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.
Experimental evidence is accumulating for the contribution of persister cells to the recalcitrance of chronic infections and the increased development of antibiotic resistance.Target inactivity and dormancy caused by energy loss, halted DNA replication, and blocked translation contribute to persister formation.Target inactivity and dormancy cannot fully explain the complex nature of persister cells because some persisters rely on intrinsic mechanisms to repair antibiotic damage or to lower the intracellular antibiotic concentration.Depending on the persistence effector at play, persisters can have distinct physiologies that contribute to their heterogeneity within bacterial populations.In vitro evolution experiments have shown that persistence is a highly evolvable trait.
Abstract 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.
Fueled by a variety of industrial applications ranging from bioplastics to cosmetics and pharmaceuticals, the global demand for unsaturated fatty acids is steadily rising. Most of these applications ...build on monounsaturated fatty acids with a chain length of 16 or 18 carbon atoms, rendering these compounds valuable industrial assets. While of high industrial interest, monounsaturated fatty acids with a chain length of 8 to 12 carbon atoms are hard to produce using conventional chemical or plant-based production ways. As a consequence, these compounds are expensive and not readily available for large-scale industrial applications. Recent advances in metabolic engineering have put forward microbes as cost-efficient factories to produce numerous chemical compounds. In this respect, the model organism
Escherichia coli
is considered an interesting species as it can grow on various feedstocks and a plethora of genetic information is available, facilitating expression of exogenous enzymes. For the purpose of shifting the fatty acid pool towards monounsaturated fatty acid, thioesterases and desaturases represent suitable candidate enzymes. The former stop chain elongation, reacting on acyl-chains of specific chain length and saturation level, whereas the latter directly target fatty acids to convert them into unsaturated analogues. In this review we summarize thioesterases and desaturases that have been introduced in
E. coli
to enrich unsaturated fatty acids. Furthermore, we discuss advantages of using bacteria for the production of designer compounds including but not limited to medium-chain monounsaturated fatty acids.
Living on a surface: swarming and biofilm formation Verstraeten, Natalie; Braeken, Kristien; Debkumari, Bachaspatimayum ...
Trends in microbiology (Regular ed.),
10/2008, Letnik:
16, Številka:
10
Journal Article
Recenzirano
Swarming is the fastest known bacterial mode of surface translocation and enables the rapid colonization of a nutrient-rich environment and host tissues. This complex multicellular behavior requires ...the integration of chemical and physical signals, which leads to the physiological and morphological differentiation of the bacteria into swarmer cells. Here, we provide a review of recent advances in the study of the regulatory pathways that lead to swarming behavior of different model bacteria. It has now become clear that many of these pathways also affect the formation of biofilms, surface-attached bacterial colonies. Decision-making between rapidly colonizing a surface and biofilm formation is central to bacterial survival among competitors. In the second part of this article, we review recent developments in the understanding of the transition between motile and sessile lifestyles of bacteria.
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
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
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.
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
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.
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.
Bactericidal antibiotics quickly kill the majority of a bacterial population. However, a small fraction of cells typically survive 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.
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
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.
Bacterial persisters constitute a small fraction of cells that transiently display multidrug tolerance, allowing them to survive antibiotic treatment and to establish a new population upon recovery ...from the persistent state. Here, we present a protocol to quantify post-antibiotic persister recovery kinetics and physiological states at the single-cell level. We describe steps for sample preparation, technical setup, and data acquisition using spectrophotometry. Our assay allows for the elucidation of genes and mechanisms involved in persister survival.
For complete details on the use and execution of this protocol, please refer to Wilmaerts et al.1
Display omitted
•Protocol for assessing persister recovery kinetics via spectrophotometry•Detailed steps to obtain persister cells using susceptibility test and time-kill assay•Analysis of physiological states in recovering cells using flow cytometry
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Bacterial persisters constitute a small fraction of cells that transiently display multidrug tolerance, allowing them to survive antibiotic treatment and to establish a new population upon recovery from the persistent state. Here, we present a protocol to quantify post-antibiotic persister recovery kinetics and physiological states at the single-cell level. We describe steps for sample preparation, technical setup, and data acquisition using spectrophotometry. Our assay allows for the elucidation of genes and mechanisms involved in persister survival.
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