Quantitative Localization Microscopy Combined with DNA smFISH Reveals New Features of the Organization of High-Copy Number Plasmids in Bacteria

Abstract

The maintenance of high-copy number (hcn) plasmids within a colony of bacteria has been commonly thought to result from simple free diffusion and random segregation. Recent microscopy experiments, however, observed hcn plasmids clustering into discrete foci, which seemed to contradict this model, and hinted at an undiscovered active mechanism, as often found in low-copy number plasmids. To address this issue, we investigated the cellular organization of hcn plasmids (ColE1-like) in bacteria using quantitative localization microscopy in combination with DNA single-molecule fluorescence in situ hybridization (smFISH). We observed that, although many hcn plasmids aggregated into large clusters, a majority of the plasmids were randomly distributed throughout the bacteria, minus an excluded volume about the chromosomal DNA. Our results indicate that neither of the previous models are complete, and suggest a “mixed distribution model” of randomly diffusing plasmids and large clustered aggregates. We also found plasmids within the chromosomal volume of the cell, suggesting that the nucleoid may not fully exclude plasmids, and that the nucleoid may be more amorphous than previously thought.