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Microfilaments (actin) and microtubules represent the extremes in eukaryotic cytoskeleton cross-sectional dimensions, raising the question of whether filament architectures are limited by protein fold. Here, we report the cryoelectron microscopy structure of a complex filament formed from 15 protofilaments of an actin-like protein. This actin-like ParM is encoded on the large pCBH Clostridium botulinum plasmid. In cross-section, the ~26 nm diameter filament comprises a central helical protofilament surrounded by intermediate and outer layers of six and eight twisted protofilaments, respectively. Alternating polarity of the layers allows for similar lateral contacts between each layer. This filament design is stiffer than the actin filament, and has likely been selected for during evolution to move large cargos. The comparable sizes of microtubule and pCBH ParM filaments indicate that larger filament architectures are not limited by the protomer fold. Instead, function appears to have been the evolutionary driving force to produce broad, complex filaments., The plasmid-segregating actin-like protein ParM is encoded on the large, toxin carrying plasmid pCBH from Clostridium botulinum. Here the authors present the cryo-EM structure of the ParM filament that is formed from the association of 15 protofilaments and discuss its architecture.

One of the well-known functions of the bacterial cytoskeleton is plasmid segregation. Type II plasmid segregation systems, among the best characterized with respect to the mechanism of action, possess an actin-like cytomotive filament as the motor component. This chapter describes the essential components of the plasmid segregation machinery and their mechanism of action, concentrating on the actin-like protein family of the bacterial cytoskeleton. The structures of the actin-like filaments depend on their nucleotide state and these in turn contribute to the dynamics of the filaments. The components that link the filaments to the plasmid DNA also regulate filament dynamics. The modulation of the dynamics facilitates the cytomotive filament to function as a mitotic spindle with a minimal number of components.

Significance Many bacteria contain large, circular DNA molecules, called plasmids, that encode physiologically, medically, and commercially important genes, including genes conferring virulence and drug resistance. The largest plasmids use active segregation systems to maintain themselves in the host bacterium. Such segregation systems provide remarkable insight into bacterial cell biology. The pLS32 plasmid, found in a commercially important strain of Bacillus subtilis , relies on a segregation system encoded by the alf operon. We show that DNA movement is driven by self-assembly of a polymeric protein, called AlfA, and that self-assembly of AlfA into a functional, DNA-segregating machine requires the activity of the accessory factor AlfB, which (unexpectedly) promotes both assembly and disassembly of AlfA polymers.

Bipolar elongation of filaments of the bacterial actin homolog ParM drives movement of newly replicated plasmid DNA to opposite poles of a bacterial cell. We used a combination of vitreous sectioning and electron cryotomography to study this DNA partitioning system directly in native, frozen cells. The diffraction patterns from overexpressed ParM bundles in electron cryotomographic reconstructions were used to unambiguously identify ParM filaments in Escherichia coli cells. Using a low–copy number plasmid encoding components required for partitioning, we observed small bundles of three to five intracellular ParM filaments that were situated close to the edge of the nucleoid. We propose that this may indicate the capture of plasmid DNA within the periphery of this loosely defined, chromosome-containing region.

Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments.

Bacterial actins polymerize in the presence of nucleotide (preferably ATP), form a common arrangement of monomeric interfaces within a protofilament, and undergo ATP hydrolysis-dependent change in stability of the filament—all of which contribute to performing their respective functions. The relative stability of the filament in the ADP-bound form compared to that of ATP and the rate of addition of monomers at the two ends decide the filament dynamics. One of the major differences between eukaryotic actin and bacterial actins is the variety in protofilament arrangements and dynamics exhibited by the latter. The filament structure and the polymerization dynamics enable them to perform various functions such as shape determination in rod-shaped bacteria (MreB), cell division (FtsA), plasmid segregation (ParM family of actin-like proteins), and organelle positioning (MamK). Though the architecture and dynamics of a few representative filaments have been studied, information on the effect of interacting partners on bacterial actin filament dynamics is not very well known. The chapter reviews some of the structural and functional aspects of bacterial actins, with special focus on the effect that interacting partners exert on the dynamics of bacterial actins, and how these assist them to carry out the functions within the bacterial cell.

