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The global interest in fatty acids is steadily rising due to their wealth of industrial potential ranging from cosmetics to biofuels. Unfortunately, certain fatty acids, such as monounsaturated C12, cannot be produced cost and energy-efficiently using conventional methods. Biosynthesis of fatty acids using microorganisms can overcome this drawback. However, rewiring a microbe’s metabolome for increased production remains challenging. To overcome this, sophisticated genome-wide metabolic network models have become available. These models predict the effect of genetic perturbations on the metabolism, thereby serving as a guide for metabolic pathways optimization. In this work, we used constraint-based modeling in combination with the algorithm Optknock to identify gene deletions in Escherichia coli that improve the C12 fatty acid production. Nine gene targets were identified that, when deleted, were predicted to increase C12 titers. Targets play a role in anaplerotic reactions, amino acid synthesis, carbon metabolism and cofactor-balancing. Subsequently, we constructed the corresponding (combinatorial) deletion mutants to validate the in silico predictions in vivo. Our highest producer (Δ maeB Δ ndk Δ pykA) reaches a titer of 6.7 mg/L, corresponding to a 7.5-fold increase in C12 fatty acid production. This study demonstrates that model-guided metabolic engineering is a useful tool to improve C12 fatty acid production.

Nucleic acids research 51(7), 3420 - 3435 (2023). doi:10.1093/nar/gkad127, Obg is a widely conserved and essential GTPase in bacteria, which plays a central role in a large range of important cellular processes, such as ribosome biogenesis, DNA replication, cell division and bacterial persistence. Nevertheless, the exact function of Obg in these processes and the interactions it makes within the associated pathways remain largely unknown. Here, we identify the DNA-binding TrpD2 protein YbiB as an interactor of the Escherichia coli Obg (ObgE). We show that both proteins interact with high affinity in a peculiar biphasic fashion, and pinpoint the intrinsically disordered and highly negatively charged C-terminal domain of ObgE as a main driver for this interaction. Molecular docking and X-ray crystallography, together with site-directed mutagenesis, are used to map the binding site of this ObgE C-terminal domain within a highly positively charged groove on the surface of the YbiB homodimer. Correspondingly, ObgE efficiently inhibits the binding of DNA to YbiB, indicating that ObgE competes with DNA for binding in the positive clefts of YbiB. This study thus forms an important step for the further elucidation of the interactome and cellular role of the essential bacterial protein Obg., Published by Oxford Univ. Press, Oxford

For long, persistence research has focused primarily on disentangling mechanisms of persister state entry. Due to the rapid advances in the field of single-cell techniques and newly obtained insights in the persister phenotype, studying persister awakening has been unlocked and it has gained much interest in the scientific community. However, a framework on how this research should be conducted is currently lacking. Therefore, we here present a method to detect and validate genes important for persister awakening.

To date, we are living in a postantibiotic era in which several human pathogens have developed multidrug resistance and very few new antibiotics are being discovered. In addition to the problem of antibiotic resistance, every bacterial population harbors a small fraction of transiently antibiotic-tolerant persister cells that can survive lethal antibiotic attack. Upon cessation of the treatment, these persister cells wake up and give rise to a new, susceptible population. Studies conducted over the past two decades have demonstrated that persister cells are key players in the recalcitrance of chronic infections and that they contribute to antibiotic resistance development. As a consequence, the scientific interest in persistence has increased tremendously and while some questions remain unanswered, many important insights have been brought to light thanks to the development of dedicated techniques. In this chapter, we provide an overview of well-established methods in the field and recent advances that have facilitated the investigation of persister cells and we highlight the challenges to be tackled in future persistence research.

Bacteria have at their disposal a battery of strategies to withstand antibiotic stress. Among these, resistance is a well-known mechanism, yet bacteria can also survive antibiotic attack by adopting a tolerant phenotype. In the case of persistence, only a small fraction within an isogenic population switches to this antibiotic-tolerant state. Persistence depends on the ecological niche and the genetic background of the strains involved. Furthermore, it has been shown to be under direct and indirect evolutionary pressure. Persister cells play a role in chronic infections and the development of resistance, and therefore a better understanding of this phenotype could contribute to the development of effective antibacterial therapies. In the current review, we discuss how ecological and evolutionary forces shape persistence. ispartof: Trends In Microbiology vol:30 issue:5 pages:1-14 ispartof: location:England status: Published online

