their mechanism of action is not yet fully understood. In particular, the dynamical response of bacterial cells to fluoroquinolone exposure has hardly been investigated, although the SOS response, triggered by DNA damage, is often thought to play a key role. Here we investigate growth inhibition of the bacterium Escherichia coli by the fluoroquinolone ciprofloxacin at low doses (up to 5x the minimum inhibitory concentration). We measure the long-term and short-term (dynamic) response of the growth rate and DNA production rate to ciprofloxacin, at both population- and single-cell level. We show that despite the molecular complexity of DNA metabolism, a simple `roadblock-and-kill’ model focusing on replication fork blockage and DNA damage by ciprofloxacin-poisoned DNA topoisomerase II (gyrase) quantitatively reproduces long-term growth rates. The model also predicts dynamical changes in DNA production rate in wild type E. coli and in an SOS-deficient mutant, following a step-up of ciprofloxacin. Our work reveals new insights into the dynamics of fluoroquinolone action, with important implications for predicting the rate of resistance evolution. Most importantly, our model explains why the response is delayed: it takes many doubling times to fragment the DNA sufficiently to inhibit gene expression. Our model also challenges the view that the SOS response plays a central role: the dynamical response is controlled by the timescale of DNA replication and gyrase binding/unbinding to the DNA rather than by the SOS response. More generally, our work highlights the importance of including biophysical processes in biochemical-systems models to fully understand bacterial response to antibiotics.

scholarly journals Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Peter E. Burby ◽  
Lyle A. Simmons
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Genome Instability ◽  
Sos Response ◽  
Bacterial Cells ◽  
Cycle Progression ◽  
Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

Applying the Fama and French three-factor model to analyze risk/reward in the Spanish REITs: an ARDL approach

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Zhenyu Su ◽  
Paloma Taltavull
Keyword(s):  
Growth Rate ◽  
Factor Model ◽  
Explanatory Power ◽  
Gdp Growth ◽  
Size Factor ◽  
Content Type ◽  
Gdp Growth Rate ◽  
Fama And French ◽  
Three Factor

Purpose This paper aims to analyse the risk and excess returns of the Spanish real estate investment trusts (S-REITs) using various methods, though focusing primarily on the Fama-French three-factor (FF3) model, over the period from 2007Q3 to 2017Q2. Design/methodology/approach The autoregressive distributed lag model is used for the empirical analysis to test long-term stable relationships between variables. Findings The findings indicate that the FF3 model is suitable for the S-REITs market, better explaining the S-REITs’ returns variation than the traditional single-index capital asset pricing model (CAPM) and the Carhart four-factor model. The empirical evidence is reasonably consistent with the FF3 model; the values for the market, size and value are highly statistically significant over the analysis period, with 68.7% variation in S-REITs’ returns explained by the model. In the long run, the market factor has less explanatory power than the size and value factors; the positive long-term multiplier of the size factor indicates that small S-REIT companies have higher returns, along with higher risk, while the negative multiplier of the value indicator suggests that S-REITs portfolios prefer to allocate growth REITs with low book-to-market ratios. The empirical findings from a modified FF3 model, which additionally incorporates Spain’s gross domestic product (GDP) growth rate, two consumer price index (CPI) macro-factors and three dummy variables, indicates that GDP growth rate and CPI also affect S-REITs’ yields, while investment funds with capital calls have a small influence on S-REITs’ returns. Practical implications The regression results of the standard and extended FF3 model can help researchers understand S-REITs’ risk and return through a general stock pattern. Potential investors are given more information to consider the new Spanish investment vehicle before making a decision. Originality/value The paper uses standard techniques but applies them for the first time to the S-REIT market.

scholarly journals Loss of Function inEscherichia coliExposed to Environmentally Relevant Concentrations of Benzalkonium Chloride

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Sarah Forbes ◽  
Nicola Morgan ◽  
Gavin J. Humphreys ◽  
Alejandro Amézquita ◽  
Hitesh Mistry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Biofilm Formation ◽  
Benzalkonium Chloride ◽  
Loss Of Function ◽  
Free Medium ◽  
Content Type ◽  
Expression Of Genes ◽  
Repeated Passage

