The Structure and Assembly Dynamics of Plasmid Actin AlfA Imply a Novel Mechanism of DNA Segregation

Abstract

Bacterial cytoskeletal proteins participate in a variety of processes, including cell division and DNA segregation. Polymerization of one plasmid-encoded, actin-like protein, ParM, segregates DNA by pushing two plasmids in opposite directions and forms the current paradigm for understanding active plasmid segregation. An essential feature of ParM assembly is its dynamically instability, the stochastic switching between growth and disassembly. It is unclear whether dynamic instability is an essential feature of all actin-like protein-based segregation mechanisms or whether bacterial filaments can segregate plasmids by different mechanisms. We expressed and purified AlfA, a plasmid-segregating actin-like protein from Bacillus subtilis, and found that it forms filaments with a unique structure and biochemistry; AlfA nucleates rapidly, polymerizes in the presence of ATP or GTP, and forms highly twisted, ribbon-like, helical filaments with a left-handed pitch and protomer nucleotide binding pockets rotated away from the filament axis. Intriguingly, AlfA filaments spontaneously associate to form uniformly sized, mixed-polarity bundles. Most surprisingly, our biochemical characterization revealed that AlfA does not display dynamic instability and is relatively stable in the presence of diphosphate nucleotides. These results (i) show that there is remarkable structural diversity among bacterial actin filaments and (ii) indicate that AlfA filaments partition DNA by a novel mechanism.Bacteria contain multiple filament-forming proteins related to eukaryotic actin (6). These actin-like proteins have multiple cellular roles, including determination of cell shape (18), arrangement of organelles (20), and segregation of DNA (36). Little is known about the assembly dynamics of most of these proteins or about the identities and activities of the factors that regulate them. The widely expressed actin-like protein MreB, for example, has been purified and studied in vitro, but its assembly appears to be strongly inhibited by physiological concentrations of monovalent cations, suggesting that its assembly in vivo is facilitated by as-yet-unknown factors (23). At present, the best-understood actin-like protein is ParM, a plasmid-encoded protein that constructs a bipolar spindle capable of pushing plasmids to opposite poles of rod-shaped cells (5,25). In contrast to the eukaryotic actin cytoskeleton, whose assembly and architecture are regulated by a variety of accessory factors, ParM dynamics are regulated by a single factor, a complex composed of multiple copies of the repressor protein ParR bound to a DNA locus, parC (17). The ParR/parC complex binds the ends of ParM filaments and is pushed through the cytoplasm by filament elongation (5, 14, 25). The ability of ParM to function with such minimal regulation appears to be due to its unique assembly dynamics, which are dramatically different from those of eukaryotic actins. One of the most important differences is that ParM filaments are dynamically unstable (13). That is, similar to...

Actin on DNA—An ancient and dynamic relationship

“…A single 3 kb DNA fragment containing alfA and the downstream gene alfB is required and sufficient for plasmid survival during growth and sporulation [Becker et al, 2006]. In an analogy to the ParMRC system, AlfB has been suggested to play the role of ParR, acting as an adapter in AlfA-DNA interactions [Polka et al, 2009]. Already these two cases, ParM and AlfA, indicate that although performing similar tasks, the bacterial actin filaments show remarkable structural diversity.…”

“…In contrast, another bacterial actin from Bacillus subtilis, AlfA, has also been shown to play a part in plasmid segregation [Becker et al, 2006] but no evidence of catastrophic disassembly has been found in this case, and instead AlfA spontaneously associates to form mixed-polarity bundles Polka et al, 2009]. A single 3 kb DNA fragment containing alfA and the downstream gene alfB is required and sufficient for plasmid survival during growth and sporulation [Becker et al, 2006].…”

“…In the absence of apparent disassembly factors, ParM filaments exhibit eukaryotic tubulin-like behavior in the sense that they are dynamically unstable and stabilize only upon ParM binding to ParRC scaffold. This enables ParM filaments to elongate only if bound to plasmids on both ends, reducing unnecessary polymerization cycles [Garner et al, 2004[Garner et al, , 2007Salje and Lowe, 2008;Galkin et al, 2009;Salje et al, 2009].In contrast, another bacterial actin from Bacillus subtilis, AlfA, has also been shown to play a part in plasmid segregation [Becker et al, 2006] but no evidence of catastrophic disassembly has been found in this case, and instead AlfA spontaneously associates to form mixed-polarity bundles Polka et al, 2009]. A single 3 kb DNA fragment containing alfA and the downstream gene alfB is required and sufficient for plasmid survival during growth and sporulation [Becker et al, 2006].…”

“…The ability to form polymers is both a conserved and required feature also in prokaryotic actin homologues, but the ABPs are not conserved. It appears that different bacteria may have evolved unique ways to regulate their actin polymerization cycle, which appears to have resulted in structurally very diverse polymer assemblies [Garner et al, 2004;Polka et al, 2009]. In the absence of common themes, it is possible that prokaryotic actin-regulators have so far been mostly missed.…”

“…NMI ¼ nuclear myosin I. [Polka et al, 2009]. A single 3 kb DNA fragment containing alfA and the downstream gene alfB is required and sufficient for plasmid survival during growth and sporulation [Becker et al, 2006].…”