Experimental Evolution Research Articles

A Lack of Mass-gap Compact Object Binaries in APOGEE

Depending principally on mass, the compact object remnant left behind after a star’s life may be a white dwarf, neutron star (NS), or black hole (BH). While we have large samples of each of these remnants, we lack knowledge of the exact conditions separating these outcomes. The boundary between low-mass BHs and massive NSs is particularly poorly understood, as few objects between 2 and 5 M ⊙ are known. To probe this regime, we search the Apache Point Observatory Galactic Evolution Experiment 17th Data Release data set of 657,000 unique stars for binary systems with one stellar component that exhibit large radial velocity shifts over multiple observations. We identify 4751 likely binary systems, and estimate a minimum mass for each system’s “invisible companion” under the assumption of tidal synchronization. Two systems have companion masses ≳2 M ⊙, although we conclude that neither are good candidates for possessing a mass-gap NS or BH companions.

Widespread fungal-bacterial competition for magnesium lowers bacterial susceptibility to polymyxin antibiotics.

Fungi and bacteria coexist in many polymicrobial communities, yet the molecular basis of their interactions remains poorly understood. Here, we show that the fungus Candida albicans sequesters essential magnesium ions from the bacterium Pseudomonas aeruginosa. To counteract fungal Mg2+ sequestration, P. aeruginosa expresses the Mg2+ transporter MgtA when Mg2+ levels are low. Thus, loss of MgtA specifically impairs P. aeruginosa in co-culture with C. albicans, but fitness can be restored by supplementing Mg2+. Using a panel of fungi and bacteria, we show that Mg2+ sequestration is a general mechanism of fungal antagonism against gram-negative bacteria. Mg2+ limitation enhances bacterial resistance to polymyxin antibiotics like colistin, which target gram-negative bacterial membranes. Indeed, experimental evolution reveals that P. aeruginosa evolves C. albicans-dependent colistin resistance via non-canonical means; antifungal treatment renders resistant bacteria colistin-sensitive. Our work suggests that fungal-bacterial competition could profoundly impact polymicrobial infection treatment with antibiotics of last resort.

Experimental evolution of an RNA virus in Caenorhabditis elegans

The discovery of Orsay virus (OrV), the first virus infecting wild populations of Caenorhabditis elegans, has boosted studies of viral immunity pathways in this nematode. Considering the many advantages that C. elegans offers for fundamental research in host-pathogen interactions, this pathosystem has high potential to become a model system for experimental virus evolution studies. However, the evolutionary constraints – i.e, the balance between genetic variation, selection, drift and historical contingency- operating in this pathosystem have barely been explored. Here we describe for the first time an evolution experiment of two different OrV strains in C. elegans. Comparison of the two ancestral strains showed differences in infectivity and sequence, and highlighted the importance of consistently normalize viral inocula for meaningful comparisons among strains. After 10 serial passages of evolution, we report slight changes in infectivity and non-synonymous mutations fixed in the evolved viral populations. In addition, we observed numerous minor variants emerging in the viral population. These minor variants were not randomly distributed along the genome but concentrated in polymorphic genomic regions. Overall, our work established the grounds for future experimental virus evolution studies using Caenorhabditis nematodes.

Biotic interactions promote local adaptation to soil in plants

Although different ecological factors shape adaptative evolution in natural habitats, we know little about how their interactions impact local adaptation. Here we used eight generations of experimental evolution with outcrossing Brassica rapa plants as a model system, in eight treatment groups that varied in soil type, herbivory (with/without aphids), and pollination mode (hand- or bumblebee-pollination), to study how biotic interactions affect local adaptation to soil. First, we show that several plant traits evolved in response to biotic interactions in a soil-specific way. Second, using a reciprocal transplant experiment, we demonstrate that significant local adaptation to soil-type evolved in the “number of open flowers”, a trait used as a fitness proxy, but only in plants that evolved with herbivory and bee pollination. Whole genome re-sequencing of experimental lines revealed that biotic interactions caused a 10-fold increase in the number of SNPs across the genome with significant allele frequency change, and that alleles with opposite allele frequency change in different soil types (antagonistic pleiotropy) were most common in plants with an evolutionary history of herbivory and bee pollination. Our results demonstrate that the interaction with mutualists and antagonists can facilitate local adaptation to soil type through antagonistic pleiotropy.