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable. For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited. This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

To date, we are living in a postantibiotic era in which several human pathogens have developed multidrug resistance and very few new antibiotics are being discovered. In addition to the problem of antibiotic resistance, every bacterial population harbors a small fraction of transiently antibiotic-tolerant persister cells that can survive lethal antibiotic attack. Upon cessation of the treatment, these persister cells wake up and give rise to a new, susceptible population. Studies conducted over the past two decades have demonstrated that persister cells are key players in the recalcitrance of chronic infections and that they contribute to antibiotic resistance development. As a consequence, the scientific interest in persistence has increased tremendously and while some questions remain unanswered, many important insights have been brought to light thanks to the development of dedicated techniques. In this chapter, we provide an overview of well-established methods in the field and recent advances that have facilitated the investigation of persister cells and we highlight the challenges to be tackled in future persistence research.

For long, persistence research has focused primarily on disentangling mechanisms of persister state entry. Due to the rapid advances in the field of single-cell techniques and newly obtained insights in the persister phenotype, studying persister awakening has been unlocked and it has gained much interest in the scientific community. However, a framework on how this research should be conducted is currently lacking. Therefore, we here present a method to detect and validate genes important for persister awakening.

The Hok/Gef family consists of structurally similar, single-span membrane peptides that all contain a positively charged N-terminal domain, an α-helix and a periplasmic C-terminal domain. Hok/Gef peptides have previously been described to play distinct physiological roles. Indeed, while HokB has been implicated in bacterial persistence, other members of the Hok/Gef family are known to induce cell lysis. However, the generalizability of previously published studies is problematic, as they have all used different expression systems. Therefore, we conducted a systematic study of the nine Hok/Gef peptides of Escherichia coli. We observed rapid cell death following expression of hokA, hokC, hokD, hokE, pndA1, hok or srnB, while expression of hokB or pndA2 does not result in cell lysis. A remarkable feature of Hok/Gef peptides is the presence of conserved periplasmic tyrosine and/or cysteine residues. For the HokB peptide, one of these residues has previously been implicated in intermolecular dimerization, which is essential for HokB to exert its role in persistence. To assess the role of the periplasmic cysteine and tyrosine residues in other Hok/Gef peptides and to decipher if these residues determine peptide toxicity, an array of substitution mutants were constructed. We found that these residues are important activators of toxicity for Hok, HokA and HokE peptides. Despite the loss of the cell killing phenotype in HokS31_Y48, HokAS29_S46 and HokES29_Y46, these peptides do not exert a persister phenotype. More research is needed to fully comprehend why HokB is the sole peptide of the Hok/Gef family that mediates persistence. ispartof: Fems Microbiology Letters vol:368 issue:11 ispartof: location:England status: Published online

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*&ndash, GTP, but not ObgE*&ndash, GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function.

Bacterial proliferation depends on the cells' capability to proceed through consecutive rounds of the cell cycle. The cell cycle consists of a series of events during which cells grow, copy their genome, partition the duplicated DNA into different cell halves and, ultimately, divide to produce two newly formed daughter cells. Cell cycle control is of the utmost importance to maintain the correct order of events and safeguard the integrity of the cell and its genomic information. This review covers insights into the regulation of individual key cell cycle events in Escherichia coli. The control of initiation of DNA replication, chromosome segregation and cell division is discussed. Furthermore, we highlight connections between these processes. Although detailed mechanistic insight into these connections is largely still emerging, it is clear that the different processes of the bacterial cell cycle are coordinated to one another. This careful coordination of events ensures that every daughter cell ends up with one complete and intact copy of the genome, which is vital for bacterial survival.

