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Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes., Whereas the toxic effects of ethanol are well-documented, the underlying mechanism is obscure. This study uses the eukaryotic model S. cerevisiae to reveal how exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate.

Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths. ispartof: CANCER CELL vol:39 issue:8 pages:1135-+ ispartof: location:United States status: published

IAMBEE is a web server designed for the Identification of Adaptive Mutations in Bacterial Evolution Experiments (IAMBEE). Input data consist of genotype information obtained from independently evolved clonal populations or strains that show the same adapted behavior (phenotype). To distinguish adaptive from passenger mutations, IAMBEE searches for neighborhoods in an organism-specific interaction network that are recurrently mutated in the adapted populations. This search for recurrently mutated network neighborhoods, as proxies for pathways is driven by additional information on the functional impact of the observed genetic changes and their dynamics during adaptive evolution. In addition, the search explicitly accounts for the differences in mutation rate between the independently evolved populations. Using this approach, IAMBEE allows exploiting parallel evolution to identify adaptive pathways. The web-server is freely available at http://bioinformatics.intec.ugent.be/iambee/ with no login requirement. ispartof: NUCLEIC ACIDS RESEARCH vol:47 issue:W1 pages:W151-W157 ispartof: location:England status: published

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia that is most frequent in children and is characterized by the presence of few chromosomal rearrangements and 10 to 20 somatic mutations in protein-coding regions at diagnosis. The majority of T-ALL cases harbor activating mutations in NOTCH1 together with mutations in genes implicated in kinase signaling, transcriptional regulation, or protein translation. To obtain more insight in the level of clonal heterogeneity at diagnosis and during treatment, we used single-cell targeted DNA sequencing with the Tapestri platform. We designed a custom ALL panel and obtained accurate single-nucleotide variant and small insertion-deletion mutation calling for 305 amplicons covering 110 genes in about 4400 cells per sample and time point. A total of 108 188 cells were analyzed for 25 samples of 8 T-ALL patients. We typically observed a major clone at diagnosis (>35% of the cells) accompanied by several minor clones of which some were less than 1% of the total number of cells. Four patients had >2 NOTCH1 mutations, some of which present in minor clones, indicating a strong pressure to acquire NOTCH1 mutations in developing T-ALL cells. By analyzing longitudinal samples, we detected the presence and clonal nature of residual leukemic cells and clones with a minor presence at diagnosis that evolved to clinically relevant major clones at later disease stages. Therefore, single-cell DNA amplicon sequencing is a sensitive assay to detect clonal architecture and evolution in T-ALL. ispartof: BLOOD vol:137 issue:6 pages:801-811 ispartof: location:United States status: published

SummaryThe ability to predict the future behaviour of an individual cancer is crucial for precision cancer medicine and, in particular, for the development of strategies that prevent acquisition of resistance to anti-cancer drugs. Therapy resistance, which often develops from a heterogeneous pool of drug-tolerant cells known as minimal residual disease (MRD), is thought to mainly occur through acquisition of genetic alterations. Increasing evidence, however, indicates that drug resistance might also be acquired though nongenetic mechanisms. A key emerging question is therefore whether specific molecular and/or cellular features of the MRD ecosystem determine which of these two distinct resistance trajectories will eventually prevail. We show herein that, in melanoma exposed to MAPK-therapeutics, the presence of a neural crest stem cell (NCSC) subpopulation in MRD concurred with the rapid development of resistance through nongenetic mechanisms. Emergence of this drug-tolerant population in MRD relies on a GDNF-dependent autocrine and paracrine signalling cascade, which activates the AKT survival pathway in a Focal-adhesion kinase-(FAK) dependent manner. Ablation of this subpopulation through inhibition of FAK/SRC-signalling delayed relapse in patient-derived tumour xenografts. Strikingly, all tumours that eventually escaped this treatment exhibited resistance-conferring genetic alterations and increased sensitivity to ERK-inhibition. These findings firmly establish that nongenetic reprogramming events contribute to therapy resistance in melanoma and identify a clinically-compatible approach that abrogates such a trajectory. Importantly, these data demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths and highlight key principles that may permit more effective pre-emptive therapeutic interventions.