Experimental Evolution Studies in Φ6 Cystovirus.

Experimental evolution studies, in which biological populations are evolved in a specific environment over time, can address questions about the nature of spontaneous mutations, responses to selection, and the origins and maintenance of novel traits. Here, we review more than 30 years of experimental evolution studies using the bacteriophage (phage) Φ6 cystovirus. Similar to many lab-studied bacteriophages, Φ6 has a high mutation rate, large population size, fast generation time, and can be genetically engineered or cryogenically frozen, which facilitates its rapid evolution in the laboratory and the subsequent characterization of the effects of its mutations. Moreover, its segmented RNA genome, outer membrane, and capacity for multiple phages to coinfect a single host cell make Φ6 a good non-pathogenic model for investigating the evolution of RNA viruses that infect humans. We describe experiments that used Φ6 to address the fitness effects of spontaneous mutations, the consequences of evolution in the presence of coinfection, the evolution of host ranges, and mechanisms and consequences of the evolution of thermostability. We highlight open areas of inquiry where further experimentation on Φ6 could inform predictions for pathogenic viruses.

The corrosion resistance and anti-bacterial performance of polyelectrolyte-embedded Ca-P coating loaded with ciprofloxacin on magnesium alloy

Polyacrylic acid (PAA) is utilized as the template for the hydrothermal fabrication of Ca-P coatings on biodegradable magnesium (Mg) alloys, and ciprofloxacin (CIP) is also adopted for the coating's anti-bacterial performance. The coating's composition and structure are characterized. The anti-bacterial performance of the coating is evaluated using plate counting method against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The corrosion rates of different samples are measured by electrochemical polarization, electrochemical impedance spectroscopy (EIS), and hydrogen evolution (HE) experiments. The long-term release behavior of the loaded antibiotics is analyzed through UV–vis. The results show that the obtained coating would display corrosion protection, anti-bacterial effect, and acceptable adhesive force, verifying that this drug-loaded coating effectively overcomes the limitations of Mg alloys in the biological environment. The biocompatibility of the coating is also characterized using the CCK-8 method. Finally, the role of the PAA in the structural transformation of the coating is explained based on the molecular dynamics (MD) simulation and this method seems to open a new pathway for understanding the reconstructing effect of the templates inside a hydrothermally synthesized coating.

Escherichia coli adaptation under prolonged resource exhaustion is characterized by extreme parallelism and frequent historical contingency.

Like many other non-sporulating bacterial species, Escherichia coli is able to survive prolonged periods of resource exhaustion, by entering a state of growth called long-term stationary phase (LTSP). In July 2015, we initiated a set of evolutionary experiments aimed at characterizing the dynamics of E. coli adaptation under LTSP. In these experiments populations of E. coli were allowed to initially grow on fresh rich media, but where not provided with any new external growth resources since their establishment. Utilizing whole genome sequencing data obtained for hundreds of clones sampled at 12 time points spanning the first six years of these experiments, we reveal several novel aspects of the dynamics of adaptation. First, we show that E. coli continuously adapts genetically, up to six years under resource exhaustion, through the highly convergent accumulation of mutations. We further show that upon entry into LTSP, long-lasting lineages are established. This lineage structure is in itself convergent, with similar lineages arising across independently evolving populations. The high parallelism with which adaptations occur under LTSP, combined with the LTSP populations' lineage structure, enable us to screen for pairs of loci displaying a significant association in the occurrence of mutations, suggestive of a historical contingency. We find that such associations are highly frequent and that a third of convergently mutated loci are involved in at least one such association. Combined our results demonstrate that LTSP adaptation is characterized by remarkably high parallelism and frequent historical contingency.

