Heavy Metal Pollution Impacts Soil Bacterial Community Structure and Antimicrobial Resistance at the Birmingham 35th Avenue Superfund Site.

Heavy metals (HMs) are known to modify bacterial communities both in the laboratory and in situ. Consequently, soils in HM-contaminated sites such as the U.S. Environmental Protection Agency (EPA) Superfund sites are predicted to have altered ecosystem functioning, with potential ramifications for the health of organisms, including humans, that live nearby. Further, several studies have shown that heavy metal-resistant (HMR) bacteria often also display antimicrobial resistance (AMR), and therefore HM-contaminated soils could potentially act as reservoirs that could disseminate AMR genes into human-associated pathogenic bacteria. To explore this possibility, topsoil samples were collected from six public locations in the zip code 35207 (the home of the North Birmingham 35th Avenue Superfund Site) and in six public areas in the neighboring zip code, 35214. 35027 soils had significantly elevated levels of the HMs As, Mn, Pb, and Zn, and sequencing of the V4 region of the bacterial 16S rRNA gene revealed that elevated HM concentrations correlated with reduced microbial diversity and altered community structure. While there was no difference between zip codes in the proportion of total culturable HMR bacteria, bacterial isolates with HMR almost always also exhibited AMR. Metagenomes inferred using PICRUSt2 also predicted significantly higher mean relative frequencies in 35207 for several AMR genes related to both specific and broad-spectrum AMR phenotypes. Together, these results support the hypothesis that chronic HM pollution alters the soil bacterial community structure in ecologically meaningful ways and may also select for bacteria with increased potential to contribute to AMR in human disease. IMPORTANCE Heavy metals cross-select for antimicrobial resistance in laboratory experiments, but few studies have documented this effect in polluted soils. Moreover, despite decades of awareness of heavy metal contamination at the EPA Superfund site in North Birmingham, Alabama, this is the first analysis of the impact of this pollution on the soil microbiome. Specifically, this work advances the understanding of the relationship between heavy metals, microbial diversity, and patterns of antibiotic resistance in North Birmingham soils. Our results suggest that polluted soils carry a risk of increased exposure to antibiotic-resistant infections in addition to the direct health consequences of heavy metals. Our work provides important information relevant to both political and scientific efforts to advance environmental justice for the communities that call Superfund neighborhoods home.

Species-specific effects and the ecological role of programmed cell death in the microalgae Ankistrodesmus (Sphaeropleales, Selenastraceae).

Reports of programmed cell death (PCD) in phytoplankton raise questions about the ecological evolutionary role of cell death in these organisms. We induced PCD by nitrogen deprivation and unregulated cell death (non-PCD) in one strain of the green microalga Ankistrodesmus densus and investigated the effects of the cell death supernatants on phylogenetically related co-occurring organisms using growth rates and maximum biomass as proxies of fitness. PCD-released materials from A. densus CCMA-UFSCar-3 significantly increased growth rates of two conspecific strains compared to healthy culture (HC) supernatants and improved the maximum biomass of all A. densus strains compared to related species. Although growth rates of non-A. densus with PCD supernatants were not statistically different from HC treatment, biomass gain was significantly reduced. Thus, the organic substances released by PCD, possibly nitrogenous compounds, could promote conspecific growth. These results support the argument that PCD may differentiate species or subtypes and increases inclusive fitness in this model unicellular chlorophyte. Further research, however, is needed to identify the responsible molecules and how they interact with cells to provide the PCD benefits.

Plasma Electroless Reduction: A Green Process for Designing Metallic Nanostructure Interfaces onto Polymeric Surfaces and 3D Scaffolds.

