Intracellular competition shapes plasmid population dynamics.

From populations of multicellular organisms to selfish genetic elements, conflicts between levels of biological organization are central to evolution. Plasmids are extrachromosomal, self-replicating genetic elements that face selective pressures from their hosts but also compete within the host cell for replication resources. Although theory indicates that within-cell selection matters for plasmid evolution, experimental measurement of these dynamics has remained elusive. We measured within-cell fitness of competing Escherichia coli plasmids and characterized their drift and selective dynamics. We made synthetic plasmid dimers that can be split in a controlled way to create balanced competition, which we probed experimentally. Incompatible plasmids coexist for an extended time owing to methylation-based replication control. Moreover, less transcriptionally active plasmids display a within-cell advantage and fix preferentially, favoring gene loss. Critically, fixation depends nontrivially on the interplay between plasmid transcription and translation. Our results show that plasmid evolution is driven by within- and between-cell dynamics. Editor's summary: How does natural selection operate at the multiple scales of organization that constitute biological organisms? To test theoretical expectations, Rossine et al. designed experiments involving two coexisting bacterial plasmids evolving in cells under various growth scenarios. To obtain controlled starting conditions, the synthetic plasmids were designed to split enzymatically, thus uncoupling their replication and freeing them to compete for host resources. Both plasmids surprisingly persisted together through many cell divisions. The consequences of within-cell evolution play out in the conflict between the costs of carrying accessory genes versus the competitive benefits of being well-armed with virulence, antimicrobial resistance, and phage resistance genes. —Caroline Ash INTRODUCTION: Conflicts between levels of biological organization underlie a broad range of fundamental evolutionary processes, from the emergence of multicellular organisms, to the development of social behaviors, and to the spread of mobile genetic elements (MGEs). In plasmids—multicopy, self-replicating MGEs that often carry antibiotic resistance genes—cross-scale conflicts may arise from trade-offs between the replication rate of a plasmid and the fitness of its host bacterial cell. However, these trade-offs are poorly understood despite their practical and theoretical importance. We measured the within- and between-cell components of plasmid genetic drift and selection. We then showed how their interplay can shape the evolution of antibiotic resistance and other plasmid-borne traits. RATIONALE: We developed an experimental system to independently measure the within- and between-cell components of competitive plasmid fitness. Plasmid pairs were joined as heterodimers and electroporated into host cells. Subsequent expression of a thermosensitive recombinase allowed for fast and controlled plasmid monomerization, yielding a population of cells synchronously carrying equal frequencies of each of the competing plasmids. Experimental environments encompassing a range of spatial structures allowed us to manipulate and track the between- and within-cell dynamics of a series of plasmids carrying different cargos under different promoters and ribosomal binding sites (RBSs). Last, we used mathematical models that combined cell- and plasmid-level fitness measurements to predict the outcomes of plasmid invasion under antibiotic stress. RESULTS: We found that within-cell genetic drift occurred more slowly than expected for ColE1-like plasmids owing to hemimethylation that reduced plasmid replication variability and therefore reduced effective competition and extended within-cell plasmid cooccurrence times. Despite this competition-dampening effect, increased plasmid transcriptional activity reduced within-cell fitness, favoring the fixation of less active plasmids. Therefore, when a plasmid carried an antibiotic resistance gene (ARG) that was beneficial for their host cell, we observed a trade-off between within-cell fitness, which was disfavored by transcription, and cell-level fitness, which was favored by transcription. Accordingly, the spread of ARG+ plasmids in an expanding bacterial population followed two phases. Initially, bacteria carried both ARG+ and ARG– plasmids, and within-cell competition was the predominant force, causing a reduction in frequency of the ARG+ plasmids. This phase lasted until stochastic plasmid replication and assortment generated a small fraction of cells carrying only ARG+ plasmids. Only then, with within-cell selection nullified, could the purely ARG+ cells outcompete others, leading to a secondary increase in ARG+ plasmid frequency. Moreover, we found that the RBS strength of ARGs could further modulate the relative importance of within- and between-cell fitness by altering the dominance of the antibiotic resistance phenotype. CONCLUSION: We have shown that within-cell competition modulates the fixation probability of novel plasmid variants, hindering the capture and spread of new, highly transcribed genes, which is the rate-limiting step for the evolution of antimicrobial resistance plasmids. Moreover, within-cell competition may promote plasmid gene loss, which explains the abundance of cryptic plasmids and suggests that they might be weaponized to displace antimicrobial resistance plasmids. More generally, we have demonstrated how conflicts between levels of selection lead to the counterintuitive, complex dynamics that lie at the core of the major transitions in evolution. Intracellular competition shapes plasmid population dynamics.: (A) Novel plasmid variants emerge as a minority and contend with within-cell competition until the appearance of homoplasmic cells. (B) To study within-cell competition, we created populations of cells with equilibrated plasmid composition by controllably splitting plasmid dimers. (C) Environmental structure modulates the relative importance of within- and between-cell competition. (D and E) Expression of a plasmid-borne ARG increases cell-level fitness at the cost of within-cell plasmid fitness. [ABSTRACT FROM AUTHOR]