Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance.

The evolution of antibiotic-resistant bacteria threatens to become the leading cause of worldwide mortality. This crisis has renewed interest in the practice of phage therapy. Yet, bacteria's capacity to evolve resistance may debilitate this therapy as well. To combat the evolution of phage resistance and improve treatment outcomes, many suggest leveraging phages' ability to counter resistance by evolving phages on target hosts before using them in therapy (phage training). We found that in vitro, λtrn, a phage trained for 28 d, suppressed bacteria ∼1,000-fold for three to eight times longer than its untrained ancestor. Prolonged suppression was due to a delay in the evolution of resistance caused by several factors. Mutations that confer resistance to λtrn are ∼100× less common, and while the target bacterium can evolve complete resistance to the untrained phage in a single step, multiple mutations are required to evolve complete resistance to λtrn. Mutations that confer resistance to λtrn are more costly than mutations for untrained phage resistance. Furthermore, when resistance does evolve, λtrn is better able to suppress these forms of resistance. One way that λtrn improved was through recombination with a gene in a defunct prophage in the host genome, which doubled phage fitness. This transfer of information from the host genome is an unexpected but highly efficient mode of training phage. Lastly, we found that many other independently trained λ phages were able to suppress bacterial populations, supporting the important role training could play during phage therapeutic development.

Limits of neutral drift: lessons from the in vitro evolution of two ribozymes.

The relative contributions of adaptive selection and neutral drift to genetic change are unknown but likely depend on the inherent abundance of functional genotypes in sequence space and how accessible those genotypes are to one another. To better understand the relative roles of selection and drift in evolution, local fitness landscapes for two different RNA ligase ribozymes were examined using a continuous in vitro evolution system under conditions that foster the capacity for neutral drift to mediate genetic change. The exploration of sequence space was accelerated by increasing the mutation rate using mutagenic nucleotide analogs. Drift was encouraged by carrying out evolution within millions of separate compartments to exploit the founder effect. Deep sequencing of individuals from the evolved populations revealed that the distribution of genotypes did not escape the starting local fitness peak, remaining clustered around the sequence used to initiate evolution. This is consistent with a fitness landscape where high-fitness genotypes are sparse and well isolated, and suggests, at least in this context, that neutral drift alone is not a primary driver of genetic change. Neutral drift does, however, provide a repository of genetic variation upon which adaptive selection can act.

Deep sequencing analysis of mutations resulting from the incorporation of dNTP analogs.

Next-generation DNA sequencing technology was used to score >100,000 mutations resulting from exposure of a nucleic acid template to a mutagenic dNTP analog during a single pass of a DNA polymerase. An RNA template of known secondary structure was reverse transcribed in the presence of 8-oxo-dGTP, dPTP or both, followed by forward transcription in the presence of standard NTPs. Each mutagen, whether used alone or in combination, resulted in a highly characteristic mutation profile. Mutations were generated at a mean frequency of 1-2% per eligible nucleotide position, but there was substantial variation in the frequency of mutation at different positions, with a SD close to the mean. This variation was partly due to the identity of the immediately surrounding nucleotides and was not significantly influenced by the secondary structure of the RNA template. Most of the variation appears to result from idiosyncratic features that derive from local sequence context, demonstrating how different genetic sequences have different chemical phenotypes.

There're CRISPRs in My Yogurt: A Discovery-Based CURE at the Intersection of Industrial Food Production and the Human Microbiome.

Support for undergraduate laboratory education based on a CURE (Course-based Undergraduate Research Experience) model is more widespread than ever. By giving students the opportunity to conduct genuine research in laboratory courses they are required to take, CUREs can expose more students to scientific practice and have the potential to make science more inclusive, especially when research topics have direct impact on students' lives. Here, I present a new microbiology CURE module where students explore the real-world intersection between industrial food production and the human microbiome. In this module, students sequence CRISPR arrays in the genomes of lactic acid bacteria they isolate from yogurt. Natural CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) act as the bacterial immune system. When a bacterial cell survives viral infection, it can incorporate a bit of that virus's DNA into its own genome, and produce small RNA guides that surveil the cell, ready to deploy virus-destroying enzymes if matching DNA from a fresh viral infection is detected. This viral immunity is of particular interest in the fermentation industry, since viral infection can destroy stocks of starter cultures and batches of product. Commercial producers of lactic acid bacteria for yogurt production often endeavor to produce strains with large CRISPR arrays and robust immunities. With this context, students are given the task of cataloging the viral immunities found in both commercial and traditionally produced yogurt, and exploring their potential impact on human health. Wet-lab practices (strain isolation, PCR, and Sanger sequencing) are combined with bioinformatic and literature sleuthing to identify the viruses to which bacteria are immune and explore whether consumption of these strains could impact human health via interactions with the human microbiome. Here, a detailed implementation of the module is presented with guides for educators and students.

Bacteriophage lambda overcomes a perturbation in its host-viral genetic network through mutualism and evolution of life history traits.

An important driver of evolution in viruses is natural selection to optimize the use of their hosts' genetic network. To learn how viruses respond to this pressure, we disrupted the genetic network of Escherichia coli to inhibit replication of its virus, bacteriophage lambda, and then observed how λ evolved to compensate. We deleted E. coli's dnaJ gene, which lambda uses to initiate DNA replication. Lambda partially restored its ability to reproduce with just two adaptive mutations associated with genes J and S. The location of the mutations was unexpected because they were not in genes that directly interact with DnaJ, rather they affected seemingly unrelated life history traits. A nonsynonymous J mutation increased lambda's adsorption rate and an S regulatory mutation delayed lysis timing. Lambda also recovered some of its reproductive potential through intracellular mutualism. This study offers two important lessons: first, viruses can rapidly adapt to disruptive changes in their host's genetic network. Second, organisms can employ mechanisms thought to operate at the population scale, such as evolution of life history traits and social interactions, in order to overcome hurdles at the molecular level. As life science research progresses and new fields become increasingly specialized, these results remind us of the importance of multiscale and interdisciplinary approaches to understand adaptation.

Gain-of-function experiments with bacteriophage lambda uncover residues under diversifying selection in nature.

Viral gain-of-function mutations frequently evolve during laboratory experiments. Whether the specific mutations that evolve in the lab also evolve in nature and whether they have the same impact on evolution in the real world is unknown. We studied a model virus, bacteriophage λ, that repeatedly evolves to exploit a new host receptor under typical laboratory conditions. Here, we demonstrate that two residues of λ's J protein are required for the new function. In natural λ variants, these amino acid sites are highly diverse and evolve at high rates. Insertions and deletions at these locations are associated with phylogenetic patterns indicative of ecological diversification. Our results show that viral evolution in the laboratory mirrors that in nature and that laboratory experiments can be coupled with protein sequence analyses to identify the causes of viral evolution in the real world. Furthermore, our results provide evidence for widespread host-shift evolution in lambdoid viruses.

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.