The development of novel fluorescence methods for the detection of key biomolecules is of great interest, both in basic research and in drug discovery. Particularly relevant and widespread molecules in cells are ADP and GDP, which are the products of a large number of cellular reactions, including reactions catalysed by nucleoside triphosphatases and kinases. Previously, biosensors for ADP were developed in this laboratory, based on fluorophore adducts with the bacterial actin homologue ParM. It is shown in the present study that one of these biosensors, tetramethylrhodamine–ParM, can also monitor GDP. The biosensor can be used to measure micromolar concentrations of GDP on the background of millimolar concentrations of GTP. The fluorescence response of the biosensor is fast, the response time being

Eubacteria and archaea contain a variety of actin-like proteins (ALPs) that form filaments with surprisingly diverse architectures, assembly dynamics, and cellular functions. Although there is much data supporting differences between ALP families, there is little data regarding conservation of structure and function within these families. We asked whether the filament architecture and biochemical properties of the best-understood prokaryotic actin, ParM from plasmid R1, are conserved in a divergent member of the ParM family from plasmid pB171. Previous work demonstrated that R1 ParM assembles into filaments that are structurally distinct from actin and the other characterized ALPs. They also display three biophysical properties thought to be essential for DNA segregation: 1) rapid spontaneous nucleation, 2) symmetrical elongation, and 3) dynamic instability. We used microscopic and biophysical techniques to compare and contrast the architecture and assembly of these related proteins. Despite being only 41% identical, R1 and pB171 ParMs polymerize into nearly identical filaments with similar assembly dynamics. Conservation of the core assembly properties argues for their importance in ParM-mediated DNA segregation and suggests that divergent DNA-segregating ALPs with different assembly properties operate via different mechanisms.

Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a polymerizing cytomotive filament. Together these components drive newly replicated plasmids to opposite ends of the dividing cell. The Bacillus thuringiensis plasmid pBToxis relies on a filament of the tubulin/FtsZ-like protein TubZ for its segregation. By combining crystallography and electron microscopy, we have determined the structure of this filament. We explain how GTP hydrolysis weakens the subunit–subunit contact and also shed light on the partitioning of the plasmid–adaptor complex. The double helical superstructure of TubZ filaments is unusual for tubulin-like proteins. Filaments of ParM, the actin-like partitioning protein, are also double helical. We suggest that convergent evolution shapes these different types of cytomotive filaments toward a general mechanism for plasmid separation.

Non-essential extra-chromosomal DNA elements such as plasmids are responsible for their own propagation in dividing host cells, and one means to ensure this is to carry a miniature active segregation system reminiscent of the mitotic spindle. Plasmids that are maintained at low numbers in prokaryotic cells have developed a range of such active partitioning systems, which are characterized by an impressive simplicity and efficiency and which are united by the use of dynamic, nucleotide-driven filaments to separate and position DNA molecules. A comparison of different plasmid segregation systems reveals (i) how unrelated filament-forming and DNA-binding proteins have been adopted and modified to create a range of simple DNA segregating complexes and (ii) how subtle changes in the few components of these DNA segregation machines has led to a remarkable diversity in the molecular mechanisms of closely related segregation systems. Here, our current understanding of plasmid segregation systems is reviewed and compared with other DNA segregation systems, and this is extended by a discussion of basic principles of plasmid segregation systems, evolutionary implications and the relationship between an autonomous DNA element and its host cell.

Fluorescence assays for ADP detection are of considerable current interest, both in basic research and in drug discovery, as they provide a generic method for measuring the activity of ATPases and kinases. The development of a novel fluorescent biosensor is described that is based on a tetramethylrhodamine-labeled, bacterial actin homologue, ParM. The design of the biosensor takes advantage of the large conformational change of ParM on ADP binding and the strong quenching of the tetramethylrhodamine fluorescence by stacking of the dye. ParM was labeled with two tetramethylrhodamines in close proximity, whereby the fluorophores are able to interact with each other. ADP binding alters the distance and relative orientation of the tetramethylrhodamines, which leads to a change in this stacking interaction and so in the fluorescence intensity. The final ADP biosensor shows approximately 15-fold fluorescence increase in response to ADP binding. It has relatively weak affinity for ADP (K(d) = 30 microM), enabling it to be used at substoichiometric concentrations relative to ADP, while reporting ADP concentration changes in a wide range around the K(d) value, namely, submicromolar to tens of micromolar. The biosensor strongly discriminates against ATP (>100-fold), allowing ADP detection against a background of millimolar ATP. At 20 degrees C, the labeled ParM binds ADP with a rate constant of 9.5 x 10(4) M(-1) s(-1) and the complex dissociates at 2.9 s(-1). Thus, the biosensor is suitable for real-time measurements, and its performance in such assays is demonstrated using a sugar kinase and a mammalian protein kinase.