The design of metal nanoparticle-modified polymer surfaces in a green and scalable way is both desirable and highly challenging. Herein, a new green low-temperature plasma-based in situ surface reduction strategy termed plasma electroless reduction (PER) is reported for achieving in situ metallic nanostructuring on polymer surfaces. Proof of concept for this new method was first demonstrated on hydrophilic cellulose papers. Cellulose papers were dip-coated with different metal ion (Ag+ and Au3+) solutions and then subjected to hydrogen plasma treatment for this PER process. Transmission electron microscopy (TEM) analysis has revealed that this PER process caused anisotropic growth of either gold or silver nanoparticles, resulting in the time-dependent formation of both distinct spherical nanoparticles (∼20 nm) and anisotropic 2D nanosheets. Furthermore, we have demonstrated the adaptability of this process by applying it to hydrophobic fibrous and 3D printed polymeric materials such as surgical face masks and 3D printed polylactic acid scaffolds. The PER process on these hydrophobic polymer surfaces was accomplished via a sequential combination of air plasma and hydrogen plasma treatment. The metallic nanostructuring caused by the PER process on these hydrophobic surfaces was systematically studied using different surface imaging techniques including 3D confocal laser surface scanning microscopy and scanning electron microscopy. We have also systematically optimized the PER process on the surface of 3D scaffolds via varying the concentration of the silver ion precursor and by different postprocessing methods such as sonication and medium soaking. These optimization processes were found to be very important in generating uniform metallic nanoparticle-modified 3D printed scaffolds while simultaneously improving cytocompatibility. Through joint disk diffusion and inhibitory concentration testing, the antibacterial efficacy of silver coatings on face masks and 3D scaffolds was established. Altogether, these results clearly suggest the excellent futuristic potential of this new PER method for designing metallic nanostructured interfaces for different biomedical applications.

Reactive oxygen species in the world ocean and their impacts on marine ecosystems.

Reactive oxygen species (ROS) are omnipresent in the ocean, originating from both biological (e.g., unbalanced metabolism or stress) and non-biological processes (e.g. photooxidation of colored dissolved organic matter). ROS can directly affect the growth of marine organisms, and can also influence marine biogeochemistry, thus indirectly impacting the availability of nutrients and food sources. Microbial communities and evolution are shaped by marine ROS, and in turn microorganisms influence steady-state ROS concentrations by acting as the predominant sink for marine ROS. Through their interactions with trace metals and organic matter, ROS can enhance microbial growth, but ROS can also attack biological macromolecules, causing extensive modifications with deleterious results. Several biogeochemically important taxa are vulnerable to very low ROS concentrations within the ranges measured in situ, including the globally distributed marine cyanobacterium Prochlorococcus and ammonia-oxidizing archaea of the phylum Thaumarchaeota. Finally, climate change may increase the amount of ROS in the ocean, especially in the most productive surface layers. In this review, we explore the sources of ROS and their roles in the oceans, how the dynamics of ROS might change in the future, and how this change might impact the ecology and chemistry of the future ocean.

Community context and pCO2 impact the transcriptome of the "helper" bacterium Alteromonas in co-culture with picocyanobacteria.

Many microbial photoautotrophs depend on heterotrophic bacteria for accomplishing essential functions. Environmental changes, however, could alter or eliminate such interactions. We investigated the effects of changing pCO2 on gene transcription in co-cultures of 3 strains of picocyanobacteria (Synechococcus strains CC9311 and WH8102 and Prochlorococcus strain MIT9312) paired with the 'helper' bacterium Alteromonas macleodii EZ55. Co-culture with cyanobacteria resulted in a much higher number of up- and down-regulated genes in EZ55 than pCO2 by itself. Pathway analysis revealed significantly different transcription of genes involved in carbohydrate metabolism, stress response, and chemotaxis, with different patterns of up- or down-regulation in co-culture with different cyanobacterial strains. Gene transcription patterns of organic and inorganic nutrient transporter and catabolism genes in EZ55 suggested resources available in the culture media were altered under elevated (800 ppm) pCO2 conditions. Altogether, changing transcription patterns were consistent with the possibility that the composition of cyanobacterial excretions changed under the two pCO2 regimes, causing extensive ecophysiological changes in both members of the co-cultures. Additionally, significant downregulation of oxidative stress genes in MIT9312/EZ55 cocultures at 800 ppm pCO2 were consistent with a link between the predicted reduced availability of photorespiratory byproducts (i.e., glycolate/2PG) under this condition and observed reductions in internal oxidative stress loads for EZ55, providing a possible explanation for the previously observed lack of "help" provided by EZ55 to MIT9312 under elevated pCO2. If similar broad alterations in microbial ecophysiology occur in the ocean as atmospheric pCO2 increases, they could lead to substantially altered ecosystem functioning and community composition.

Agar Art: a CURE for the Microbiology Laboratory.

