Molecular kinetics dictate population dynamics in CRISPR-based plasmid defense

Abstract

Understanding and manipulating the spread of mobile genetic elements represents a great challenge with potential benefits across synthetic biology, agriculture, and medicine. A key part of this challenge is the multiple scales in play, from the molecular kinetics of defense systems such as CRISPR-Cas, to single-cell variability in immunity levels, to spatial structure in bacterial populations. In this work, we use a time-lapse, imaging-based approach to characterize conjugative plasmid dynamics at the molecular, single-cell, and population levels. By fluorescently tagging the conjugative plasmid RP4 and CRISPR-Cascade complexes, we quantify population dynamics as a function of spacer target number, Cascade expression level, and the presence of plasmid addiction modules. Using single-cell tracking, we report conjugation rate per neighboring donor cell, estimate the latent period between plasmid uptake and subsequent onward transmission, and quantify the effect of Cascade expression variability on plasmid clearance kinetics. Finally, using a spatially resolved, agent-based model, we show that plasmid population dynamics can be successfully predicted using these single-cell biophysical parameters as inputs. 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 imaging and analysis techniques used here will facilitate the disentanglement of how these factors coordinate to realize community-wide plasmid dynamics in diverse contexts.