Molecular kinetics dictate population dynamics in CRISPR-based plasmid defense

Understanding and manipulating the complexity of microbial community diversity, including the mobile genetic elements contained within, represents a great challenge that has wide-ranging potential benefits across synthetic biology, agriculture and medicine. An important component to this complexity is the acquisition of genetic material via conjugative plasmids, which can encode new functions such as antibiotic resistance, and the degradation of those plasmids by CRISPR interference. In this work we use single-cell tracking of E. coli populations in microfluidic devices coupled with high resolution fluorescence microscopy to characterize plasmid dynamics at the single-cell and population level. On a population level we find that the ability of cells to clear plasmids is highly dependent on both the number of spacer targets present and the defense expression level. Additionally we assess the impact that counter-defense mechanisms such as plasmid addiction have on plasmid population dynamics and show that CRISPR may be an ineffective method to counter plasmids with such mechanisms. By leveraging single-cell tracking, we were also able to report conjugation rate per neighboring donor cell, estimate the latent period between plasmid uptake and subsequent onward transmission, and corroborate existing estimates of cascade search time. This synthesis of population and single-cell measurements suggests that plasmids are the subject of a dynamic tug-of-war between defense expression, spacer distribution, neighboring cell identity and plasmid cost-benefit tradeoffs. The use of imaging and analysis techniques used here and subsequent multi-scale measurements will facilitate the disentanglement of how these factors coordinate to realize community-wide plasmid dynamics in diverse contexts.