High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS

Abstract

The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB a protein that binds to centromeric-like parS sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We identify ParA hopping on the nucleoid as the primary determinant of this geometry-sensing and develop a unified computational model that quantitatively explains the observed behaviour. Furthermore, we confirm our model prediction that cells with the lowest plasmid concentration transition to oscillatory dynamics and find similar behaviour in a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, minimising ATP consumption. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.

★Track: Inferred counting and tracking of replicating DNA loci

“…This has been a topic of active research for several decades [23][24][25][26][27] but progress has more recently slowed due, in part, to the inability to accurately measure plasmid copy numbers in individual cells 28 . Having recently performed a high-throughput study of the partitioning mechanism of the low copy number F plasmid using a microfluidic 'mother machine' device 19 , we decided to revisit our data in the context of copy number control. F plasmid is a tractable system in this regard since duplicated plasmids segregate beyond the diffraction limit within about a minute of replication 29,30 .…”

“…To assess the accuracy of ★ Track, we generated ground truth data using a stochastic model of plasmid positioning 19 and mimicked the presence of false positive and false negative foci by randomly adding and removing data points respectively. We found that even after removing a substantial fraction of localisations and adding many false positives, ★ Track could accurately reproduce the ground truth tracking including correctly identifying the timepoints of plasmid replication (Fig.…”

“…Our image processing pipeline, Mothersegger (https://gitlab.gwdg.de/murray-group/MotherSegger) has been described previously 19 . Briefly, it consists of four parts: I) preprocessing, II) segmentation III) cell tracking and IV) foci detection.…”

“…The trajectories used in Fig. 1c-f were generated with our previous published model of plasmid positioning 19 . This is a stochastic model of plasmid positioning by the interaction of plasmid-bound ParB with nucleoid associated ParA.…”

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes

“…First, regarding the plasmid, while both oscillatory and defined positioning have been described, the prevalence of either mode within and across species was unclear (17,18,29,30). In this direction, we recently used high-throughput imaging and analysis to quantify the dynamics of F-plasmid and found that it is elastically 'pulled' to defined locations within the cell (mid-cell for a single plasmid, the quarter positions for two plasmid) (31). However, at the lowest plasmid concentrations (a single plasmid in a long cell) the dynamics become oscillatory in nature.…”