Evolution of polyamine resistance in Staphylococcus aureus through modulation of potassium transport.

Microbes must adapt to diverse biotic and abiotic factors encountered in host environments. Polyamines are an abundant class of aliphatic molecules that play essential roles in fundamental cellular processes across the tree of life. Surprisingly, the bacterial pathogen Staphylococcus aureus is highly sensitive to polyamines encountered during infection, and acquisition of a polyamine resistance locus has been implicated in spread of the prominent USA300 methicillin-resistant S. aureus lineage. At present, alternative pathways of polyamine resistance in staphylococci are largely unknown. Here we applied experimental evolution to identify novel mechanisms and consequences of S. aureus adaption when exposed to increasing concentrations of the polyamine spermine. Evolved populations of S. aureus exhibited striking evidence of parallel adaptation, accumulating independent mutations in the potassium transporter genes ktrA and ktrD. Mutations in either ktrA or ktrD are sufficient to confer polyamine resistance and function in an additive manner. Moreover, we find that ktr mutations provide increased resistance to multiple classes of unrelated cationic antibiotics, suggesting a common mechanism of resistance. Consistent with this hypothesis, ktr mutants exhibit alterations in cell surface charge indicative of reduced affinity and uptake of cationic molecules. Finally, we observe that laboratory-evolved ktr mutations are also present in diverse natural S. aureus isolates, suggesting these mutations may contribute to antimicrobial resistance during human infections. Collectively this study identifies a new role for potassium transport in S. aureus polyamine resistance with consequences for susceptibility to both host-derived and clinically-used antimicrobials.

Synthesis of mixed-ligand two-dimensional zeolite imidazole framework (ZIF-L) via water-assisted strategy for enhanced photocatalytic hydrogen evolution

Metal organic frameworks (MOFs) are regarded as the promising potential photocatalytic materials because of its high specific surface area, regular morphology and highly adjustable structure. Herein, introducing different addition percentage of 1-vinylimidazole to two-dimensional zeolite imidazole framework (ZIF-L) with leaf-shaped morphology was successfully prepared via water-assisted strategy and applied in the photocatalytic hydrogen evolution experiments. The results indicate that VZ-12 shows the highest photocatalytic activity with hydrogen production rate of 162.7 μmol g−1 h−1, which is 3 times than that in VZ-0 (pure ZIF-L, 56.33 μmol g−1 h−1) under simulated sunlight. The enhanced performance is ascribed to the increased specific surface area observed via BET, narrow band gap (Eg) explored through UV–vis DRS and decreased recombination of photo-generated charge carriers measured by photoluminescence (PL). In a word, the effort of our study provides a new idea for developing and fabricating efficient hydrogen production photocatalyst via water splitting.

Boosting hydrogen production in Rhodospirillum rubrum by syngas-driven photoheterotrophic adaptive evolution

Rhodospirillum rubrum is a photosynthetic purple non-sulphur bacterium with great potential to be used for complex waste valorisation in biotechnological applications due to its metabolic versatility. This study investigates the production of hydrogen (H2) and polyhydroxyalkanoates (PHA) by R. rubrum from syngas under photoheterotrophic conditions. An adaptive laboratory evolution strategy (ALE) has been carried out to improve the yield of the process. After 200 generations, two evolved strains were selected that showed reduced lag phase and enhanced poly-3-hydroxybutyrate (PHB) and H2 synthesis compared to the parental strain. Genomic analysis of the photo-adapted (PA) variants showed four genes with single point mutations, including the photosynthesis gene expression regulator PpsR. The proteome of the variants suggested that the adapted variants overproduced H2 due to a more efficient CO oxidation through the CO-dehydrogenase enzyme complex and confirmed that energy acquisition was enhanced through overexpression of the photosynthetic system and metal cofactors essential for pigment biosynthesis.

Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named “LAbrini”, exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain “LAbrini” to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.

