Classification of Features across Five CURE Networks Reveals Opportunities to Improve Course Design, Instruction, and Equity.

Course-based undergraduate research experiences (CUREs) are tools used to introduce students to authentic participation in science. Several specific CUREs have been shown to benefit students' interest and retention in the biological sciences. Nevertheless, CUREs vary greatly in terms of their context, methodology, and degree of research authenticity, so different types of CUREs may differently influence student outcomes. This programmatic diversity poses a challenge to educators who want to better understand which course components and features are reliably present in a CURE curriculum. To address these issues, we identified, catalogued, and classified 112 potential features of CUREs across the biosciences. To develop the list, we interviewed instructors experienced with teaching individual and large networked CUREs across a diversity of the biological disciplines, including: Squirrel-Net (field-based animal behavior), SEA-PHAGES (wet lab microbiology and computational microbiology), Tiny Earth (environmental and wet lab microbiology), PARE (environmental microbiology), and the Genomics Education Partnership (eukaryotic computational biology). Twenty-five interviewees contributed expert content in terms of CURE features and classification of those items into an organized list. The resulting list's categories encompasses student experiences with the following: (i) the scientific process; (ii) technical aspects of science; (iii) the professional development associated with research; and (iv) building scientific identity. The most striking insight was that CUREs vary widely in terms of which features they contain, since different CUREs will by necessity have different approaches to science and student involvement. We also identified several features commonly thought to be crucial to CUREs yet have ambiguous definitions. This ambiguity can potentially confound efforts to make CUREs research-authentic and aligned with the central goals of science. We disambiguate these terms and represent their varied meanings throughout the classification. We also provide instructor-friendly supplementary worksheets along with considerations for instructors interested in expanding their CURE course design, instruction, and equity.

Complete Genome Assembly and Annotation of Escherichia coli Bacteriophage 55 from Rivière la Quint in Gonaïves, Haiti.

We present the structural and functional annotation of Escherichia coli bacteriophage 55, which has a genome length of 170,393 bp, with 219 predicted genes.

Complete Genome Assembly and Annotation of Escherichia coli Bacteriophage 107.

We present the annotated genome sequence of Escherichia coli bacteriophage 107, a T4-like bacteriophage. Phage 107 has a genome length of 167,509 bp and 287 predicted genes.

Experimental Evolution of the TolC-Receptor Phage U136B Functionally Identifies a Tail Fiber Protein Involved in Adsorption through Strong Parallel Adaptation.

Bacteriophages have received recent attention for their therapeutic potential to treat antibiotic-resistant bacterial infections. One particular idea in phage therapy is to use phages that not only directly kill their bacterial hosts but also rely on particular bacterial receptors, such as proteins involved in virulence or antibiotic resistance. In such cases, the evolution of phage resistance would correspond to the loss of those receptors, an approach termed evolutionary steering. We previously found that during experimental evolution, phage U136B can exert selection pressure on Escherichia coli to lose or modify its receptor, the antibiotic efflux protein TolC, often resulting in reduced antibiotic resistance. However, for TolC-reliant phages like U136B to be used therapeutically, we also need to study their own evolutionary potential. Understanding phage evolution is critical for the development of improved phage therapies as well as the tracking of phage populations during infection. Here, we characterized phage U136B evolution in 10 replicate experimental populations. We quantified phage dynamics that resulted in five surviving phage populations at the end of the 10-day experiment. We found that phages from all five surviving populations had evolved higher rates of adsorption on either ancestral or coevolved E. coli hosts. Using whole-genome and whole-population sequencing, we established that these higher rates of adsorption were associated with parallel molecular evolution in phage tail protein genes. These findings will be useful in future studies to predict how key phage genotypes and phenotypes influence phage efficacy and survival despite the evolution of host resistance. IMPORTANCE Antibiotic resistance is a persistent problem in health care and a factor that may help maintain bacterial diversity in natural environments. Bacteriophages ("phages") are viruses that specifically infect bacteria. We previously discovered and characterized a phage called U136B, which infects bacteria through TolC. TolC is an antibiotic resistance protein that helps bacteria pump antibiotics out of the cell. Over short timescales, phage U136B can be used to evolutionarily "steer" bacterial populations to lose or modify the TolC protein, sometimes reducing antibiotic resistance. In this study, we investigate whether U136B itself evolves to better infect bacterial cells. We discovered that the phage can readily evolve specific mutations that increase its infection rate. This work will be useful for understanding how phages can be used to treat bacterial infections.

