Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling

Abstract

In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNAsegregating spindles.DNA segregation | bacterial cytoskeleton | Bacillus subtilis | reconstitution T he first filament-forming actin-like protein (ALP) was identified in bacteria in 2001 (1), and subsequent work identified more than 30 additional classes of ALPs in eubacteria and archaea (2). These proteins are involved in a variety of cellular processes, including assembly of the cell wall (1, 3-5), positioning of organelles (6), anchoring of cytokinesis machinery (7), and segregation of DNA (8). Most DNA-segregating ALPs participate in type II plasmid partitioning systems, which consist of three components: (i) a centromeric DNA sequence; (ii) a DNAbinding protein that interacts with the centromeric sequence to form a segrosome complex; and (iii) a polymer-forming ALP, whose self-assembly moves the segrosome through the cytoplasm (SI Appendix, Fig. S1). In the best understood type II segregation system, bidirectional polymerization of the ALP, ParM, pushes pairs of plasmids to opposite poles of rod-shaped cells. Extensive studies, both in vivo and in vitro, have produced detailed models of ParM-mediated DNA segregation (9, 10), but it is unclear to what extent these models can be generalized to describe other ALP-dependent DNA segregation systems.To better understand the diversity of molecular mechanisms underlying plasmid segregation, we studied a type II plasmid partitioning system encoded by the alf operon from the Bacillus subtilis plasmid pLS32 (11). The alf operon was first identified based on its ability to maintain plasmids through the process of sporulation, presumably by actively pushing a plasmid into the forespore at one end of the cell. In addition, the alf operon confers approximately eightfold greater stability to plasmids in rapidly dividing cells. The alf operon itself contains a centromeric sequence, parN, with three short, repeated sequences, and it encodes both an ALP, AlfA, and a DNA-binding protein, AlfB. The AlfA protein shares 15% sequence identity with ParM (2), whereas AlfB shares only 8% identity with ParR and has no significant BLAST hits (expect value < 1). In addition to sequence differences, AlfA...

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

“…4C, Inset). The initial slope when plotting scattering signal vs. time on a double-logarithmic scale yields the number of steps required for the formation of a growing filament (19,25). Indeed, TubRC changed the slope of the initial polymerization signal, further supporting the finding that TubZ filaments nucleate by fewer steps when TubRC is present than with TubZ alone (Fig.…”

“…In vitro, TubZ assembles into two-and four-stranded polymers (13)(14)(15)(16), and structural studies suggested that the centromeric TubRC complex forms a ring-like structure (17), interacting with the long C-terminal TubZ extensions (18). Thus, it was proposed that the TubRC complex tracks growing ends of TubZ filaments in analogy to the centromeric complex that follows growing filament ends of the actin-like partitioning systems (5,6,19). In bulk assays, TubRC has been reported to enhance TubZ filament formation, possibly indicating a switch in dynamic behavior (17,20).…”

Bacterial Actin-Like Proteins

“…As an example, wild-type actin elongates bidirectionally from meromyosin-labeled stable seeds (left) (Woodrum, Rich, & Pollard, 1975). AlfA (right) seeds with wild-type protein growing from a streptavidin-labeled seed (Polka, Kollman, Mullins, 2014). (A) AlfA images from Polka et al (2009).…”

“…This inherent instability of ParM filaments produces a high concentration of monomeric ParM that is available to do polymerization-coupled work. Interestingly, another DNA-segregating ALP, AlfA, has a remarkably different set of assembly properties from ParM: (1) it is not inherently dynamically unstable (Polka et al, 2009), (2) its nucleation is controlled by accessory factors (Polka, Kollman, Mullins, 2014), (3) its elongation is polarized (Polka, Kollman, Mullins, 2014), and (4) it strongly self-associates and forms large filament bundles (Polka et al, 2009). …”

Bacterial Filament Systems: Toward Understanding Their Emergent Behavior and Cellular Functions

“…Reconstitution of all three components in vitro demonstrated that AlfB/parN complexes ride on the ends of elongating bundles and also move along existing bundles in both directions. Continued growth at AlfB/parN is ensured by free AlfB repressing AlfA polymerization elsewhere in the cell (32) (Fig. 1).…”