ABSTRACTAssessing the risk of resistance associated with biocide exposure commonly involves exposing microorganisms to biocides at concentrations close to the MIC. With the aim of representing exposure to environmental biocide residues,Escherichia coliMG1655 was grown for 20 passages in the presence or absence of benzalkonium chloride (BAC) at 100 ng/liter and 1,000 ng/liter (0.0002% and 0.002% of the MIC, respectively). BAC susceptibility, planktonic growth rates, motility, and biofilm formation were assessed, and differentially expressed genes were determined via transcriptome sequencing. Planktonic growth rate and biofilm formation were significantly reduced (P< 0.001) following BAC adaptation, while BAC minimum bactericidal concentration increased 2-fold. Transcriptomic analysis identified 289 upregulated and 391 downregulated genes after long-term BAC adaptation compared with the respective control organism passaged in BAC-free medium. When the BAC-adapted bacterium was grown in BAC-free medium, 1,052 genes were upregulated and 753 were downregulated. Repeated passage solely in biocide-free medium resulted in 460 upregulated and 476 downregulated genes compared with unexposed bacteria. Long-term exposure to environmentally relevant BAC concentrations increased the expression of genes associated with efflux and reduced the expression of genes associated with outer-membrane porins, motility, and chemotaxis. This was manifested phenotypically through the loss of function (motility). Repeated passage in a BAC-free environment resulted in the upregulation of multiple respiration-associated genes, which was reflected by increased growth rate. In summary, repeated exposure ofE. colito BAC residues resulted in significant alterations in global gene expression that were associated with minor decreases in biocide susceptibility, reductions in growth rate and biofilm formation, and loss of motility.IMPORTANCEExposure to very low concentrations of biocides in the environment is a poorly understood risk factor for antimicrobial resistance. Repeated exposure to trace levels of the biocide benzalkonium chloride (BAC) resulted in loss of function (motility) and a general reduction in bacterial fitness but relatively minor decreases in susceptibility. These changes were accompanied by widespread changes in theEscherichia colitranscriptome. These results demonstrate the importance of including phenotypic characterization in studies designed to assess the risks of biocide exposure.

scholarly journals SOS-Independent Pyocin Production in P. aeruginosa Is Induced by XerC Recombinase Deficiency

mBio ◽  
2021 ◽  
Author(s):  
Nina S. Baggett ◽  
Adam S. Bronson ◽  
Matthew T. Cabeen
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Damage ◽  
Sos Response ◽  
Opportunistic Pathogen ◽  
Content Type

Pseudomonas aeruginosa is a versatile and ubiquitous bacterium that frequently infects humans as an opportunistic pathogen. P. aeruginosa competes with other strains within the species by producing killing complexes termed pyocins, which are only known to be induced by cells experiencing DNA damage and the subsequent SOS response. Here, we discovered that strains lacking a recombinase enzyme called XerC strongly produce pyocins independently of the SOS response.

The growth oxymoron: strategic execution

2019 ◽  
Vol 35 (6) ◽  
pp. 1-4
Author(s):  
Carol M. Connell ◽  
Christine Lemyze
Keyword(s):  
Growth Rate ◽  
Financial Crisis ◽  
Annual Growth ◽  
Annual Growth Rate ◽  
Strategy Execution ◽  
Content Type ◽  
Compound Annual Growth Rate ◽  
The Past ◽  
Strategic Execution