Host-parasite coevolution promotes innovation through deformations in fitness landscapes.

During the struggle for survival, populations occasionally evolve new functions that give them access to untapped ecological opportunities. Theory suggests that coevolution between species can promote the evolution of such innovations by deforming fitness landscapes in ways that open new adaptive pathways. We directly tested this idea by using high-throughput gene editing-phenotyping technology (MAGE-Seq) to measure the fitness landscape of a virus, bacteriophage λ, as it coevolved with its host, the bacterium Escherichia coli. An analysis of the empirical fitness landscape revealed mutation-by-mutation-by-host-genotype interactions that demonstrate coevolution modified the contours of λ's landscape. Computer simulations of λ's evolution on a static versus shifting fitness landscape showed that the changes in contours increased λ's chances of evolving the ability to use a new host receptor. By coupling sequencing and pairwise competition experiments, we demonstrated that the first mutation λ evolved en route to the innovation would only evolve in the presence of the ancestral host, whereas later steps in λ's evolution required the shift to a resistant host. When time-shift replays of the coevolution experiment were run where host evolution was artificially accelerated, λ did not innovate to use the new receptor. This study provides direct evidence for the role of coevolution in driving evolutionary novelty and provides a quantitative framework for predicting evolution in coevolving ecological communities.

Assembly and Annotation of Escherichia coli Bacteriophage U115.

We present the annotated genome sequence of Escherichia coli bacteriophage U115, a T4-like bacteriophage. Phage U115 has a genome length of 166,986 bp and has 286 predicted genes.

Bridging Trade-Offs between Traditional and Course-Based Undergraduate Research Experiences by Building Student Communication Skills, Identity, and Interest.

Undergraduate research plays an important role in the development of science students. The two most common forms of undergraduate research are those in traditional settings (such as internships and research-for-credit in academic research labs) and course-based undergraduate research experiences (CUREs). Both of these settings offer many benefits to students, yet they have unique strengths and weaknesses that lead to trade-offs. Traditional undergraduate research experiences (UREs) offer the benefits of personalized mentorship and experience in a professional setting, which help build students' professional communication skills, interest, and scientific identity. However, UREs can reach only a limited number of students. On the other end of the trade-off, CUREs offer research authenticity in a many-to-one classroom research environment that reaches more students. CUREs provide real research experience in a collaborative context, but CUREs are not yet necessarily equipping students with all of the experiences needed to transition into a research lab environment outside the classroom. We propose that CURE instructors can bridge trade-offs between UREs and CUREs by deliberately including learning goals and activities in CUREs that recreate the benefits of UREs, specifically in the areas of professional communication, scientific identify, and student interest. To help instructors implement this approach, we provide experience- and evidence-based guidance for student-centered, collaborative learning opportunities.

Sustained coevolution of phage Lambda and Escherichia coli involves inner- as well as outer-membrane defences and counter-defences.

Bacteria often evolve resistance to phage through the loss or modification of cell surface receptors. In Escherichia coli and phage λ, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyse another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair λ's ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that λ evolved an additional counter-defence that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral λ, but some evolved phage grew well on the deletion mutants, indicating that they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfils the model of an inverse gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated.

Fighting microbial pathogens by integrating host ecosystem interactions and evolution.

