No abstract

To ensure their stable inheritance by daughter cells during cell division, bacterial low copynumber plasmids make simple DNA segregating machines that use an elongating protein filament between sister plasmids. In the ParMRC system of Escherichia coli R1 plasmid, ParM, an actinlike protein, forms the spindle between ParRC complexes on sister plasmids. Using a combination of structural work and total internal reflection fluorescence microscopy, we show that ParRC bound and could accelerate growth at only one end of polar ParM filaments, mechanistically resembling eukaryotic formins. The architecture of ParM filaments enabled two ParRC-bound filaments to associate in an antiparallel orientation, forming a bipolar spindle. The spindle elongated as a bundle of at least two antiparallel filaments, thereby pushing two plasmid clusters towards the poles.During bacterial cell division, equal distribution of replicated plasmids to daughter cells ensures their stable inheritance. Low copy-number plasmids encode the simplest known DNA segregation machines to perform this task. They comprise a nucleotide-driven (cytomotive) protein filament and a centromere-like DNA region, linked by an adaptor protein. The ParMRC segregation system of E. coli R1 plasmid consists of ParM, an actinlike cytomotive protein (1) that forms polar, left-handed double-helical filaments (2), ParR, an adaptor protein, and parC, a centromeric region (3, 4). Dynamic instability of ParM filaments enables plasmid segregation by a 'search and capture' mechanism (5, 6), with ParRC (7, 8) stabilizing the filaments. It has been reported that ParRC binds to both ends of a single ParM filament (9,10). This leads to a conundrum -how does ParRC bind to two different ends of a polar ParM filament?Here, we provide a comprehensive description of ParM in the monomeric and filament states. An electron cryomicroscopy (cryoEM) reconstruction (11) (Fig. 1A) provided a subnanometer-resolution map of the polar filament of ParM (resolution of 8.5 Å at FSC 0.5;

Mutual gliding motility A (MglA), a small Ras-like GTPase; Mutual gliding motility B (MglB), its GTPase activating protein (GAP); and Required for Motility Response Regulator (RomR), a protein that contains a response regulator receiver domain, are major components of a GTPase-dependent biochemical oscillator that drives cell polarity reversals in the bacterium Myxococcus xanthus. We report the crystal structure of a complex of M. xanthus MglA and MglB, which reveals that the C-terminal helix (Ct-helix) from one protomer of the dimeric MglB binds to a pocket distal to the active site of MglA. MglB increases the GTPase activity of MglA by reorientation of key catalytic residues of MglA (a GAP function) combined with allosteric regulation of nucleotide exchange by the Ct-helix (a guanine nucleotide exchange factor [GEF] function). The dual GAP-GEF activities of MglB accelerate the rate of GTP hydrolysis over multiple enzymatic cycles. Consistent with its GAP and GEF activities, MglB interacts with MglA bound to either GTP or GDP. The regulation is essential for cell polarity, because deletion of the Ct-helix causes bipolar localization of MglA, MglB, and RomR, thereby causing reversal defects in M. xanthus. A bioinformatics analysis reveals the presence of Ct-helix in homologues of MglB in other bacterial phyla, suggestive of the prevalence of the allosteric mechanism among other prokaryotic small Ras-like GTPases.

The ParMRC plasmid partitioning apparatus is one of the best characterized systems for bacterial DNA segregation. Bundles of actin-like filaments are used to push plasmids to opposite poles of the cell, whereupon they are stably inherited on cell division. This plasmid-encoded system comprises just three components: an actin-like protein, ParM, a DNA-binding adaptor protein, ParR, and a centromere-like region, parC. The properties and interactions of these components have been finely tuned to enable ParM filaments to search the cell space for plasmids and then move ParR-parC-bound DNA molecules apart. In this Review, we look at some of the most exciting questions in the field concerning the exact molecular mechanisms by which the components of this self-contained system modulate one another's activity to achieve bipolar DNA segregation.