The actin homolog ParM plays a microtubule-like role in segregating DNA prior to bacterial cell division. Fluorescence and cryo-electron microscopy have shown that ParM forms filament bundles between separating DNA plasmids in vivo. Given the lack of ParM bundling proteins it remains unknown how ParM bundles form at the molecular level. Here we show using time-lapse TIRF microscopy, under in vitro molecular crowding conditions, that ParM-bundle formation consists of two distinct phases. At the onset of polymerization bundle thickness and shape are determined in the form of nuclei of short helically disordered filaments arranged in a liquid-like lattice. These nuclei then undergo an elongation phase whereby they rapidly increase in length. At steady state, ParM bundles fuse into one single large aggregate. This behavior had been predicted by theory but has not been observed for any other cytomotive biopolymer, including F-actin. We employed electron micrographs of ParM rafts, which are 2-D analogs of 3-D bundles, to identify the main molecular interfilament contacts within these suprastructures. The interface between filaments is similar for both parallel and anti-parallel orientations and the distribution of filament polarity is random within a bundle. We suggest that the interfilament interactions are not due to the interactions of specific residues but rather to long-range, counter ion mediated, electrostatic attractive forces. A randomly oriented bundle ensures that the assembly is rigid and that DNA may be captured with equal efficiency at both ends of the bundle via the ParR binding protein.

Nearly every cellular process requires the presence of ATP. This is reflected in the vast number of enzymes like kinases or ATP hydrolases, both of which cleave the terminal phosphate from ATP, thereby releasing ADP. Despite the fact that ATP hydrolysis is one of the most fundamental reactions in biological systems, there are only a few methods available for direct measurements of enzymatic-driven ATP conversion. Here we describe the development of a reagentless biosensor for ADP, the common product of all ATPases and kinases, which allows the real-time detection of ADP, produced enzymatically. The biosensor is derived from a bacterial actin homologue, ParM, as protein framework. A single fluorophore (a diethylaminocoumarin), attached to ParM at the edge of the nucleotide binding site, couples ADP binding to a3.5-fold increase in fluorescence intensity. The labeled ParM variant has high affinity for ADP (0.46 mum) and a fast signal response, controlled by the rate of ADP binding to the sensor (0.65 microm(-1)s(-1)). Amino acids in the active site were mutated to reduce ATP affinity and achieve a400-fold discrimination against triphosphate binding. A further mutation ensured that the final sensor did not form filaments and, as a consequence, has extremely low ATPase activity. The broad applicability of N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC)-ParM as a sensitive probe for ADP is demonstrated in real-time kinetic assays on two different ATPases and a protein kinase.

To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. We found that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmids segregating as a unit.

A crystal structure of the plasmid partition protein ParR bound to centromeric DNA is described. ParR binds the centromeric DNA repeats as a dimer-of-dimers, which assemble in a super-helical array to form a large segrosome with a solenoid-shaped structure. The stable inheritance of genetic material depends on accurate DNA partition. Plasmids serve as tractable model systems to study DNA segregation because they require only a DNA centromere, a centromere-binding protein and a force-generating ATPase. The centromeres of partition (par) systems typically consist of a tandem arrangement of direct repeats1,2,3,4,5,6,7. The best-characterized par system contains a centromere-binding protein called ParR and an ATPase called ParM. In the first step of segregation, multiple ParR proteins interact with the centromere repeats to form a large nucleoprotein complex of unknown structure called the segrosome, which binds ParM filaments4,8,9,10. pSK41 ParR binds a centromere consisting of multiple 20-base-pair (bp) tandem repeats to mediate both transcription autoregulation and segregation. Here we report the structure of the pSK41 segrosome revealed in the crystal structure of a ParR–DNA complex. In the crystals, the 20-mer tandem repeats stack pseudo-continuously to generate the full-length centromere with the ribbon–helix–helix (RHH) fold of ParR binding successive DNA repeats as dimer-of-dimers. Remarkably, the dimer-of-dimers assemble in a continuous protein super-helical array, wrapping the DNA about its positive convex surface to form a large segrosome with an open, solenoid-shaped structure, suggesting a mechanism for ParM capture and subsequent plasmid segregation.

