Abstract
Precise and rapid DNA segregation is required for proper inheritance of genetic material. In most bacteria and archaea, this process is assured by a broadly conserved mitotic-like apparatus in which a NTPase (ParA) displaces the partition complex. Competing observations and models imply starkly different 3D localization patterns of the components of the partition machinery during segregation. Here we use super-resolution microscopies to localize in 3D each component of the segregation apparatus with respect to the bacterial chromosome. We show that Par proteins locate within the nucleoid volume and reveal that proper volumetric localization and segregation of partition complexes requires ATPase and DNA-binding activities of ParA. Finally, we find that the localization patterns of the different components of the partition system highly correlate with dense chromosomal regions. We propose a new mechanism in which the nucleoid provides a scaffold to guide the proper segregation of partition complexes.
Highlights
Precise and rapid DNA segregation is required for proper inheritance of genetic material
We combined high-density whole-chromosome labelling with high-throughput, 3D structured illumination microscopy (3D-SIM)[26], a super-resolution method that provides a twofold increase in both lateral and axial resolution as compared with diffraction-limited microscopies
DNA was either fluorescently labelled by the nonspecific DNA (ns-DNA)-binding protein histone-like U93 (HU) tagged with monomeric fluorescent protein mCherry or by the intercalating agent 4,6-diamidino-2phenylindole, while localization of partition complexes was detected by tagging ParB with a fluorescent tag
Summary
Precise and rapid DNA segregation is required for proper inheritance of genetic material. A variation of this mechanism (‘DNA relay’ model) was recently proposed in which chromosome elasticity plays a role by partly powering movement of the partition complex[25] (Fig. 1a, lower panel) All these models were largely based on biochemical assays (for example, polymerization, DNA binding and ATPase activity assays), two-dimensional (2D) imaging and in vitro microscopy assays monitoring the dynamics of ParBbound plasmids on a 2D ParA-bound DNA carpet, and proposed that partition components segregate through the space between the nucleoid surface and the membrane. These various models proposed distinct localization patterns of Par components within or around the nucleoid.
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