We previously developed and assessed "The Art of Microbiology," a course-based undergraduate research experience (CURE) which uses agar art to spur student experimentation, where we found student outcomes related to science persistence. However, these outcomes were not correlated with specific activities and gains were not reported from more than one class. In this study, we explored which of the three major activities in this CURE-agar art, experimental design, or poster presentations-affected student engagement and outcomes associated with improved understanding of the nature of science (NOS). The Art of Microbiology was studied in three microbiology teaching laboratories: at a research university with either the CURE developer (18 students) or a CURE implementer (39 students) and at a community college with a CURE implementer (25 students). Our quasi-experimental mixed methods study used pre/post-NOS surveys and semi-structured class-wide interviews. Community college students had lower baseline NOS responses but had gains in NOS similar to research university students post-CURE. We surveyed research university students following each major activity using the Assessing Student Perspective of Engagement in Class Tool (ASPECT) survey but did not find a correlation between NOS and activity engagement. Of the three activities, we found the highest engagement with agar art, especially in the CURE developer class. Interviewed students in all classes described agar art as a fun, relevant, and low-stakes assignment. This work contributes to the evidence supporting agar art as a curricular tool, especially in ways that can add research to classrooms in and beyond the research university.

Market forces determine the distribution of a leaky function in a simple microbial community.

Many biological functions are leaky, and organisms that perform them contribute some of their products to a community "marketplace" in which nonperforming individuals may compete for them. Leaky functions are partitioned unequally in microbial communities, and the evolutionary forces determining which species perform them and which become beneficiaries are poorly understood. Here, we demonstrate that the market principle of comparative advantage determines the distribution of a leaky antibiotic resistance gene in an environment occupied by two "species"-strains of Escherichia coli growing on mutually exclusive resources and thus occupying separate niches. Communities comprised of antibiotic-resistant cells were rapidly invaded by sensitive cells of both types. While the two phenotypes coexisted stably for 500 generations, in 15/18 replicates, antibiotic sensitivity became fixed in one species. Fixation always occurred in the same species despite both species being genetically identical except for their niche-defining mutation. In the absence of antibiotic, the fitness cost of resistance was identical in both species. However, the intrinsic resistance of the species that ultimately became the sole helper was significantly lower, and thus its reward for expressing the resistance gene was higher. Opportunity cost of resistance, not absolute cost or efficiency of antibiotic removal, determined which species became the helper, consistent with the economic theory of comparative advantage. We present a model that suggests that this market-like dynamic is a general property of Black Queen systems and, in communities dependent on multiple leaky functions, could lead to the spontaneous development of an equitable and efficient division of labor.

Structure and Long-Term Stability of the Microbiome in Diverse Diatom Cultures.

Microalgal cultures are often maintained in xenic conditions, i.e., with associated bacteria, and many studies indicate that these communities both are complex and have significant impacts on the physiology of the target photoautotroph. Here, we investigated the structure and stability of microbiomes associated with a diverse sampling of diatoms during long-term maintenance in serial batch culture. We found that, counter to our initial expectation, evenness diversity increased with time since cultivation, driven by a decrease in dominance by the most abundant taxa in each culture. We also found that the site from which and time at which a culture was initially collected had a stronger impact on microbiome structure than the diatom species; however, some bacterial taxa were commonly present in most cultures despite having widely geographically separated collection sites. Our results support the conclusion that stochastic initial conditions (i.e., the local microbial community at the collection site) are important for the long-term structure of these microbiomes, but deterministic forces such as negative frequency dependence and natural selection exerted by the diatom are also at work. IMPORTANCE Natural microbial communities are extremely complex, with many more species coexisting in the same place than there are different resources to support them. Understanding the forces that allow this high level of diversity has been a central focus of ecological and evolutionary theory for many decades. Here, we used stock cultures of diatoms, which were maintained for years in continuous growth alongside populations of bacteria, as proxies for natural communities. We show that the bacterial communities remained relatively stable for years, and there is evidence that ecological forces worked to stabilize coexistence instead of favoring competition and exclusion. We also show evidence that, despite some important regional differences in bacterial communities, there was a globally present core microbiome potentially selected for in these diatom cultures. Understanding interactions between bacteria and diatoms is important both for basic ecological science and for practical science, such as industrial biofuel production.

A Tale of Two Institutions: Analyzing the Impact of Gamified Student Response Systems on Student Anxiety in Two Different Introductory Biology Courses.

Anxiety can impact overall performance and persistence in college. Student response systems (SRSs), real-time active-learning technologies used to engage students and gauge their understanding, have been shown to elicit anxiety for some students. Kahoot! is an SRS technology that differs from others in that it involves gamification, the use of gamelike elements. Recent studies have explored the impact of active-learning strategies on student anxiety across different institutions, but there is little known about how Kahoot! impacts student perceived anxiety, especially in comparison with other active-learning strategies. In two complementary yet parallel studies of introductory biology courses at a western research-intensive institution (n = 694) and a southeastern research-intensive institution (n = 60), we measured students' perceived anxiety. We then explored how students were influenced by nongraded Kahoot! play and other elements of instruction. Using previously developed and course-specific pre- and post-course surveys, we found students at both universities agreed that nongraded Kahoot! play caused less anxiety compared with other pedagogical practices, such as working in small groups or reading the textbook. After playing Kahoot!, lower-performing students demonstrated greater engagement and lower levels of anxiety compared with their peers, suggesting that Kahoot! may be a particularly engaging active-learning strategy for these students.

