Genetic and functional basis of the reduction effect in bacteriophage ΦX174.

The ΦX174 reduction effect describes a plasmid-based inhibitory phenomenon that mimics the superinfection inhibition found in wild phage populations. In this effect, when a portion of the ΦX174 genome - the 3' end of the pilot protein gene (H), the 5' end of the replication gene (A), and the H-A intergenic region - is present on a plasmid in the host cell, almost complete protection from phage infection occurs. Here we demonstrate that only the phage pilot protein H portion of the plasmid is sufficient for the observed inhibition, that protein synthesis is necessary for inhibition to occur, that inserting the entire H gene in the plasmid may also impart a blocking effect, and that partial to complete recovery from this inhibition is possible with minimal viral evolution.

Directed evolution of bacteriophages: thwarted by prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny.

Directed evolution of bacteriophages: impacts of prolific prophage.

Unlabelled: Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods is to propagate phage on multiple bacterial hosts, pool the lysate, and repeat the propagation process until phage(s) can form plaques on the target host(s). In theory, this propagation process produces a phage lysate that contains input phages and their evolved phage progeny.

Controlling Recombination to Evolve Bacteriophages.

Recombination among different phages sometimes facilitates their ability to grow on new hosts. Protocols to direct the evolution of phage host range, as might be used in the application of phage therapy, would then benefit from including steps to enable recombination. Applying mathematical and computational models, in addition to experiments using phages T3 and T7, we consider ways that a protocol may influence recombination levels.

Modeling the Directed Evolution of Broad Host Range Phages.

The host ranges of individual phages tend to be narrow, yet many applications of phages would benefit from expanded host ranges. Empirical methods have been developed to direct the evolution of phages to attack new strains, but the methods have not been evaluated or compared for their consequences. In particular, how do different methods favor generalist (broad host range) phages over specialist phages? All methods involve exposing phages to two or more novel bacterial strains, but the methods differ in the order in which those hosts are presented through time: Parallel presentation, Sequential presentation, and Mixed presentation.

Ecological Approach to Understanding Superinfection Inhibition in Bacteriophage.

In microbial communities, viruses compete with each other for host cells to infect. As a consequence of competition for hosts, viruses evolve inhibitory mechanisms to suppress their competitors. One such mechanism is superinfection exclusion, in which a preexisting viral infection prevents a secondary infection.

ΦX174 Attenuation by Whole-Genome Codon Deoptimization.

Natural selection acting on synonymous mutations in protein-coding genes influences genome composition and evolution. In viruses, introducing synonymous mutations in genes encoding structural proteins can drastically reduce viral growth, providing a means to generate potent, live-attenuated vaccine candidates. However, an improved understanding of what compositional features are under selection and how combinations of synonymous mutations affect viral growth is needed to predictably attenuate viruses and make them resistant to reversion.

Flying Around in the Genome: Characterization of LINE-1 in Chiroptera.

L1s are transposable elements that move by a copy-and-paste mechanism that continuously increases their copy number in the genome, such that each genome has a record of the L1 history in that host lineage. They make up about 20% of the genomes of eutherian mammals and have played a major role in shaping genome evolution. Chiroptera has the lowest average genome size among mammalian orders and the only documented case of L1 extinction affecting an entire mammalian family.

Saturation Mutagenesis Genome Engineering of Infective ΦX174 Bacteriophage Unamplified Oligo Pools and Golden Gate Assembly.

Here we present a novel protocol for the construction of saturation single-site-and massive multisite-mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 nontoxic and nonreplicative fragments compatible with Golden Gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned).

Tracing the history of LINE and SINE extinction in sigmodontine rodents.

Background: L1 retrotransposons have co-evolved with their mammalian hosts for the entire history of mammals and currently compose ~ 20% of a mammalian genome. B1 retrotransposons are dependent on L1 for retrotransposition and span the evolutionary history of rodents since their radiation. L1s were found to have lost their activity in a group of South American rodents, the Sigmodontinae, and B1 inactivation preceded the extinction of L1 in the same group.

Selecting among three basic fitness landscape models: Additive, multiplicative and stickbreaking.

Fitness landscapes map genotypes to organismal fitness. Their topographies depend on how mutational effects interact - epistasis - andare important for understanding evolutionary processes such as speciation, the rate of adaptation, the advantage of recombination, and the predictability versus stochasticity of evolution. The growing amount of data has made it possible to better test landscape models empirically.

Love the one you're with: replicate viral adaptations converge on the same phenotypic change.

Parallelism is important because it reveals how inherently stochastic adaptation is. Even as we come to better understand evolutionary forces, stochasticity limits how well we can predict evolutionary outcomes. Here we sought to quantify parallelism and some of its underlying causes by adapting a bacteriophage (ID11) with nine different first-step mutations, each with eight-fold replication, for 100 passages.

New Perspectives on Ebola Virus Evolution.