Efficient production of ethanol for use as a renewable fuel requires organisms with a high level of ethanol tolerance. However, this trait is complex and increased tolerance therefore requires mutations in multiple genes and pathways. Here, we use experimental evolution for a system-level analysis of adaptation of Escherichia coli to high ethanol stress. As adaptation to extreme stress often results in complex mutational data sets consisting of both causal and noncausal passenger mutations, identifying the true adaptive mutations in these settings is not trivial. Therefore, we developed a novel method named IAMBEE (Identification of Adaptive Mutations in Bacterial Evolution Experiments). IAMBEE exploits the temporal profile of the acquisition of mutations during evolution in combination with the functional implications of each mutation at the protein level. These data are mapped to a genome-wide interaction network to search for adaptive mutations at the level of pathways. The 16 evolved populations in our data set together harbored 2,286 mutated genes with 4,470 unique mutations. Analysis by IAMBEE significantly reduced this number and resulted in identification of 90 mutated genes and 345 unique mutations that are most likely to be adaptive. Moreover, IAMBEE not only enabled the identification of previously known pathways involved in ethanol tolerance, but also identified novel systems such as the AcrAB-TolC efflux pump and fatty acids biosynthesis and even allowed to gain insight into the temporal profile of adaptation to ethanol stress. Furthermore, this method offers a solid framework for identifying the molecular underpinnings of other complex traits as well. ispartof: Molecular Biology and Evolution vol:34 issue:11 pages:2927-2943 ispartof: location:United States status: published

In experimental evolution, laboratory-controlled conditions select for the adaptation of species, which can be monitored in real time. Despite the current popularity of such experiments, nature's most pervasive biological force was long believed to be observable only on time scales that transcend a researcher's life-span, and studying evolution by natural selection was therefore carried out solely by comparative means. Eventually, microorganisms' propensity for fast evolutionary changes proved us wrong, displaying strong evolutionary adaptations over a limited time, nowadays massively exploited in laboratory evolution experiments. Here, we formulate a guide to experimental evolution with microorganisms, explaining experimental design and discussing evolutionary dynamics and outcomes and how it is used to assess ecoevolutionary theories, improve industrially important traits, and untangle complex phenotypes. Specifically, we give a comprehensive overview of the setups used in experimental evolution. Additionally, we address population dynamics and genetic or phenotypic diversity during evolution experiments and expand upon contributing factors, such as epistasis and the consequences of (a)sexual reproduction. Dynamics and outcomes of evolution are most profoundly affected by the spatiotemporal nature of the selective environment, where changing environments might lead to generalists and structured environments could foster diversity, aided by, for example, clonal interference and negative frequency-dependent selection. We conclude with future perspectives, with an emphasis on possibilities offered by fast-paced technological progress. This work is meant to serve as an introduction to those new to the field of experimental evolution, as a guide to the budding experimentalist, and as a reference work to the seasoned expert. ispartof: MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS vol:82 issue:3 ispartof: location:United States status: published

CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule (gRNA). For precisely targeting a gene for modification, each genetic construct requires a unique gRNA. By generating a gRNA against the flippase recognition target (FRT) site, a common genetic element shared by multiple genetic collections, CRISPR-FRT circumvents this design constraint to provide a broad platform for fast, scarless, off-the-shelf genome engineering., Genome editing requires precise targeting of loci with specific gRNAs. Here the authors introduce CRISPR-FRT, which targets flippase recognition sites, common in bacterial genetic collections, for fast off-the-shelf genome engineering.

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies. ispartof: Frontiers in Microbiology vol:9 issue:FEB ispartof: location:Switzerland status: published

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 μg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.