The Escherichia coli low-copy-number plasmid R1 contains a segregation machinery composed of parC, ParR and parM. The R1 centromere-like site parC contains two separate sets of repeats. By atomic force microscopy (AFM) we show here that ParR molecules bind to each of the 5-fold repeated iterons separately with the intervening sequence unbound by ParR. The two ParR protein complexes on parC do not complex with each other. ParR binds with a stoichiometry of about one ParR dimer per each single iteron. The measured DNA fragment lengths agreed with B-form DNA and each of the two parC 5-fold interon DNA stretches adopts a linear path in its complex with ParR. However, the overall parC/ParR complex with both iteron repeats bound by ParR forms an overall U-shaped structure: the DNA folds back on itself nearly completely, including an angle of approximately 150 degrees . Analysing linear DNA fragments, we never observed dimerized ParR complexes on one parC DNA molecule (intramolecular) nor a dimerization between ParR complexes bound to two different parC DNA molecules (intermolecular). This bacterial segrosome is compared to other bacterial segregation complexes. We speculate that partition complexes might have a similar overall structural organization and, at least in part, common functional properties.

Udgivelsesdato: 2007-Oct-17 Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA-binding protein ParR and its cognate centromere site parC on the DNA. The partition complex is recognized by a second partition protein, the actin-like ATPase ParM, which forms filaments required for the active bidirectional movement of DNA replicates. Here, we present the 2.8 A crystal structure of ParR from E. coli plasmid pB171. ParR forms a tight dimer resembling a large family of dimeric ribbon-helix-helix (RHH)2 site-specific DNA-binding proteins. Crystallographic and electron microscopic data further indicate that ParR dimers assemble into a helix structure with DNA-binding sites facing outward. Genetic and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition.

Using in vitro TIRF- and electron-microscopy, we reinvestigated the dynamics of native ParM, a prokaryotic DNA segregation protein and actin homolog. In contrast to a previous study, which used a cysteine ParM mutant, we find that the polymerization process of wild type ATP-ParM filaments consists of a polymerization phase and a subsequent steady state phase, which is dynamically unstable, like that of microtubules. We find that the apparent bidirectional polymerization of ParM, is not due to the intrinsic nature of this filament, but results from ParM forming randomly oriented bundles in the presence of crowding agents. Our results imply, that in the bacterium, ParM filaments spontaneously form bipolar bundles. Due to their intrinsic dynamic instability, ParM bundles can efficiently "search" the cytoplasmic lumen for DNA, bind it equally well at the bipolar ends and segregate it approximately symmetrically, by the insertion of ParM subunits at either end.

Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

Unlike the mitotic segregation of eukaryotic sister chromatids, DNA partitioning in bacteria is still not well understood. Bacterial high–copy-number plasmids can be stably maintained by random distribution of their copies during cell division. In contrast, the faithful transmission of low–copy-number plasmids and many chromosomes depends on an active process mediated by conserved, tripartite segregation systems (1). A central component of these machineries is a nucleoside triphosphatase driving the partitioning reaction, which can be classified as an actin-like ATPase (ParM), a tubulin-like GTPase (TubZ), or a Walker-type ATPase (ParA). Systems using an actin or tubulin homolog function by means of a filament-based pushing or pulling mechanism (2, 3). Most low–copy-number plasmids and chromosomes are, however, segregated by Walker-type ATPases. Despite extensive research, it is not yet unambiguously established how this third group of proteins harnesses the energy released during ATP hydrolysis for plasmid movement. Based on previous analyses, two competing models have been put forward. In the filament-pulling model, ParA is assumed to form polymers that move DNA by repeated polymerization/depolymerization cycles. In contrast, the diffusion-ratchet model proposes a concentration gradient of ParA dimers on the nucleoid as the driving force for DNA segregation. In PNAS, Vecchiarelli et al. (4) now provide direct evidence in support of the latter model by fully reconstituting in vitro the segregation system of the Escherichia coli F plasmid.