Since the recent devastating outbreak of Ebola virus disease in western Africa, there has been significant effort to understand the evolution of the deadly virus that caused the outbreak. There has been a considerable investment in sequencing Ebola virus (EBOV) isolates, and the results paint an important picture of how the virus has spread in western Africa. EBOV evolution cannot be understood outside the context of previous outbreaks, however.

Initiating a watch list for Ebola virus antibody escape mutations.

The 2014 Ebola virus (EBOV) outbreak in West Africa is the largest in recorded history and resulted in over 11,000 deaths. It is essential that strategies for treatment and containment be developed to avoid future epidemics of this magnitude. With the development of vaccines and antibody-based therapies using the envelope glycoprotein (GP) of the 1976 Mayinga strain, one important strategy is to anticipate how the evolution of EBOV might compromise these efforts.

Integration of molecular cytogenetics, dated molecular phylogeny, and model-based predictions to understand the extreme chromosome reorganization in the Neotropical genus Tonatia (Chiroptera: Phyllostomidae).

Background: Defining factors that contributed to the fixation of a high number of underdominant chromosomal rearrangements is a complex task because not only molecular mechanisms must be considered, but also the uniqueness of natural history attributes of each taxon. Ideally, detailed investigation of the chromosome architecture of an organism and related groups, placed within a phylogenetic context, is required. We used multiple approaches to investigate the dynamics of chromosomal evolution in lineages of bats with considerable karyotypic variation, focusing on the different facets contributing to fixation of the exceptional chromosomal changes in Tonatia saurophila.

Changing folding and binding stability in a viral coat protein: a comparison between substitutions accessible through mutation and those fixed by natural selection.

Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy).

Positive selection and multiple losses of the LINE-1-derived L1TD1 gene in mammals suggest a dual role in genome defense and pluripotency.

Mammalian genomes comprise many active and fossilized retroelements. The obligate requirement for retroelement integration affords host genomes an opportunity to 'domesticate' retroelement genes for their own purpose, leading to important innovations in genome defense and placentation. While many such exaptations involve retroviruses, the L1TD1 gene is the only known domesticated gene whose protein-coding sequence is almost entirely derived from a LINE-1 (L1) retroelement.

Reviving the dead: history and reactivation of an extinct l1.

Although L1 sequences are present in the genomes of all placental mammals and marsupials examined to date, their activity was lost in the megabat family, Pteropodidae, ∼24 million years ago. To examine the characteristics of L1s prior to their extinction, we analyzed the evolutionary history of L1s in the genome of a megabat, Pteropus vampyrus, and found a pattern of periodic L1 expansion and quiescence. In contrast to the well-characterized L1s in human and mouse, megabat genomes have accommodated two or more simultaneously active L1 families throughout their evolutionary history, and major peaks of L1 deposition into the genome always involved multiple families.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development.

Background: Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution--some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 - 75 Gb, 12-74 Gb of which are lost from pre-somatic cell lineages at germline--soma differentiation.

The impact of spatial structure on viral genomic diversity generated during adaptation to thermal stress.

Background: Most clinical and natural microbial communities live and evolve in spatially structured environments. When changes in environmental conditions trigger evolutionary responses, spatial structure can impact the types of adaptive response and the extent to which they spread. In particular, localized competition in a spatial landscape can lead to the emergence of a larger number of different adaptive trajectories than would be found in well-mixed populations.

Selection affects genes involved in replication during long-term evolution in experimental populations of the bacteriophage φX174.

Observing organisms that evolve in response to strong selection over very short time scales allows the determination of the molecular mechanisms underlying adaptation. Although dissecting these molecular mechanisms is expensive and time-consuming, general patterns can be detected from repeated experiments, illuminating the biological processes involved in evolutionary adaptation. The bacteriophage φX174 was grown for 50 days in replicate chemostats under two culture conditions: Escherichia coli C as host growing at 37°C and Salmonella typhimurium as host growing at 43.

Stickbreaking: a novel fitness landscape model that harbors epistasis and is consistent with commonly observed patterns of adaptive evolution.

In relating genotypes to fitness, models of adaptation need to both be computationally tractable and qualitatively match observed data. One reason that tractability is not a trivial problem comes from a combinatoric problem whereby no matter in what order a set of mutations occurs, it must yield the same fitness. We refer to this as the bookkeeping problem.

First-step mutations for adaptation at elevated temperature increase capsid stability in a virus.

The relationship between mutation, protein stability and protein function plays a central role in molecular evolution. Mutations tend to be destabilizing, including those that would confer novel functions such as host-switching or antibiotic resistance. Elevated temperature may play an important role in preadapting a protein for such novel functions by selecting for stabilizing mutations.

Retrofitting the genome: L1 extinction follows endogenous retroviral expansion in a group of muroid rodents.

Long interspersed nuclear element 1 (LINE-1; L1) retrotransposons are the most common retroelements in mammalian genomes. Unlike individual families of endogenous retroviruses (ERVs), they have remained active throughout the mammalian radiation and are responsible for most of the retroelement movement and much genome rearrangement within mammals. They can be viewed as occupying a substantial niche within mammalian genomes.