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

Obg is a versatile GTPase that plays a pivotal role in bacterial persistence. We previously showed that the Escherichia coli homolog ObgE exerts this activity through transcriptional activation of a toxin-antitoxin module and subsequent membrane depolarization. Here, we assessed the role of G-domain functionality in ObgE-mediated persistence. Through screening of a mutant library, we identified five obgE alleles (with substitutions G166V, D246G, S270I, N283I and I313N) that have lost their persistence function and no longer activate hokB expression. These alleles support viability of a strain otherwise deprived of ObgE, indicating that ObgE's persistence function can be uncoupled from its essential role. Based on the ObgE crystal structure, we designed two additional mutant proteins (T193A and D286Y), one of which (D286Y) no longer affects persistence. Using isothermal titration calorimetry, stopped-flow experiments and kinetics, we subsequently assessed nucleotide binding and GTPase activity in all mutants. With the exception of the S270I mutant that is possibly affected in protein-protein interactions, all mutants that have lost their persistence function display severely reduced binding to GDP or the alarmone ppGpp. However, we find no clear relation between persistence and GTP or pppGpp binding nor with GTP hydrolysis. Combined, our results signify an important step toward understanding biochemical determinants underlying persistence. ispartof: MOLECULAR MICROBIOLOGY vol:112 issue:5 pages:1593-1608 ispartof: location:England status: Published online

Every bacterial population harbors a small subpopulation of so-called persisters that are transiently antibiotic tolerant. These persisters are associated with the recalcitrance of chronic infections because they can recolonize the host after antibiotic removal. Although several effectors have been described to induce persistence, persister cell awakening is poorly understood. We previously reported that the toxin HokB induces persistence via pore formation, resulting in membrane depolarization and ATP leakage. We now delineate mechanisms responsible for the awakening of HokB-induced persisters. We show that HokB dimerization by the oxidoreductase DsbA is essential for pore formation and peptide stability. Pores are disassembled via DsbC-mediated monomerization, which targets HokB for DegQ-mediated degradation. Finally, pore disassembly allows membrane repolarization by the electron transport chain, supporting cells to resume growth. These results provide a detailed view of both the formation and awakening of HokB-induced persister cells. ispartof: MOLECULAR CELL vol:75 issue:5 pages:1031-1042 ispartof: location:United States status: published

All bacterial populations harbor a small fraction of transiently antibiotic-tolerant cells called persisters. These phenotypic variants compromise successful antibiotic treatment because they are held responsible for the relapse of many chronic infections. In addition, studies employing experimental evolution have demonstrated that persistence contributes to the development of antibiotic resistance. Persisters are typically described as dormant cells. However, recent findings indicate a role for active mechanisms in the formation and maintenance of the persister phenotype. This review summarizes novel insights into the molecular mechanisms of persister formation and awakening, focusing on changes in cell physiology mediated by persistence effectors. ispartof: Trends In Genetics vol:35 issue:6 pages:401-411 ispartof: location:England status: published

Persisters comprise a small fraction of cells within a bacterial population that transiently are tolerant to lethal doses of antibiotics. Following their discovery, persister cells went unheeded for nearly 40 years until Moyed and Bertrand revived the field of persister research in 1983. Ever since, an increasing body of literature has reported on genetic determinants of persistence. We here present a comprehensive overview of all currently known genes affecting persistence in Escherichia coli. We systematically group persister genes according to the biological processes they are involved in, more specifically a variety of stress responses and energy metabolism. We also briefly touch upon the role of toxin-antitoxin systems in persistence. In general, persister levels are positively correlated with expression levels of genes that yield protection against nutrient stress (e.g., dksA, relA), DNA damage (e.g., recA, lexA, umuDC), heat shock (e.g., dnaJ, dnaK), or oxidative stress (e.g., soxS, oxyR). This underlines the importance of these stress responses in the formation of persister cells. However, both elevated and decreased persister levels are found upon impeding the general stress response and energy metabolism, emphasizing the need for further research. Combined with additional persister genes that undoubtedly await discovery, the information presented in this work will support the development of new persister models that will in turn greatly contribute to our understanding of this intriguing phenomenon.