Impacts of a COVID-19 E-Service-Learning Module in a Non-Major Biology Course.

Educators need to create an informed scientifically aware citizenry, especially in the era of the COVID-19 pandemic, where public health measures have focused on increasing adoption of safe behaviors for reducing the transmission of COVID-19. Non-major science students make up an important, yet understudied, part of our public, given that they constitute tomorrow's voters, workers, consumers, and policy-makers. Expecting that non-majors may benefit from a module connecting COVID-19 to community education, we implemented a novel E-service-learning module in light of the transition from an in-person course to an online platform. Our 4-week module included expert-led lectures, assigned digital infographics about COVID-19 safety precautions, and a required post-reflection assignment summarizing their learning gains. Out of 112 enrolled students, 87 consented to have their reflections analyzed and 8 students chose to participate in additional one-on-one online interviews. In an effort to determine which parts of our module garnered the most student commentary, we grouped post-reflection and interview data into four categories: service-learning infographic, service-learning guest lectures, information on COVID-19, and the broader implications of COVID-19. While 13% of students explicitly referenced infographics in their reflections, a far greater proportion (37%) explicitly referenced learning gains related to the expert-led lectures. Based on these findings, we encourage other educators to continue to explore the impact of E-service-learning content and assignments to help maximize learning in an online classroom environment during the COVID-19 pandemic and beyond.

Co-culture with Synechococcus facilitates growth of Prochlorococcus under ocean acidification conditions.

Anthropogenic CO2 emissions are projected to lower the pH of the ocean 0.3 units by 2100. Previous studies suggested that Prochlorococcus and Synechococcus, the numerically dominant phytoplankton in the oceans, have different responses to elevated CO2 that may result in a dramatic shift in their relative abundances in future oceans. Here we showed that the exponential growth rates of these two genera respond to future CO2 conditions in a manner similar to other cyanobacteria, but Prochlorococcus strains had significantly lower realized growth rates under elevated CO2 regimes due to poor survival after exposure to fresh culture media. Despite this, a Synechococcus strain was unable to outcompete a Prochlorococcus strain in co-culture at elevated CO2 . Under these conditions, Prochlorococcus' poor response to elevated CO2 disappeared, and Prochlorococcus' relative fitness showed negative frequency dependence, with both competitors having significant fitness advantages when initially rare. These experiments suggested that the two strains should be able to coexist indefinitely in co-culture despite sharing nearly identical nutritional requirements. We speculate that negative frequency dependence exists due to reductive Black Queen evolution that has resulted in a passively mutualistic relationship analogous to that connecting Prochlorococcus with the 'helper' heterotrophic microbes in its environment.

Is Community Relevance Enough? Civic and Science Identity Impact of Microbiology CUREs Focused on Community Environmental Justice.

Course-based undergraduate research experiences (CUREs) often involve a component where the outcomes of student research are broadly relevant to outside stakeholders. We wanted to see if building courses around an environmental justice issue relevant to the local community would impact students' sense of civic engagement and appreciation of the relevance of scientific research to the community. In this quasi-experimental study, we assessed civic engagement and scientific identity gains (N = 98) using pre- and post-semester surveys and open-ended interview responses in three different CUREs taught simultaneously at three different universities. All three CURES were focused on an environmental heavy metal pollution issue predominantly affecting African-Americans in Birmingham, Alabama. While we found increases in students' sense of science efficacy and identity, our team was unable to detect meaningful changes in civic engagement levels, all of which were initially quite high. However, interviews suggested that students were motivated to do well in their research because the project was of interest to outside stakeholders. Our observations suggest that rather than directly influencing students' civic engagement, the "broadly relevant" component of our CUREs engaged their pre-existing high levels of engagement to increase their engagement with the material, possibly influencing gains in science efficacy and science identity. Our observations are consistent with broader community relevance being an important component of CURE success, but do not support our initial hypothesis that CURE participation would influence students' attitudes toward the civic importance of science.

What is the hologenome concept of evolution?