State of the engineered barrier system and host rock of the HE-E experiment after 12 years of heating

This paper presents the status of the HE-E heater experiment, which is currently running at the Mont Terri Underground Research Laboratory (URL) in Switzerland. The experiment is located in a 10 m-long section of a micro-tunnel in the shaly facies of the Opalinus Clay and includes two 4 m-long heater sections isolated by plugs. Heater 1 is located in a sand/bentonite (65/35) buffer section and Heater 2 is located in a granular bentonite buffer section. Heating started on 28 June 2011, and this paper reports results up to 30 June 2023. During the 12 years of heating the system has otherwise been left undisturbed, with ongoing but very slow resaturation from the Opalinus Clay. The observed response to heating is considered in two phases: (1) the initial transient period of about 500 days when heater temperatures rose to the target 140°C and (2) the subsequent evolution. Recently a novel telescoped drilling procedure has been performed to recover and characterize material from the bentonite buffer without interruption of the heating. This paper describes the experimental long-term evolution and status prior to this disturbance, together with expectations regarding the current state of the buffer.

Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs.

There is growing interest in designing multidrug therapies that leverage tradeoffs to combat resistance. Tradeoffs are common in evolution and occur when, for example, resistance to one drug results in sensitivity to another. Major questions remain about the extent to which tradeoffs are reliable, specifically, whether the mutants that provide resistance to a given drug all suffer similar tradeoffs. This question is difficult because the drug-resistant mutants observed in the clinic, and even those evolved in controlled laboratory settings, are often biased towards those that provide large fitness benefits. Thus, the mutations (and mechanisms) that provide drug resistance may be more diverse than current data suggests. Here, we perform evolution experiments utilizing lineage-tracking to capture a fuller spectrum of mutations that give yeast cells a fitness advantage in fluconazole, a common antifungal drug. We then quantify fitness tradeoffs for each of 774 evolved mutants across 12 environments, finding these mutants group into 6 classes with characteristically different tradeoffs. Their unique tradeoffs may imply that each group of mutants affects fitness through different underlying mechanisms. Some of the groupings we find are surprising. For example, we find some mutants that resist single drugs do not resist their combination, while others do. And some mutants to the same gene have different tradeoffs than others. These findings, on one hand, demonstrate the difficulty in relying on consistent or intuitive tradeoffs when designing multidrug treatments. On the other hand, by demonstrating that hundreds of adaptive mutations can be reduced to a few groups with characteristic tradeoffs, our findings may yet empower multidrug strategies that leverage tradeoffs to combat resistance. More generally speaking, by grouping mutants that likely affect fitness through similar underlying mechanisms, our work guides efforts to map the phenotypic effects of mutation.

Performance of Saccharomyces cerevisiae strains against the application of adaptive laboratory evolution strategies for butanol tolerance

Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds.

A library-on-library screen reveals the breadth expansion landscape of a broadly neutralizing betacoronavirus antibody.

Broadly neutralizing antibodies (bnAbs) typically evolve cross-reactivity breadth through acquiring somatic hypermutations. While evolution of breadth requires improvement of binding to multiple antigenic variants, most experimental evolution platforms select against only one antigenic variant at a time. In this study, a yeast display library-on-library approach was applied to delineate the affinity maturation of a betacoronavirus bnAb, S2P6, against 27 spike stem helix peptides in a single experiment. Our results revealed that the binding affinity landscape of S2P6 varies among different stem helix peptides. However, somatic hypermutations that confer general improvement in binding affinity across different stem helix peptides could also be identified. We further showed that a key somatic hypermutation for breadth expansion involves long-range interaction. Overall, our work not only provides a proof-of-concept for using a library-on-library approach to analyze the evolution of antibody breadth, but also has important implications for the development of broadly protective vaccines.