We previously synthesized several series of compounds, based on the 5-aryl-2-aminoimidazole scaffold, that showed activity preventing the formation of Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa biofilms. Here, we further studied the activity spectrum of a number of the most active N 1- and 2 N -substituted 5-aryl-2-aminoimidazoles against a broad panel of biofilms formed by monospecies and mixed species of bacteria and fungi. An N 1-substituted compound showed very strong activity against the biofilms formed by Gram-negative and Gram-positive bacteria and the fungus Candida albicans but was previously shown to be toxic against various eukaryotic cell lines. In contrast, 2 N -substituted compounds were nontoxic and active against biofilms formed by Gram-negative bacteria and C. albicans but had reduced activity against biofilms formed by Gram-positive bacteria. In an attempt to develop nontoxic compounds with potent activity against biofilms formed by Gram-positive bacteria for application in antibiofilm coatings for medical implants, we synthesized novel compounds with substituents at both the N 1 and 2 N positions and tested these compounds for antibiofilm activity and toxicity. Interestingly, most of these N 1-,2 N -disubstituted 5-aryl-2-aminoimidazoles showed very strong activity against biofilms formed by Gram-positive bacteria and C. albicans in various setups with biofilms formed by monospecies and mixed species but lost activity against biofilms formed by Gram-negative bacteria. In light of application of these compounds as anti-infective coatings on orthopedic implants, toxicity against two bone cell lines and the functionality of these cells were tested. The N 1-,2 N -disubstituted 5-aryl-2-aminoimidazoles in general did not affect the viability of bone cells and even induced calcium deposition. This indicates that modulating the substitution pattern on positions N 1 and 2 N of the 5-aryl-2-aminoimidazole scaffold allows fine-tuning of both the antibiofilm activity spectrum and toxicity.

Any bacterial population harbors a small number of phenotypic variants that survive exposure to high concentrations of antibiotic. Importantly, these so-called 'persister cells' compromise successful antibiotic therapy of bacterial infections and are thought to contribute to the development of antibiotic resistance. Intriguingly, drug-tolerant persisters have also been identified as a factor underlying failure of chemotherapy in tumor cell populations. Recent studies have begun to unravel the complex molecular mechanisms underlying persister formation and revolve around stress responses and toxin-antitoxin modules. Additionally, in vitro evolution experiments are revealing insights into the evolutionary and adaptive aspects of this phenotype. Furthermore, ever-improving experimental techniques are stimulating efforts to investigate persisters in their natural, infection-associated, in vivo environment. This review summarizes recent insights into the molecular mechanisms of persister formation, explains how persisters complicate antibiotic treatment of infections, and outlines emerging strategies to combat these tolerant cells.

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.

Bacterial populations harbor a small fraction of cells that display transient multidrug tolerance. These so-called persister cells are extremely difficult to eradicate and contribute to the recalcitrance of chronic infections. Several signaling pathways leading to persistence have been identified. However, it is poorly understood how the effectors of these pathways function at the molecular level. In a previous study, we reported that the conserved GTPase Obg induces persistence in Escherichia coli via transcriptional upregulation of the toxin HokB. In the present study, we demonstrate that HokB inserts in the cytoplasmic membrane where it forms pores. The pore-forming capacity of the HokB peptide is demonstrated by in vitro conductance measurements on synthetic and natural lipid bilayers, revealing an asymmetrical conductance profile. Pore formation is directly linked to persistence and results in leakage of intracellular ATP. HokB-induced persistence is strongly impeded in the presence of a channel blocker, thereby providing a direct link between pore functioning and persistence. Furthermore, the activity of HokB pores is sensitive to the membrane potential. This sensitivity presumably results from the formation of either intermediate or mature pore types depending on the membrane potential. Taken together, these results provide a detailed view on the mechanistic basis of persister formation through the effector HokB., IMPORTANCE There is increasing awareness of the clinical importance of persistence. Indeed, persistence is linked to the recalcitrance of chronic infections, and evidence is accumulating that persister cells constitute a pool of viable cells from which resistant mutants can emerge. Unfortunately, persistence is a poorly understood process at the mechanistic level. In this study, we unraveled the pore-forming activity of HokB in E. coli and discovered that these pores lead to leakage of intracellular ATP, which is correlated with the induction of persistence. Moreover, we established a link between persistence and pore activity, as the number of HokB-induced persister cells was strongly reduced using a channel blocker. The latter opens opportunities to reduce the number of persister cells in a clinical setting.

In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli / C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the β-1,3-glucan-degrading enzyme lyticase. In line with a role for β-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-β-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-β-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that β-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.

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.