All multicellular organisms are colonized by microbes, but a gestalt study of the composition of microbiome communities and their influence on the ecology and evolution of their macroscopic hosts has only recently become possible. One approach to thinking about the topic is to view the host-microbiome ecosystem as a "holobiont". Because natural selection acts on an organism's realized phenotype, and the phenotype of a holobiont is the result of the integrated activities of both the host and all of its microbiome inhabitants, it is reasonable to think that evolution can act at the level of the holobiont and cause changes in the "hologenome", or the collective genomic content of all the individual bionts within the holobiont. This relatively simple assertion has nevertheless been controversial within the microbiome community. Here, I provide a review of recent work on the hologenome concept of evolution. I attempt to provide a clear definition of the concept and its implications and to clarify common points of disagreement.

Anthropogenic N Deposition Alters the Composition of Expressed Class II Fungal Peroxidases.

Here, we present evidence that ca. 20 years of experimental N deposition altered the composition of lignin-decaying class II peroxidases expressed by forest floor fungi, a response which has occurred concurrently with reductions in plant litter decomposition and a rapid accumulation of soil organic matter. This finding suggests that anthropogenic N deposition has induced changes in the biological mediation of lignin decay, the rate limiting step in plant litter decomposition. Thus, an altered composition of transcripts for a critical gene that is associated with terrestrial C cycling may explain the increased soil C storage under long-term increases in anthropogenic N deposition.IMPORTANCE Fungal class II peroxidases are enzymes that mediate the rate-limiting step in the decomposition of plant material, which involves the oxidation of lignin and other polyphenols. In field experiments, anthropogenic N deposition has increased soil C storage in forests, a result which could potentially arise from anthropogenic N-induced changes in the composition of class II peroxidases expressed by the fungal community. In this study, we have gained unique insight into how anthropogenic N deposition, a widespread agent of global change, affects the expression of a functional gene encoding an enzyme that plays a critical role in a biologically mediated ecosystem process.

Interdisciplinary STEM education reform: dishing out art in a microbiology laboratory.

In the modern educational framework, life science and visual art are usually presented as mutually exclusive subjects. Despite this perceived disciplinary contrast, visual art has the ability to engage and provoke students in ways that can have important downstream effects on scientific discovery, especially when applied in a practical setting such as a laboratory course. This review broadly examines the benefit of interdisciplinary fusions of science and art as well as recent ways in which art strategies have been used in undergraduate biology classrooms. In a case study, we found that undergraduate students in an introductory microbiology laboratory course who participated in open-inquiry activities involving agar art had greater confidence in their personal efficacy as scientists compared to a control class. Collectively, these observations suggest that visual art can be a useful enhancement in the course-based undergraduate research setting, and science educators at all levels should consider incorporating artistic creativity in their own classroom strategies.

Degradation of hydrogen peroxide at the ocean's surface: the influence of the microbial community on the realized thermal niche of Prochlorococcus.

Prochlorococcus, the smallest and most abundant phytoplankter in the ocean, is highly sensitive to hydrogen peroxide (HOOH), and co-occurring heterotrophs such as Alteromonas facilitate the growth of Prochlorococcus by scavenging HOOH. Temperature is also a major influence on Prochlorococcus abundance and distribution in the ocean, and studies in other photosynthetic organisms have shown that HOOH and temperature extremes can act together as synergistic stressors. To address potential synergistic effects of temperature and HOOH on Prochlorococcus growth, high- and low-temperature-adapted representative strains were cultured at ecologically relevant concentrations under a range of HOOH concentrations and temperatures. Higher concentrations of HOOH severely diminished the permissive temperature range for growth of both Prochlorococcus strains. At the permissive temperatures, the growth rates of both Prochlorococcus strains decreased as a function of HOOH, and cold temperature increased susceptibility of photosystem II to HOOH-mediated damage. Serving as a proxy for the natural community, co-cultured heterotrophic bacteria increased the Prochlorococcus growth rate under these temperatures, and expanded the permissive range of temperature for growth. These studies indicate that in the ocean, the cross-protective function of the microbial community may confer a fitness increase for Prochlorococcus at its temperature extremes, especially near the ocean surface where oxidative stress is highest. This interaction may play a substantial role in defining the realized thermal niche and habitat range of Prochlorococcus with respect to latitude.The ISME Journal advance online publication, 31 October 2017; doi:10.1038/ismej.2017.182.

The impact of elevated CO2 on Prochlorococcus and microbial interactions with 'helper' bacterium Alteromonas.