Purpose The purpose of this paper is to present a viewpoint on aligning strategy and execution to produce superior business results. Design/methodology/approach The paper examines the long-term financials of the top ten growers to reveal companies that have continued to grow in good economic times and bad, including the Great Recession. While some companies dug deeper into their core businesses during the financial crisis, others continued to innovate. Findings Where companies continued to focus on strategy execution, they were rewarded, for example, Amazon’s compound annual growth rate for the ten-year period that included the financial crisis was 36.45 per cent; in the past three years, Amazon’s compound annual growth rate (CAGR) has been 56.76 per cent. Most of the top ten long-term growers are headed by the same founder/entrepreneur. Research limitations/implications Look beyond the past three years for models of successful strategy execution. Practical implications For long-term company leaders, entrepreneurs, or turnaround experts, strategic execution is no oxymoron, but a requirement for growth and, ultimately, their unique responsibility. Social implications The paper identifies three major focus areas for strategy teams and company leadership: 1. customer centricity and strategy execution; 2. learning from survivors; and 3. rethinking capabilities and talent. Originality/value As a professor of strategic management and as a consultant to organizations on strategy and marketing transformation, we have focused on the activities that are necessary for leaders to create effective strategy and to execute successfully. We have also been responsible for equipping the larger teams of strategy professionals (and future strategy professionals) who support these leaders with the approaches, the methods, and the tools necessary to plan effectively, to assess effectiveness, and to correct problems in strategy and execution. We bring that perspective to this viewpoint paper.

scholarly journals Intracellular d-Serine Accumulation Promotes Genetic Diversity via Modulated Induction of RecA in Enterohemorrhagic Escherichia coli

2016 ◽  
Vol 198 (24) ◽  
pp. 3318-3328 ◽  
Author(s):  
James P. R. Connolly ◽  
Andrew J. Roe
Keyword(s):  
Escherichia Coli ◽  
Genetic Diversity ◽  
Dna Damage ◽  
Mutation Frequency ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Lytic Bacteriophage ◽  
Content Type ◽  
Lexa Repressor ◽  
Physiological Relevance

ABSTRACTWe recently discovered that exposure of enterohemorrhagicEscherichia coli(EHEC) tod-serine resulted in accumulation of this unusual amino acid, induction of the SOS regulon, and downregulation of the type III secretion system that is essential for efficient colonization of the host. Here, we have investigated the physiological relevance of this elevated SOS response, which is of particular interest given the presence of Stx toxin-carrying lysogenic prophages on the EHEC chromosome that are activated during the SOS response. We found that RecA elevation in response tod-serine, while being significant, was heterogeneous and not capable of activatingstxexpression orstxphage transduction to a nonlysogenic recipient. This “SOS-like response” was, however, capable of increasing the mutation frequency associated with low-level RecA activity, thus promoting genetic diversity. Furthermore, this response was entirely dependent on RecA and enhanced in the presence of a DNA-damaging agent, indicating a functional SOS response, but did not result in observable cleavage of the LexA repressor alone, indicating a controlled mechanism of induction. This work demonstrates that environmental factors not usually associated with DNA damage are capable of promoting an SOS-like response. We propose that this modulated induction of RecA allows EHEC to adapt to environmental insults such asd-serine while avoiding unwanted phage-induced lysis.IMPORTANCEThe SOS response is a global stress network that is triggered by the presence of DNA damage due to breakage or stalled replication forks. Activation of the SOS response can trigger the replication of lytic bacteriophages and promote genetic diversification through error-prone polymerases. We have demonstrated that the host-associated metabolited-serine contributes toEscherichia coliniche specification and accumulates inside cells that cannot catabolize it. This results in a modulated activation of the SOS antirepressor RecA that is insufficient to trigger lytic bacteriophage but capable of increasing the SOS-associated mutation frequency. These findings describe how relevant signals not normally associated with DNA damage can hijack the SOS response, promoting diversity asE. colistrains adapt while avoiding unwanted phage lysis.

scholarly journals Genome-Wide Functional Screen for Calcium Transients in Escherichia coli Identifies Increased Membrane Potential Adaptation to Persistent DNA Damage

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Rose Luder ◽  
Giancarlo N. Bruni ◽  
Joel M. Kralj
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Membrane Potential ◽  
Calcium Transients ◽  
Eukaryotic Cells ◽  
Calcium Dynamics ◽  
Content Type ◽  
Genome Wide ◽  
Gene Knockouts