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

The Obg protein family belongs to the TRAFAC (translation factor) class of P-loop GTPases and is conserved from bacteria to eukaryotes. Essential roles in many different cellular processes have been suggested for the Obg protein from Escherichia coli (ObgE), and we recently showed that it is a central regulator of bacterial persistence. Here, we report the first crystal structure of ObgE at 1.85 Å resolution in the GDP-bound state, showing the characteristic N-terminal domain and a central G domain that are common to all Obg proteins. ObgE also contains an intrinsically disordered C-terminal domain, and we show here that this domain specifically contributed to GTP binding, while it did not influence GDP binding or GTP hydrolysis. Biophysical analysis, using small angle X-ray scattering and multi-angle light scattering experiments, revealed that ObgE is a monomer in solution, regardless of the bound nucleotide. In contrast to recent suggestions, our biochemical analyses further indicate that ObgE is neither activated by K+ ions nor by homodimerization. However, the ObgE GTPase activity was stimulated upon binding to the ribosome, confirming the ribosome-dependent GTPase activity of the Obg family. Combined, our data represent an important step toward further unraveling the detailed molecular mechanism of ObgE, which might pave the way to further studies into how this GTPase regulates bacterial physiology, including persistence. ispartof: Journal of Biological Chemistry vol:292 issue:14 pages:5871-5883 ispartof: location:United States status: published

The spread of antibiotic resistance and the challenges associated with antiseptics such as chlorhexidine have necessitated a search for new antibacterial agents against oral bacterial pathogens. As a result of failing traditional approaches, drug repurposing has emerged as a novel paradigm to find new antibacterial agents. In this study, we examined the effects of the FDA-approved anticancer agent toremifene against the oral bacteria Porphyromonas gingivalis and Streptococcus mutans . We found that the drug was able to inhibit the growth of both pathogens, as well as prevent biofilm formation, at concentrations ranging from 12.5 to 25 μM. Moreover, toremifene was shown to eradicate preformed biofilms at concentrations ranging from 25 to 50 μM. In addition, we found that toremifene prevents P. gingivalis and S. mutans biofilm formation on titanium surfaces. A time-kill study indicated that toremifene is bactericidal against S. mutans . Macromolecular synthesis assays revealed that treatment with toremifene does not cause preferential inhibition of DNA, RNA, or protein synthesis pathways, indicating membrane-damaging activity. Biophysical studies using fluorescent probes and fluorescence microscopy further confirmed the membrane-damaging mode of action. Taken together, our results suggest that the anticancer agent toremifene is a suitable candidate for further investigation for the development of new treatment strategies for oral bacterial infections.

Oral infections are among the most common diseases worldwide. Many protocols for the prevention and treatment of oral infections have been described, yet no golden standard has been developed so far. The antiseptic chlorhexidine and antibiotics are often used in these treatment procedures. However, long-term use of chlorhexidine can lead to side effects and extensive use of antibiotics can promote the development of antibiotic-resistant bacteria, which in turn can compromise the effectiveness of the treatment. Consequently, it remains important to search for new antibacterial agents for the treatment of oral infections. In this study, we report on the antibacterial activity of the anti-asthma drug zafirlukast against oral pathogens Porphyromonas gingivalis and Streptococcus mutans. Furthermore, its activity against oral biofilms grown on titanium surfaces was confirmed. In addition, we demonstrated that zafirlukast displays no cytotoxicity against human osteoblasts. Combined, this study paves the way for further research to determine the potential of zafirlukast to be used as a new antibiotic against oral pathogens. ispartof: FEMS Microbiology Letters vol:364 issue:2 ispartof: location:England status: published

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. ispartof: Journal of Orthopaedic Research vol:34 issue:12 pages:2191-2198 ispartof: location:United States status: published

Persister cells display tolerance to high doses of bactericidal antibiotics and typically comprise a small fraction of a bacterial population. Recently, evidence was provided for a causal link between therapy failure and the presence of persister cells in chronic infections, underscoring the need for research on bacterial persistence. A series of recent breakthroughs have shed light on the multiplicity of persister genes, the contribution of gene expression noise to persister formation, the importance of active responses to antibiotic tolerance and heterogeneity among persister cells. Moreover, the development of in vivo model systems has highlighted the clinical relevance of persistence. This review discusses these recent advances and how this knowledge fundamentally changes the way in which we will perceive the problem of antibiotic tolerance in years to come. ispartof: Trends in Microbiology vol:20 issue:12 pages:577-585 ispartof: location:England status: published