Bacterial plasmid partitioning systems segregate plasmids into each daughter cell. In the well-understood ParMR C plasmid partitioning system, adapter protein ParR binds to centromere parC , forming a helix around which the DNA is externally wrapped. This complex stabilizes the growth of a filament of actin-like ParM protein, which pushes the plasmids to the poles. The TubZR C plasmid partitioning system consists of two proteins, tubulin-like TubZ and TubR, and a DNA centromere, tubC , which perform analogous roles to those in ParMR C , despite being unrelated in sequence and structure. We have dissected in detail the binding sites that comprise Bacillus thuringiensis tubC , visualized the TubR C complex by electron microscopy, and determined a crystal structure of TubR bound to the tubC repeat. We show that the TubR C complex takes the form of a flexible DNA–protein filament, formed by lateral coating along the plasmid from tubC , the full length of which is required for the successful in vitro stabilization of TubZ filaments. We also show that TubR from Bacillus megaterium forms a helical superstructure resembling that of ParR. We suggest that the TubR C DNA–protein filament may bind to, and stabilize, the TubZ filament by forming such a ring-like structure around it. The helical superstructure of this TubR C may indicate convergent evolution between the actin-containing ParMR C and tubulin-containing TubZR C systems.

Plasmid Partitioning Bacterial plasmids need to be able to partition faithfully into daughter cells. Combining structural data and advanced microscopy, Gayathri et al. (p. 1334 , published online 25 October; see the cover) describe how actin-like ParM filaments form a bipolar spindle for the faithful segregation of low-copy-number plasmids to the two cell poles of Escherichia coli , despite being polar, with two distinct ends. ParM filaments are elongated by a small adaptor protein ParR that physically links the filament end(s) to a centromere-like region on the plasmid. Antiparallel filament bundling and sliding appear to produce a bipolar spindle made of filaments that elongate unidirectionally so that all the plasmids are segregated together in one large bundle.

Filamentous actin (F-actin) is a core component of the cytoskeleton that is active in both homeostatic and dynamic cellular processes and interacts with a broad range of actin-binding proteins that regulate its stability, higher-order structure, and mechanical properties. Recent structural studies utilizing cryo-electron microscopy and 3D particle reconstruction have provided novel structural models of F-actin in isolation as well as with various bound actin-binding proteins, offering new opportunities to understand the molecular basis of F-actin stability and mechanics. Here we apply a recently introduced computational framework based on the finite element method to model the bending, twisting, and stretching deformation of 150 nm F-actin in experimentally observed states at near atomic resolution. We use the model to address two questions: (1) are bare F-actin modes of deformation conserved among the experimentally observed models and (2) do actin-binding proteins change F-actin's flexibility along one or more low modes of deformation? We find that the lowest mode shapes of the molecule are conserved across distinct F-actin models, as well as the bacterial homolog ParM, and that the actin-bundling protein fimbrin decreases significantly the torsional stiffness of F-actin while the cross-linking protein α-actinin does not. Because F-actin is itself highly conserved, these actin-binding proteins may provide a means to tune its stability and mechanical properties for specific cellular processes.

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.

Based on Waiters' GERM (Genetic Evolved Receptor Model) algorithm, an improved algorithm FARM (Pseudo Atomic Receptor Model) was put forward. PARM uses a combination of a genetic algorithm and a cross-validation technique to produce an atomic-level pseudoreceptor model, based on a set of known structure-activity relationships. During the genetic process, an artificial interfering method, which is based on a complementary principle of ligand-receptor interaction, was used to accelerate the search speed. The evolved models show a high correlation between intermolecular energy and bioactivity and can predict the bioactivity of an unknown molecule by interpolating in the regression equation of the structure-activity relationship. This algorithm was applied to two systems and produced reasonable results.