ABSTRACT Calcium plays numerous critical roles in signaling and homeostasis in eukaryotic cells. Far less is known about calcium signaling in bacteria than in eukaryotic cells, and few genes controlling influx and efflux have been identified. Previous work in Escherichia coli showed that calcium influx was induced by voltage depolarization, which was enhanced by mechanical stimulation, which suggested a role in bacterial mechanosensation. To identify proteins and pathways affecting calcium handling in bacteria, we designed a live-cell screen to monitor calcium dynamics in single cells across a genome-wide knockout panel in E. coli. The screen measured cells from the Keio collection of knockouts and quantified calcium transients across the population. Overall, we found 143 gene knockouts that decreased levels of calcium transients and 32 gene knockouts that increased levels of transients. Knockouts of proteins involved in energy production and regulation appeared, as expected, as well as knockouts of proteins of a voltage sink, F1Fo-ATPase. Knockouts of exopolysaccharide and outer membrane synthesis proteins showed reduced transients which refined our model of electrophysiology-mediated mechanosensation. Additionally, knockouts of proteins associated with DNA repair had reduced calcium transients and voltage. However, acute DNA damage did not affect voltage, and the results suggested that only long-term adaptation to DNA damage decreased membrane potential and calcium transients. Our work showed a distinct separation between the acute and long-term DNA damage responses in bacteria, which also has implications for mitochondrial DNA damage in eukaryotes. IMPORTANCE All eukaryotic cells use calcium as a critical signaling molecule. There is tantalizing evidence that bacteria also use calcium for cellular signaling, but much less is known about the molecular actors and physiological roles. To identify genes regulating cytoplasmic calcium in Escherichia coli, we created a single-cell screen for modulators of calcium dynamics. The genes uncovered in this screen helped refine a model for voltage-mediated bacterial mechanosensation. Additionally, we were able to more carefully dissect the mechanisms of adaptation to long-term DNA damage, which has implications for both bacteria and mitochondria in the face of unrepaired DNA.

scholarly journals Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Esha Atolia ◽  
Spencer Cesar ◽  
Heidi A. Arjes ◽  
Manohary Rajendram ◽  
Handuo Shi ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Growth Rate ◽  
High Throughput ◽  
Bacterial Growth ◽  
Physiological State ◽  
Maximal Growth ◽  
Content Type ◽  
Initial Cell ◽  
Maximal Growth Rate

ABSTRACT Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth. IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

scholarly journals RexAB Is Essential for the Mutagenic Repair of Staphylococcus aureus DNA Damage Caused by Co-trimoxazole

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Rebecca S. Clarke ◽  
Maya S. Bruderer ◽  
Kam Pou Ha ◽  
Andrew M. Edwards
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Damage ◽  
Dna Repair ◽  
Sos Response ◽  
Dependent Manner ◽  
Content Type ◽  
Sos Induction ◽  
Therapeutic Value ◽  
Emergence Of Resistance

ABSTRACT Co-trimoxazole (SXT) is a combination therapeutic that consists of sulfamethoxazole and trimethoprim that is increasingly used to treat skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the use of SXT is limited to the treatment of low-burden, superficial S. aureus infections and its therapeutic value is compromised by the frequent emergence of resistance. As a first step toward the identification of approaches to enhance the efficacy of SXT, we examined the role of bacterial DNA repair in antibiotic susceptibility and mutagenesis. We found that mutants lacking the DNA repair complex RexAB had a modest SXT MIC that was 2-fold lower than that seen with wild-type strains but were killed 50-fold to 5,000-fold more efficiently by the combination antibiotic at the breakpoint concentration. SXT-mediated DNA damage occurred via both thymidine limitation and the generation of reactive oxygen species and triggered induction of the SOS response in a RexAB-dependent manner. SOS induction was associated with a 50% increase in the mutation rate, which may contribute to emergence of resistant strains during SXT therapy. In summary, this work determined that SXT caused DNA damage in S. aureus via both thymidine limitation and oxidative stress and that the damage was repaired by the RexAB complex, leading to induction of the mutagenic SOS response. Small-molecule inhibitors of RexAB could therefore have therapeutic value by increasing the efficacy of SXT and decreasing the emergence of drug resistance during treatment of infections caused by S. aureus.

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