Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

Abstract

Segregation of DNA is a fundamental process during cell division. The mechanism of prokaryotic DNA segregation is largely unknown, but several low-copy-number plasmids encode cytomotive filament systems of the actin type and tubulin type important for plasmid inheritance. Of these cytomotive filaments, only actin-like systems are mechanistically well characterized. In contrast, the mechanism by which filaments of tubulin-like TubZ protein mediate DNA motility is unknown. To understand polymer-driven DNA transport, we reconstituted the filaments of TubZ protein (TubZ filaments) from Bacillus thuringiensis pBtoxis plasmid with their centromeric TubRC complexes containing adaptor protein TubR and tubC DNA. TubZ alone assembled into polar filaments, which annealed laterally and treadmilled. Using single-molecule imaging, we show that TubRC complexes were not pushed by filament polymerization; instead, they processively tracked shrinking, depolymerizing minus ends. Additionally, the TubRC complex nucleated TubZ filaments and allowed for treadmilling. Overall, our results indicate a pulling mechanism for DNA transport by the TubZRC system. The discovered minus end-tracking property of the TubRC complex expands the mechanistic diversity of the prokaryotic cytoskeleton.cytoskeleton | tubulin homologue | DNA segregation

Structures of partition protein ParA with nonspecific DNA and ParB effector reveal molecular insights into principles governing Walker-box DNA segregation

“…In these systems, the NTPases form polymers to mediate DNA segregation. The actinlike polymers bind and push apart replicated DNA plasmid cargo in a process termed insertional polymerization, while tubulin-like NTPase filaments undergo treadmilling and pull CBP-bound cargo DNA to cell poles (Egelman 2003;Møller-Jensen et al 2003;Pogliano 2004;Garner et al 2007;Schumacher et al 2007;Gerdes et al 2010;Ni et al 2010;Gayathri et al 2012;Schumacher 2012;Bharat et al 2015;Fink and Löwe 2015). The less well-understood type I Walker-box systems are used by bacterial and archaeal chromosomes and plasmids and hence are arguably the most ubiquitous type of partition system in nature (Gerdes et al 2000;Schumacher et al 2015;Barillà 2016).…”

Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

“…5B and C). Treadmilling has also been observed in the plasmid-encoded bacterial tubulin TubZ (28,29), FtsZ filaments (30,31), as well as in eukaryotic MTs. BtubA/B filament treadmilling indicates that GDP/GTP exchange can only occur in the free heterodimers because it occurs when monomers at the plus end are GTP bound and monomers at the minus end are GDP bound.…”

The IntXO-PSL Recombination System Is a Key Component of the Second Maintenance System for Bacillus anthracis Plasmid pXO1

“…The first region carries tubZ and tubR genes that are homologous to those of the maintenance system of the B. thuringiensis virulence plasmid pBtoxis (4). These genes along with a tubC site encode a type III PPS that provides a mechanism for pBtoxis movement and, ultimately, segregation (5,22). However, the data for pXO1 presented here demonstrate that a classical type III PPS like that in pBtoxis is not sufficient for stability of either the pXO1K derivative plasmids present in strains Ames 35K(pL6) and Ames 35K(pL3) (Fig.…”