A common misconception is that evolution is a linear ‘march of progress’, where each organism along a line of descent is more fit than all those that came before it. Rejecting this misconception implies that evolution is nontransitive: a series of adaptive events will, on occasion, produce organisms that are less fit compared to a distant ancestor. Here we identify a nontransitive evolutionary sequence in a 1000-generation yeast evolution experiment. We show that nontransitivity arises due to adaptation in the yeast nuclear genome combined with the stepwise deterioration of an intracellular virus, which provides an advantage over viral competitors within host cells. Extending our analysis, we find that nearly half of our ~140 populations experience multilevel selection, fixing adaptive mutations in both the nuclear and viral genomes. Our results provide a mechanistic case-study for the adaptive evolution of nontransitivity due to multilevel selection in a 1000-generation host/virus evolution experiment.
New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five phages were engineered for nonlysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, killed P. aeruginosa PAO1 in liquid culture as well as in a waxworm (Galleria mellonella) model of infection. IMPORTANCE The antibiotic resistance crisis has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that computationally identified prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.
Non-transitivity -commonly illustrated by the rock-paper-scissors game -is purported to be common in evolution despite a lack of examples of non-transitive interactions arising along a single line of descent. We identify a non-transitive evolutionary sequence in the context of yeast experimental evolution in which a 1,000-generation evolved clone loses in direct competition with its ancestor. We show that non-transitivity arises due to the combined effects of adaptation mediated by the evolving nuclear genome combined with the stepwise deterioration of an intracellular virus. We show that multilevel selection is widespread: nearly half of all populations fix adaptive mutations in both the nuclear and viral genomes, and clonal interference and genetic hitchhiking occur at both levels. Surprisingly, we find no evidence that viral mutations increase the fitness of their host. Instead, the evolutionary success of evolved viral variants results from their selective advantage over viral competitors within the context of individual cells. Overall, our results show that widespread multilevel selection is capable of producing complex evolutionary dynamicsincluding non-transitivity -under simple laboratory conditions.A common misconception is that evolution is a linear "march of progress," where each genotype along a line of decent is more fit than all those that came before (1). Rejecting this misconception implies that evolution is non-transitive and evolutionary succession will, on occasion, produce organisms that are less fit compared to a distant ancestor. In ecology, nontransitive interactions are well-documented in response to resource (2) or interference competition (3). Early studies in experimental evolution have suggested that non-transitive interactions can arise (4); however, little is known regarding how specific
The annotation of six cluster N Mycobacterium smegmatis phages (Kevin1, Nenae, Parmesanjohn, ShrimpFriedEgg, Smurph, and SpongeBob) reveals regions of genomic diversity, particularly within the central region of the genome. The genome of Kevin1 includes two orphams (genes with no similarity to other phage genes), with one predicted to encode an AAA-ATPase.
New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements (IGEs) in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five of these were engineered for non-lysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The ∆int phages, individually and in cocktails, showed killing of P. aeruginosa PAO1 in vitro as well as in a waxworm (Galleria mellonella) model of infection. SIGNIFICANCE STATEMENTThe antibiotic-resistance crisis in medicine and agriculture has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.
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