Timescales of Autogenic Noise in River Bedform Evolution and Stratigraphy

AbstractBedform evolution and preserved cross strata are known to respond to floods. However, it is unclear if autogenic dynamics mask the flood signal in bedform evolution and cross strata. To address this, we characterize the temporal structure of autogenic noise in steady‐state bedform evolution in a physical experiment. Results reveal the existence of bedform groups—quasi‐stable collections of bedforms—that migrate at a similar speed as bedforms. We find that bedform and bedform‐group turnover timescales are the key autogenic timescales of bed evolution that set the transition time‐periods between different noise regimes in bedform evolution. Results suggest that bedform‐group turnover timescale sets the lower limit for detecting flood signals in bedform evolution, and floods with duration shorter than bedform turnover timescale can be severely degraded in bedform evolution and cross strata. Our work provides a new framework for interrogating fluvial cross strata for reconstruction of past floods.

Adaptive evolution of Clostridium thermocellum ATCC 27405 on alternate carbon sources leads to altered fermentation profiles.

Consolidated bioprocessing candidate, Clostridium thermocellum, is a cellulose hydrolysis specialist, with the ability to ferment the released sugars to produce bioethanol. C. thermocellum is generally studied with model substrates Avicel and cellobiose to understand the metabolic pathway leading to ethanol. In the present study, adaptive laboratory evolution, allowing C.thermocellum DSM 1237 to adapt to growth on glucose, fructose, and sorbitol, with the prospect that some strains will adapt their metabolism to yield more ethanol. Adaptive growth on glucose and sorbitol resulted in an approximately 1mM and 2mM increase in ethanol yield per millimolar glucose equivalent, respectively, accompanied by a shift in the production of the other expected fermentation end products. The increase in ethanol yield observed for sorbitol adapted cells was due to the carbon source being more reduced compared to cellobiose. Glucose and cellobiose have similar oxidation states thus the increase in ethanol yield is due to the rerouting of electrons from other reduced metabolic products excluding H2 which did not decrease in yield. There was no increase in ethanol yield observed for fructose adapted cells, but there was an unanticipated elimination of formate production, also observed in sorbitol adapted cells suggesting that fructose has regulatory implications on formate production either at the transcription or protein level.

Use of Viscous medium to study anthelmintic drug action in Caenorhabditis elegans

Caenorhabditis elegans is an appealing tool for experimental evolution and for working with antiparasitic drugs, from understanding the molecular mechanisms of drug action and resistance to uncover new drug targets. We present a new methodology for studying the impact of antiparasitic drugs in C. elegans. Viscous medium was initially designed for C. elegans maintenance during long-term evolution experiments. Viscous medium provides a less structured environment than the standard nematode growth media agar, yet the bacteria food source remains suspended. Further, the Viscous medium offers the worm population enough support to move freely, mate, and reproduce at a rate comparable to standard agar cultures. Here, the Viscous medium was adapted for use in antiparasitic research. We observed a similar sensitivity of C. elegans to anthelmintic drugs as in standard liquid media and statistical difference to the standard agar media through a larval development assay. Using Viscous medium in C. elegans studies will considerably improve antiparasitic resistance research, and this medium could be used in studies aimed at understanding long-term multigenerational drug activity.

Pseudomonas putida Dynamics of Adaptation under Prolonged Resource Exhaustion.

Many nonsporulating bacterial species survive prolonged resource exhaustion, by entering a state termed long-term stationary phase. Here, we performed long-term stationary phase evolutionary experiments on the bacterium Pseudomonas putida, followed by whole-genome sequencing of evolved clones. We show that P. putida is able to persist and adapt genetically under long-term stationary phase. We observed an accumulation of mutations within the evolving P. putida populations. Within each population, independently evolving lineages are established early on and persist throughout the 4-month-long experiment. Mutations accumulate in a highly convergent manner, with similar loci being mutated across independently evolving populations. Across populations, mutators emerge, that due to mutations within mismatch repair genes developed a much higher rate of mutation than other clones with which they coexisted within their respective populations. While these general dynamics of the adaptive process are quite similar to those we previously observed in the model bacterium Escherichia coli, the specific loci that are involved in adaptation only partially overlap between P. putida and E. coli.

The hallmarks of a tradeoff in transcriptomes that balances stress and growth functions.

Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the protein's central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations' impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.