Quantitative Localization Microscopy Reveals a Novel Organization of a High-Copy Number Plasmid

Abstract

The maintenance of high-copy number plasmids within bacteria had been commonly thought to result from free diffusion and random segregation. Recent microscopy experiments, however, observed high-copy number 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. We recently investigated the cellular organization of a ColE1-derivative plasmid in Escherichia coli bacteria using quantitative superresolved microscopy based on single-molecule localization in combination with single-molecule fluorescence in situ hybridization (smFISH). We observed that many of the plasmids aggregated into large clusters, although most 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 completely encompasses the behavior of high-copy number plasmids. We also found many plasmids within the chromosomal volume, providing further evidence that the nucleoid does not fully exclude DNA and RNA.