Porphyromonas gingivalis is a major pathogen involved in oral diseases such as periodontitis and peri‐implantitis. Management of these diseases typically includes mechanical debridement of the colonized surfaces followed by application of antiseptics or antibiotics. Disadvantages associated with the use of antiseptics and the growing worldwide problem of antibiotic resistance have necessitated the search for alternative agents. In this study, the antibacterial and antibiofilm properties of AM404, an active metabolite of paracetamol, were tested against P. gingivalis and other bacterial pathogens. The activity of AM404 was tested against 10 bacteria, including both oral and nonoral human pathogens. The minimal inhibitory concentration (MIC) of AM404 was determined by measuring optical density (OD) values. The minimum biofilm inhibitory concentration (MBIC) was detected by crystal violet staining. The activity of structural analogs of AM404 was tested by MIC determinations. The effect of AM404 on P. gingivalis biofilms formed on titanium disks as a model for dental implants was evaluated by colony forming unit counting. Potential cytotoxicity of AM404 towards HEK‐293 (human embryonic kidney cells), HepG2 (human hepatoma cells), IEC‐6 (rat intestinal cells), and Panc‐1 cells (pancreatic cancer cells) was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays. To get more insight in the mode of action of AM404, we used the fluorescent dyes N‐phenyl‐1‐napthylamine and SYTOX green to investigate outer and inner membrane damage of P. gingivalis induced by AM404, respectively. Of all tested pathogens, AM404 only inhibited growth and biofilm formation of P. gingivalis. Moreover, it showed potent activity against P. gingivalis biofilms formed on titanium surfaces. A structure–activity analysis demonstrated that the unsaturated carbon chain is essential for its antibacterial activity. Importantly, AM404 was not toxic towards the tested mammalian cells up to concentrations approaching 4× the MIC. Membrane damage assays using fluorescent probes N‐phenyl‐1‐napthylamine and SYTOX green revealed that membrane permeabilization presumably is the primary antibacterial mode of action of AM404. Collectively, our results suggest that AM404 has the potential to be used for the development of new drugs specifically targeting P. gingivalis‐related infections.

The Obg protein family belongs to the TRAFAC (translation factor) class of P-loop GTPases and is conserved from bacteria to eukaryotes. Essential roles in many different cellular processes have been suggested for the Obg protein from Escherichia coli (ObgE), and we recently showed that it is a central regulator of bacterial persistence. Here, we report the first crystal structure of ObgE at 1.85-Å resolution in the GDP-bound state, showing the characteristic N-terminal domain and a central G domain that are common to all Obg proteins. ObgE also contains an intrinsically disordered C-terminal domain, and we show here that this domain specifically contributed to GTP binding, whereas it did not influence GDP binding or GTP hydrolysis. Biophysical analysis, using small angle X-ray scattering and multi-angle light scattering experiments, revealed that ObgE is a monomer in solution, regardless of the bound nucleotide. In contrast to recent suggestions, our biochemical analyses further indicate that ObgE is neither activated by K+ ions nor by homodimerization. However, the ObgE GTPase activity was stimulated upon binding to the ribosome, confirming the ribosome-dependent GTPase activity of the Obg family. Combined, our data represent an important step toward further unraveling the detailed molecular mechanism of ObgE, which might pave the way to further studies into how this GTPase regulates bacterial physiology, including persistence.

Programmed cell death (PCD) in bacteria is considered an important target for developing novel antimicrobials. Development of PCD-specific therapies requires a deeper understanding of what drives this process. We recently discovered a new mode of PCD in Escherichia coli that is triggered by expression of a mutant isoform of the essential ObgE protein, ObgE*. Our previous findings demonstrate that ObgE*-mediated cell death shares key characteristics with apoptosis in eukaryotic cells. It is well-known that reactive oxygen species (ROS) are formed during PCD in eukaryotes and play a pivotal role as signaling molecules in the progression of apoptosis. Therefore, we explored a possible role for ROS in bacterial killing by ObgE*. Using fluorescent probes and genetic reporters, we found that expression of ObgE* induces formation of ROS. Neutralizing ROS by chemical scavenging or by overproduction of ROS-neutralizing enzymes did not influence toxicity of ObgE*. Moreover, expression of ObgE* under anaerobic conditions proved to be as detrimental to bacterial viability as expression under aerobic conditions. In conclusion, ROS are byproducts of ObgE* expression that do not play a role in the execution or progression of ObgE*-mediated PCD. Targeted therapies should therefore look to exploit other aspects of ObgE*-mediated PCD. ispartof: Scientific Reports vol:6 issue:1 ispartof: location:England status: published