Antibiotics typically fail to completely eradicate a bacterial population, leaving a small fraction of transiently antibiotic-tolerant persister cells intact. Persisters are therefore seen to be a major cause of treatment failure and greatly contribute to the recalcitrant nature of chronic infections. The current study focused on Pseudomonas aeruginosa , a Gram-negative pathogen belonging to the notorious ESKAPE group of pathogens ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter species) and, due to increasing resistance against most conventional antibiotics, posing a serious threat to human health. Greatly contributing to the difficult treatment of P. aeruginosa infections is the presence of persister cells, and elimination of these cells would therefore significantly improve patient outcomes. In this study, a small-molecule library was screened for compounds that, in combination with the fluoroquinolone antibiotic ofloxacin, reduced the number of P. aeruginosa persisters compared to the number achieved with treatment with the antibiotic alone. Based on the early structure-activity relationship, 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009) was selected for further characterization. Combination of SPI009 with mechanistically distinct classes of antibiotics reduced the number of persisters up to 10 6 -fold in both lab strains and clinical isolates of P. aeruginosa . Further characterization of the compound revealed a direct and efficient killing of persister cells. SPI009 caused no erythrocyte damage and demonstrated minor cytotoxicity. In conclusion, we identified a novel antipersister compound active against P. aeruginosa with promising applications for the design of novel, case-specific combination therapies in the fight against chronic infections.

While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.22939.001, eLife digest A cell’s DNA can acquire errors over the course of its lifetime. These errors, known as mutations, are often harmful and can cripple the cell. However, some mutations are needed to enable a cell or organism to adapt to changes in its environment. Since there is a trade-off between acquiring beneficial mutations versus harmful ones, cells carefully balance how often they acquire new mutations. Cells have several mechanisms that limit the number of mutations by correcting errors in DNA. Occasionally these repair mechanisms may fail so that a small number of cells in a population accumulate mutations more quickly than other cells. This process is known as “hypermutation” and it enables some cells to rapidly adapt to changing conditions in order to avoid the entire population from becoming extinct. So far, studies on hypermutation have largely been carried out under conditions that are mildly stressful to the cells, which only cause low frequency of hypermutation. However, little is known about the role of this process in cells under near-lethal levels of stress, for example, when drugs target bacteria or cancer cells in the human body. Swings et al. studied hypermutation in populations of a bacterium called Escherichia coli exposed to levels of alcohol that cause the bacteria to experience extreme stress. The experiments show that hypermutation occurs rapidly in these conditions and is essential for bacteria to adapt to the level of alcohol and avoid extinction. Populations of bacteria in which hypermutation did not occur were unable to develop tolerance to the alcohol and perished. Further experiments show that an individual population of bacteria can alter the rate of mutation (that is, how often new mutations arise) several times as a result of changing stress levels. The findings of Swings et al. suggest that hypermutation can rapidly arise in populations of cells that are experiencing extreme stress. Therefore, this process may pose a serious risk in the development of drug resistant bacteria and cancer cells. In the future, developing new drugs that target hypermutation may help to fight bacterial infections and cancer. DOI: http://dx.doi.org/10.7554/eLife.22939.002

In this study, we report the draft genome of Pseudomonas putida BW11M1, a banana rhizosphere isolate producing various antimicrobial compounds, including a lectin-like bacteriocin, an R-type tailocin, the cyclic lipopeptide xantholysin, and the fatty acid–derived pseudopyronine.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies. ispartof: PLoS One vol:11 issue:5 ispartof: location:United States status: published

Conventional fluorescence microscopy studies of the Escherichia coli SeqA protein have shown that it is localized to discrete foci in cells. In this study we have used PALM to determine the localization of SeqA, tagged with fluorescent proteins, with a localization precision of 20 - 30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA-PAmCherry was imaged in wild type E. coli, expressed from plasmid, or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA-PAmCherry was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA-PAmCherry protein numbers and the cell cycle. The numbers of SeqA-PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA-PAmCherry no longer formed foci, was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique.

Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information.Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples. ispartof: Nucleic Acids Research vol:43 issue:16 ispartof: location:England status: published

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20-30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA-PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA-PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA-PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA-PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA-PAmCherry no longer formed foci, and was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique. crosscheck: This document is CrossCheck deposited related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_data: Supplementary Information copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: This article is freely available. This article is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported Licence (CC BY-NC 3.0) history: Received 4 May 2015; Accepted 10 July 2015; Accepted Manuscript published 13 July 2015; Advance Article published 9 October 2015; Version of Record published 12 December 2015 ispartof: Faraday Discussions vol:184 pages:425-450 ispartof: location:England status: published