Actin was once considered an exclusive hallmark of eukaryotic cells. In 2001, however, a filament-forming actin homolog was discovered in eubacteria as a regulator of cell shape (Jones, 2001). Since that time, other bacterial actin filaments have been shown to segregate DNA (Moller-Jensen, 2002; Becker, 2006; Derman, 2009) and position organelles (Komelli, 2006). Recent bioinformatic analysis has identified dozens of families of Actin Like Proteins (ALPs) encoded on prokaryotic chromosomes, plasmids, and phages. While the overwhelming majority of these proteins remains uncharacterized, two plasmid-segregating bacterial actins, ParM and AlfA, have been studied in vitro. The architecture and assembly dynamics of these filaments and their mechanisms of force generation and cargo translocation are examined in this chapter.

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.

Summary Segregation of the R1 plasmid in bacteria relies on ParM, an actin homolog that segregates plasmids by switching between cycles of polymerization and depolymerization. We find similar polymerization kinetics and stability in the presence of either ATP or GTP and a 10-fold affinity preference for ATP over GTP. We used electron cryo-microscopy to evaluate the heterogeneity within ParM filaments. In addition to variable twist, ParM has variable axial rise, and both parameters are coupled. Subunits in the same ParM filaments can exist in two different structural states, with the nucleotide-binding cleft closed or open, and the bound nucleotide biases the distribution of states. The interface between protomers is different between these states, and in neither state is it similar to F-actin. Our results suggest that the closed state of the cleft is required but not sufficient for ParM polymerization, and provide a structural basis for the dynamic instability of ParM filaments.

During eukaryotic cell division, the DNA molecules are partitioned to opposite poles of the cell, ensuring accurate distribution of chromosomes between the dividing cells. In prokaryotes, the mechanism for accurate segregation is unknown. Previous studies have focused on partitioning of a plasmid, Rl, which encodes a stability operon, par, that is required for proper segregation of this low-copy-number plasmid. The par operon of Rl encodes three components: ParM, an actin-like ATPase, ParR, a DNA-bind- ing protein, and parC, a centromere-like DNA sequence element to which ParR binds. In the presence of ATP, ParM polymerizes and then disassembles. Disassembly is prevented, however, by the binding of ParR to parC, which stabilizes the ParM filaments. In the current model for DNA segregation, the ATP-dependent polymerization of ParM is believed to move newly replicated plasmids to the opposite poles. This study therefore determined how the ParM filaments are formed and stabilized and ascertained the sites involved to effect both mechanisms. Mutational studies, pull-down and gel- shift assays for point and deletion mutants of Rl ParR, and electron microscopy at varying concentrations of labeled parC established that the ParR- parC complex forms the clamp that binds at both the C-terminal ends of ParM, forming ring structures that bind ATP and stabilize the ParM filaments. ParR binds through its N-terminus to parC, forming a rigid scaffold of protein (ParR) wrapped by a DNA helix (parC). This binding does not include the Rl promoter region within the parC domain, because the promoter region extends out as a loop from the ParR binding region. Additionally, studies with mutants of ParM also identified the binding sites of the ParR-parC complex on ParM. A model for ParM polymerization consistent with electron micrograph images was thus proposed whereby ATP binding polymerizes ParM. Binding of the ParR-parC complex clamps the C-terminal ParM-ATP complex stabilizing filament formation. Hydrolysis of ATP to ADP dislocates ParR-parC and translocates ParR to one side to allow binding of a new ParM-ATP monomer. ParR translocates back, rocking the clamp, and the cycle of hydrolysis, translocation, and monomer addition is repeated until elongation is completed (Fig. 1). © 2008 Data Trace Publishing Company.

The length distribution of cytoskeletal filaments is an important physical parameter, which can modulate physiological cell functions. In both eukaryotic and prokaryotic cells various biological cytoskeletal polymers form supramolecular structures due to short-range forces induced mainly by molecular crowding or cross linking proteins, but their in vivo length distribution remains difficult to measure. In general, based on experimental evidence and mathematical modeling of actin filaments in aqueous solutions, the steady state length distribution of fibrous proteins is believed to be exponential. We performed in vitro TIRF- and electron-microscopy to demonstrate that in the presence of short-range forces, which are an integral part of any living cell, the steady state length distributions of the eukaryotic cytoskeletal biopolymer actin, its prokaryotic homolog ParM and microtubule homolog FtsZ deviate from the classical exponential and are either double-exponential or Gaussian, as recent theoretical modeling predicts. Double exponential or Gaussian distributions opposed to exponential can change for example the visco-elastic properties of actin networks within the cell, influence cell motility by decreasing the amount of free ends at the leading edge of the cell or effect the assembly of FtsZ into the bacterial Z-ring thus modulating membrane constriction.

