Molecular kinetics dictate population dynamics in CRISPR-based plasmid defense

Abstract

Understanding and manipulating mobile genetic element (MGE) spread within bacterial communities represents a great challenge with wide-ranging potential benefits. It requires a detailed understanding of spatial effects and cell-to-cell variability in defence systems such as CRISPR-Cas. Here, we report a time-lapse imaging-based assay with simultaneous fluorescent labeling of CRISPR-Cascade complexes and the conjugative plasmid RP4, enabling direct, single-cell-resolution observation of conjugation and CRISPR interference. We find that Cascade, under wild-type-like expression scenarios, provides a nudge towards plasmid clearance rather than full immunity, and can be counteracted by the ParDE plasmid addiction system. These limitations could be overcome by higher Cascade expression, yet by quantifying single-cell variability, we observe a tradeoff between plasmid clearance and cell growth. We synthesize these measurements into a spatially-resolved, agent-based model of plasmid population dynamics. The imaging and analysis techniques used here will facilitate disentanglement of how single-cell molecular events result in community-wide plasmid dynamics.