The phenomenon of programmed cell death (PCD), in which cells initiate their own demise, is not restricted to multicellular organisms. Unicellular organisms, both eukaryotes and prokaryotes, also possess pathways that mediate PCD. We recently identified a PCD mechanism in Escherichia coli that is triggered by a mutant isoform of the essential GTPase ObgE (Obg of E. coli). Importantly, the PCD pathway mediated by mutant Obg (Obg*) differs fundamentally from other previously described bacterial PCD pathways and thus constitutes a new mode of PCD. ObgE was previously proposed to act as a cell cycle checkpoint protein able to halt cell division. The implication of ObgE in the regulation of PCD further increases the similarity between this protein and eukaryotic cell cycle regulators that are capable of doing both. Moreover, since Obg is conserved in eukaryotes, the elucidation of this cell death mechanism might contribute to the understanding of PCD in higher organisms. Additionally, if Obg*-mediated PCD is conserved among different bacterial species, it will be a prime target for the development of innovative antibacterials that artificially induce this pathway. ispartof: Microbial Cell vol:3 issue:6 pages:255-256 ispartof: location:Austria status: published

The evolution of antibiotic resistance is a major threat to society and has been predicted to lead to 10 million casualties annually by 2050(1). Further aggravating the problem, multidrug tolerance in bacteria not only relies on the build-up of resistance mutations, but also on some cells epigenetically switching to a non-growing antibiotic-tolerant 'persister' state(2-6). Yet, despite its importance, we know little of how persistence evolves in the face of antibiotic treatment(7). Our evolution experiments in Escherichia coli demonstrate that extremely high levels of multidrug tolerance (20-100%) are achieved by single point mutations in one of several genes and readily emerge under conditions approximating clinical, once-daily dosing schemes. In contrast, reversion to low persistence in the absence of antibiotic treatment is relatively slow and only partially effective. Moreover, and in support of previous mathematical models(8-10), we show that bacterial persistence quickly adapts to drug treatment frequency and that the observed rates of switching to the persister state can be understood in the context of 'bet-hedging' theory. We conclude that persistence is a major component of the evolutionary response to antibiotics that urgently needs to be considered in both diagnostic testing and treatment design in the battle against multidrug tolerance.

Bactericidal antibiotics quickly kill the majority of a bacterial population. However, a small fraction of cells typically survives through entering the so-called persister state. Persister cells are increasingly being viewed as a major cause of the recurrence of chronic infectious disease and could be an important factor in the emergence of antibiotic resistance. The phenomenon of persistence was first described in the 1940s, but remained poorly understood for decades afterwards. Only recently, a series of breakthrough discoveries has started to shed light on persister physiology and the molecular and genetic underpinnings of persister formation. We here provide an overview of the key studies that have paved the way for the current boom in persistence research, with a special focus on the technological and methodological advances that have enabled this progress. ispartof: Bacterial Persistence pages:3-13 ispartof: Methods in Molecular Biology vol:1333 pages:3-13 ispartof: location:United States status: published

Objectives: Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. Methods: Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. Results: In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. Conclusions: These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Obg proteins are universally conserved GTP-binding proteins that are essential for viability in bacteria. Homologs in different organisms are involved in various cellular processes, including DNA replication. The goal of this study was to analyse the structure–function relationship of Escherichia coli ObgE with regard to DNA replication in general and sensitivity to stalled replication forks in particular. Defined C-terminal chromosomal deletion mutants of obgE were constructed and tested for sensitivity to the replication inhibitor hydroxyurea. The ObgE C-terminal domain was shown to be dispensable for normal growth of E. coli. However, a region within this domain is involved in the cellular response to replication fork stress. In addition, a mutant obgE over-expression library was constructed by error-prone PCR and screened for increased hydroxyurea sensitivity. ObgE proteins with substitutions L159Q, G163V, P168V, G216A or R237C, located within distinct domains of ObgE, display dominant-negative effects leading to hydroxyurea hypersensitivity when over-expressed. These effects are abolished in strains with a single deletion of the iron transporter TonB or combined deletions the toxin/antitoxin modules RelBE/MazEF, strains both of which have been shown to be involved in a pathway that stimulates hydroxyl radical formation following hydroxyurea treatment. Moreover, the observed dominant-negative effects are lost in the presence of the hydroxyl radical scavenger thiourea. Together, these results indicate involvement of hydroxyl radical toxicity in ObgE-mediated protection against replication fork stress.