Prochlorococcus is a globally important marine cyanobacterium that lacks the gene catalase and relies on 'helper' bacteria such as Alteromonas to remove reactive oxygen species. Increasing atmospheric CO2 decreases the need for carbon concentrating mechanisms and photorespiration in phytoplankton, potentially altering their metabolism and microbial interactions even when carbon is not limiting growth. Here, Prochlorococcus (VOL4, MIT9312) was co-cultured with Alteromonas (strain EZ55) under ambient (400 p.p.m.) and elevated CO2 (800 p.p.m.). Under elevated CO2, Prochlorococcus had a significantly longer lag phase and greater apparent die-offs after transfers suggesting an increase in oxidative stress. Whole-transcriptome analysis of Prochlorococcus revealed decreased expression of the carbon fixation operon, including carboxysome subunits, corresponding with significantly fewer carboxysome structures observed by electron microscopy. Prochlorococcus co-culture responsive gene 1 had significantly increased expression in elevated CO2, potentially indicating a shift in the microbial interaction. Transcriptome analysis of Alteromonas in co-culture with Prochlorococcus revealed decreased expression of the catalase gene, known to be critical in relieving oxidative stress in Prochlorococcus by removing hydrogen peroxide. The decrease in catalase gene expression was corroborated by a significant ~6-fold decrease in removal rates of hydrogen peroxide from co-cultures. These data suggest Prochlorococcus may be more vulnerable to oxidative stress under elevated CO2 in part from a decrease in ecosystem services provided by heterotrophs like Alteromonas. This work highlights the importance of considering microbial interactions in the context of a changing ocean.The ISME Journal advance online publication, 31 October 2017; doi:10.1038/ismej.2017.189.

Transitions in individuality through symbiosis.

When a more complex, functionally integrated entity emerges from the association of simpler, initially independent entities, a major evolutionary transition has occurred. Transitions that result from the association of different species include the evolution of the eukaryotic cell and some obligate mutualisms. Recent studies are revolutionizing our understanding of how these intimate interspecific associations come to be, revealing how and to what extent each partner contributes to the relationship, and how partners mediate conflict. Here, we review work on the evolution of mutualistic symbioses in the context of transitions in individuality and highlight how a better mechanistic understanding of the ecological drivers of host-symbiont interdependencies can help elucidate the evolutionary path to symbiotic organismality.

Private benefits and metabolic conflicts shape the emergence of microbial interdependencies.

Microbes perform many costly biological functions that benefit themselves, and may also benefit neighbouring cells. Losing the ability to perform such functions can be advantageous due to cost savings, but when they are essential for growth, organisms become dependent on ecological partners to compensate for those losses. When multiple functions may be lost, the ecological outcomes are potentially diverse, including independent organisms only; one-way dependency, where one partner performs all functions and others none; or mutual interdependency where partners perform complementary essential functions. What drives these different outcomes? We develop a model where organisms perform 'leaky' functions that provide both private and public benefits to explore the consequences of privatization level, costs and essentiality on influencing these outcomes. We show that mutual interdependency is favoured at intermediate levels of privatization for a broad range of conditions. One-way dependency, in contrast, is only favoured when privatization is low and loss-of-function benefits are accelerating. Our results suggest an interplay between privatization level and shape of benefits from loss in driving microbial dependencies. Given the ubiquity of microbial functions that are inevitably leaked and the ease of mutational inactivation, our findings may help to explain why microbial interdependencies are common in nature.

Sustained fitness gains and variability in fitness trajectories in the long-term evolution experiment with Escherichia coli.

Many populations live in environments subject to frequent biotic and abiotic changes. Nonetheless, it is interesting to ask whether an evolving population's mean fitness can increase indefinitely, and potentially without any limit, even in a constant environment. A recent study showed that fitness trajectories of Escherichia coli populations over 50 000 generations were better described by a power-law model than by a hyperbolic model. According to the power-law model, the rate of fitness gain declines over time but fitness has no upper limit, whereas the hyperbolic model implies a hard limit. Here, we examine whether the previously estimated power-law model predicts the fitness trajectory for an additional 10 000 generations. To that end, we conducted more than 1100 new competitive fitness assays. Consistent with the previous study, the power-law model fits the new data better than the hyperbolic model. We also analysed the variability in fitness among populations, finding subtle, but significant, heterogeneity in mean fitness. Some, but not all, of this variation reflects differences in mutation rate that evolved over time. Taken together, our results imply that both adaptation and divergence can continue indefinitely--or at least for a long time--even in a constant environment.