Successful therapies to combat microbial diseases and cancers require incorporating ecological and evolutionary principles. Drawing upon the fields of ecology and evolutionary biology, we present a systems-based approach in which host and disease-causing factors are considered as part of a complex network of interactions, analogous to studies of "classical" ecosystems. Centering this approach around empirical examples of disease treatment, we present evidence that successful therapies invariably engage multiple interactions with other components of the host ecosystem. Many of these factors interact nonlinearly to yield synergistic benefits and curative outcomes. We argue that these synergies and nonlinear feedbacks must be leveraged to improve the study of pathogenesis in situ and to develop more effective therapies. An eco-evolutionary systems perspective has surprising and important consequences, and we use it to articulate areas of high research priority for improving treatment strategies.

Evolution along the parasitism-mutualism continuum determines the genetic repertoire of prophages.

Integrated into their bacterial hosts' genomes, prophage sequences exhibit a wide diversity of length and gene content, from highly degraded cryptic sequences to intact, functional prophages that retain a full complement of lytic-function genes. We apply three approaches-bioinformatics, analytical modelling and computational simulation-to understand the diverse gene content of prophages. In the bioinformatics work, we examine the distributions of over 50,000 annotated prophage genes identified in 1384 prophage sequences, comparing the gene repertoires of intact and incomplete prophages. These data indicate that genes involved in the replication, packaging, and release of phage particles have been preferentially lost in incomplete prophages, while tail fiber, transposase and integrase genes are significantly enriched. Consistent with these results, our mathematical and computational approaches predict that genes involved in phage lytic function are preferentially lost, resulting in shorter prophages that often retain genes that benefit the host. Informed by these models, we offer novel hypotheses for the enrichment of integrase and transposase genes in cryptic prophages. Overall, we demonstrate that functional and cryptic prophages represent a diversity of genetic sequences that evolve along a parasitism-mutualism continuum.

Trading-off and trading-up in the world of bacteria-phage evolution.

In 1979, Richard Law introduced the conceptual idea of the 'Darwinian Demon': an organism that simultaneously maximizes all fitness traits [1]. Such an organism would dominate an ecosystem, displacing any competitors and collapsing biodiversity to only a singular species. Surveying the tremendous species diversity of bacteria in the microbial world reveals that Darwinian Demons do not exist on Earth, and the popular notion is that fitness trade-offs generally constrain such possible evolution. However, the trade-offs faced by evolving bacterial populations presumably hinder their adaptation in ways that are not fully understood. In some cases, bacteria show evolved trade-ups, whereby selection causes multiple fitness components to improve simultaneously. Understanding these trade-offs and trade-ups, as well as their prevalence and roles in shaping microbial fitness, is key to elucidating how the incredible diversity of the Bacteria domain came to be, what maintains that diversity, and whether such diversity can be leveraged for technologies that improve human health and protect environments.

Pleiotropy complicates a trade-off between phage resistance and antibiotic resistance.

Bacteria frequently encounter selection by both antibiotics and lytic bacteriophages. However, the evolutionary interactions between antibiotics and phages remain unclear, in particular, whether and when phages can drive evolutionary trade-offs with antibiotic resistance. Here, we describe Escherichia coli phage U136B, showing it relies on two host factors involved in different antibiotic resistance mechanisms: 1) the efflux pump protein TolC and 2) the structural barrier molecule lipopolysaccharide (LPS). Since TolC and LPS contribute to antibiotic resistance, phage U136B should select for their loss or modification, thereby driving a trade-off between phage resistance and either of the antibiotic resistance mechanisms. To test this hypothesis, we used fluctuation experiments and experimental evolution to obtain phage-resistant mutants. Using these mutants, we compared the accessibility of specific mutations (revealed in the fluctuation experiments) to their actual success during ecological competition and coevolution (revealed in the evolution experiments). Both tolC and LPS-related mutants arise readily during fluctuation assays, with tolC mutations becoming more common during the evolution experiments. In support of the trade-off hypothesis, phage resistance via tolC mutations occurs with a corresponding reduction in antibiotic resistance in many cases. However, contrary to the hypothesis, some phage resistance mutations pleiotropically confer increased antibiotic resistance. We discuss the molecular mechanisms underlying this surprising pleiotropic result, consideration for applied phage biology, and the importance of ecology in evolution of phage resistance. We envision that phages may be useful for the reversal of antibiotic resistance, but such applications will need to account for unexpected pleiotropy and evolutionary context.