ParM is a prokaryotic actin homologue, which ensures even plasmid segregation before bacterial cell division. In vivo, ParM forms a labile filament bundle that is reminiscent of the more complex spindle formed by microtubules partitioning chromosomes in eukaryotic cells. However, little is known about the underlying structural mechanism of DNA segregation by ParM filaments and the accompanying dynamic instability. Our biochemical, TIRF microscopy and high-pressure SAX observations indicate that polymerization and disintegration of ParM filaments is driven by GTP rather than ATP and that ParM acts as a GTP-driven molecular switch similar to a G protein. Image analysis of electron micrographs reveals that the ParM filament is a left-handed helix, opposed to the right-handed actin polymer. Nevertheless, the intersubunit contacts are similar to those of actin. Our atomic model of the ParM-GMPPNP filament, which also fits well to X-ray fibre diffraction patterns from oriented gels, can explain why after nucleotide release, large conformational changes of the protomer lead to a breakage of intra- and interstrand interactions, and thus to the observed disintegration of the ParM filament after DNA segregation.

Multiple unrelated polymer systems have evolved to partition DNA molecules between daughter cells at division. To better understand polymer-driven DNA segregation, we reconstituted the three-component segregation system of the R1 plasmid from purified components. We found that the ParR/ parC complex can construct a simple bipolar spindle by binding the ends of ParM filaments, inhibiting dynamic instability, and acting as a ratchet permitting incorporation of new monomers and riding on the elongating filament ends. Under steady-state conditions, the dynamic instability of unattached ParM filaments provides the energy required to drive DNA segregation.

The precise function of the 5-HT receptors remains undefined, and progress toward this has been hampered by the lack of selective ligands.

The parA locus of plasmid R1 encodes a prokaryotic centromere-like system that mediates genetic stabilization of plasmids by an unknown mechanism. The locus codes for two proteins, ParM and ParR, and a centromere-like DNA region (parC) to which the ParR protein binds. We showed recently that ParR mediates specific pairing of parC-containing DNA molecules in vitro. To obtain further insight into the mechanism of plasmid stabilization, we examined the intracellular localization of the components of the parA system. We found that ParM forms discrete foci that localize to specific cellular regions in a simple, yet dynamic pattern. In newborn cells, ParM foci were present close to both cell poles. Concomitant with cell growth, new foci formed at mid-cell. A point mutation that abolished the ATPase activity of ParM simultaneously prevented cellular localization and plasmid partitioning. A parA-containing plasmid localized to similar sites, i.e. close to the poles and at mid-cell, thus indicating that the plasmid co-localizes with ParM. Double labelling of single cells showed that plasmid DNA and ParM indeed co-localize. Thus, our data indicate that parA is a true partitioning system that mediates pairing of plasmids at mid-cell and subsequently moves them to the cell poles before cell division.

Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC. The ParR protein binds to parC in vivo and in vitro . The ParM protein is an ATPase that interacts with ParR specifically bound to parC . Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of parC -containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro . These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.

The parA system of plasmid R1 consists of two genes, parM and parR, and a cis-acting centromere-like site parC. The ParM protein exhibits similarity with a superfamily of ATPases that includes actin, hsp70 and hexokinase. ParM was purified to near-homogeneity and assayed for in vitro ATPase activity. The wild-type ParM protein was found to posses ATPase activity. Mutant ParM derivatives that exhibited decreased in vitro ATPase activity were non-functional in vivo, indicating that the ATP turnover by ParM is essential for correct plasmid partitioning. The mutant ParM proteins exhibited trans-dominance, suggesting that ParM participates as a structural component of the partitioning apparatus. The ATPase activity of ParM was activated slightly by the presence of ParR and activated to a much greater extent when ParR was bound to the centromere-like parC region. An analysis using the yeast two-hybrid system indicated that ParM and ParR interact, and demonstrated that ParR interacts with itself. Thus our results suggest a direct interaction of ParM and ParR at the natural partition site parC, and that the ATPase activity of ParM is specifically stimulated by this interaction.

