Insights on Minimalistic Control Systems from in vivo Visualization of Plasmids

Abstract

Plasmids are under strong selective pressure to control fluctuations in their copy number to prevent extinction from plasmid loss or low host cell fitness. Under this pressure, plasmids have therefore evolved robust yet minimalistic copy number control systems to regulate their replication and segregation. To evaluate the ability of these control system mechanisms to control copy number fluctuations, plasmid copy number must be analyzed in single cells, but doing so reliably has been a challenging constraint for the field. Here, we present an improved, minimally perturbative labeling method for visualizing plasmids in vivo. We use this method to construct a copy number distribution of plasmid mini-R1 and find that the variability is unexpectedly low and even sub-Poissonian, surpassing theoretical predictions. Interestingly, we find that low copy number states, which are the most dangerous because of their disproportionate likelihoods of plasmid loss, are observed with sub-Poissonian frequency. We also evaluate copy number control mutants to determine whether their mechanisms contribute to the sub-Poissonian copy number distribution. Our findings suggest that the CopB-mediated and partitioning mechanisms aid the primary copA-mediated mechanism with steady-state copy number control. Finally, we use our technique for labeling plasmids in combination with HaloTag-labeled filaments to visualize the R1 partitioning system, which forms filaments between plasmid pairs to push them to the poles for equipartitioning. This work provides ample avenues for future work and has implications for the field of plasmid biology as well as biological control theory and synthetic biology.