Destabilizing mutations encode nongenetic variation that drives evolutionary innovation.

Evolutionary innovations are often achieved by repurposing existing genes to perform new functions; however, the mechanisms enabling the transition from old to new remain controversial. We identified mutations in bacteriophage λ's host-recognition gene J that confer enhanced adsorption to λ's native receptor, LamB, and the ability to access a new receptor, OmpF. The mutations destabilize λ particles and cause conformational bistability of J, which yields progeny of multiple phenotypic forms, each proficient at different receptors. This work provides an example of how nongenetic protein variation can catalyze an evolutionary innovation. We propose that cases where a single genotype can manifest as multiple phenotypes may be more common than previously expected and offer a general mechanism for evolutionary innovation.

Host coevolution alters the adaptive landscape of a virus.

The origin of new and complex structures and functions is fundamental for shaping the diversity of life. Such key innovations are rare because they require multiple interacting changes. We sought to understand how the adaptive landscape led to an innovation whereby bacteriophage λ evolved the new ability to exploit a receptor, OmpF, on Escherichia coli cells. Previous work showed that this ability evolved repeatedly, despite requiring four mutations in one virus gene. Here, we examine how this innovation evolved by studying six intermediate genotypes of λ isolated during independent transitions to exploit OmpF and comparing them to their ancestor. All six intermediates showed large increases in their adsorption rates on the ancestral host. Improvements in adsorption were offset, in large part, by the evolution of host resistance, which occurred by reduced expression of LamB, the usual receptor for λ. As a consequence of host coevolution, the adaptive landscape of the virus changed such that selection favouring four of the six virus intermediates became stronger after the host evolved resistance, thereby accelerating virus populations along the path to using the new OmpF receptor. This dependency of viral fitness on host genotype thus shows an important role for coevolution in the origin of the new viral function.

Evolved Populations of Shigella flexneri Phage Sf6 Acquire Large Deletions, Altered Genomic Architecture, and Faster Life Cycles.

Genomic architecture is the framework within which genes and regulatory elements evolve and where specific constructs may constrain or potentiate particular adaptations. One such construct is evident in phages that use a headful packaging strategy that results in progeny phage heads packaged with DNA until full rather than encapsidating a simple unit-length genome. Here, we investigate the evolution of the headful packaging phage Sf6 in response to barriers that impede efficient phage adsorption to the host cell. Ten replicate populations evolved faster Sf6 life cycles by parallel mutations found in a phage lysis gene and/or by large, 1.2- to 4.0-kb deletions that remove a mobile genetic IS911 element present in the ancestral phage genome. The fastest life cycles were found in phages that acquired both mutations. No mutations were found in genes encoding phage structural proteins, which were a priori expected from the experimental design that imposed a challenge for phage adsorption by using a Shigella flexneri host lacking receptors preferred by Sf6. We used DNA sequencing, molecular approaches, and physiological experiments on 82 clonal isolates taken from all 10 populations to reveal the genetic basis of the faster Sf6 life cycle. The majority of our isolates acquired deletions in the phage genome. Our results suggest that deletions are adaptive and can influence the duration of the phage life cycle while acting in conjunction with other lysis time-determining point mutations.

Evolution across the Curriculum: Microbiology.

An integrated understanding of microbiology and evolutionary biology is essential for students pursuing careers in microbiology and healthcare fields. In this Perspective, we discuss the usefulness of evolutionary concepts and an overall evolutionary framework for students enrolled in microbiology courses. Further, we propose a set of learning goals for students studying microbial evolution concepts. We then describe some barriers to microbial evolution teaching and learning and encourage the continued incorporation of evidence-based teaching practices into microbiology courses at all levels. Next, we review the current status of microbial evolution assessment tools and describe some education resources available for teaching microbial evolution. Successful microbial evolution education will require that evolution be taught across the undergraduate biology curriculum, with a continued focus on applications and applied careers, while aligning with national biology education reform initiatives. Journal of Microbiology & Biology Education.

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.