The bacterial actin homolog ParM catalyzes segregation of plasmid DNA in E. coli. Recent studies now suggest a model in which ParM forms actin-like filaments between two plasmid molecules, thereby providing the driving force for plasmid DNA separation.

Biochemistry In a dividing cell, DNA must be replicated and accurately partitioned between the two daughter cells. In eukaryotes, microtubules, which are linked to the centromeric DNA by kinetochores, separate the replicated chromosomes with the help of motor proteins. Prokaryotic plasmid-partitioning systems are similar in that they involve an adaptor protein that links DNA to a filament-forming protein; however, no motor proteins are involved, and nucleotidehydrolysis dependent filament dynamics are responsible for movement. To gain insight into the segregation of the Bacillus thuringiensis pBToxis plasmid, Aylett et al. determined the structure of two protofilaments of the tubulin/FtsZ-like protein, TubZ, which mediates plasmid segregation. A combination of crystallography and electron microscopy revealed that instead of forming tubulin-like straight protofilaments that organize into microtubules, TubZ forms twisted double filaments that can bundle, reminiscent of structures formed by ParM, an actin-like prokaryotic partitioning filament. That two disparate proteins have evolved to perform a similar function using similar superstructures suggests strong evolutionary constraints on partitioning systems. Proc. Natl. Acad. Sci. U.S.A. 107 , 19766 (2010).

Type II plasmid partition systems utilize ParM NTPases in coordination with a centromere-binding protein called ParR to mediate accurate DNA segregation, a process critical for plasmid retention. The Staphylococcus aureus pSK41 plasmid is a medically important plasmid that confers resistance to multiple antibiotics, disinfectants, and antiseptics. In the first step of partition, the pSK41 ParR binds its DNA centromere to form a superhelical partition complex that recruits ParM, which then mediates plasmid separation. pSK41 ParM is homologous to R1 ParM, a known actin homologue, suggesting that it may also form filaments to drive partition. To gain insight into the partition function of ParM, we examined its ability to form filaments and determined the crystal structure of apoParM to 1.95 Å. The structure shows that pSK41 ParM belongs to the actin/Hsp70 superfamily. Unexpectedly, however, pSK41 ParM shows the strongest structural homology to the archaeal actin-like protein Thermoplasma acidophilum Ta0583, rather than its functional homologue, R1 ParM. Consistent with this divergence, we find that regions shown to be involved in R1 ParM filament formation are not important in formation of pSK41 ParM polymers. These data are also consonant with our finding that pSK41 ParM forms 1-start 10/4 helices very different from the 37/17 symmetry of R1 ParM. The polymerization kinetics of pSK41 ParM also differed from that of R1 ParM. These results indicate that type II NTPases utilize different polymeric structures to drive plasmid segregation.

Biochemistry![Figure][1] CREDIT: DUNHAM ET AL., EMBO J. 28 , 10.1038/EMBOJ.2009.120 (2009) In bacteria, low–copy number plasmids provide a model system for studying the segregation of DNA into daughter cells—partitioning requires only a centromere-like DNA site, a partition NTPase, and a centromere-binding protein. The type II systems use the actinlike partition protein ParM to drive plasmid separation. The Escherichia coli plasmid P1 uses the type I system of the partition ATPase ParA, the centromere binding protein ParB, and the DNA site parS . ParA with ATP bound interacts with centromere-bound ParB to mediate segregation; however, ADP-bound ParA acts as a transcription factor that represses parAB transcription. Using crystallography and biochemistry, Dunham et al. found that ParA forms a conformationally flexible dimer that relies on an interaction involving the N-terminal α helix. ADP binding locked the monomers (blue, yellow) into agreeable conformations for DNA binding and induced the folding of two positively charged regions (red) that docked onto the DNA (gray). In contrast, electron microscopy showed that ATP-bound ParA forms filaments that likely facilitate segregation. EMBO J. 28 , 10.1038/emboj.2009.120 (2009). [1]: pending:yes

The F-actin filament has been constructed from the atomic structure of the actin monomer to fit the observed X-ray fibre diagram from oriented gels of F-actin. A unique orientation of the monomer with respect to the actin helix has been found. The main interactions are along the two-start helix with a contribution from a loop extending across the filament axis provided by the molecule in the adjacent strand. There are also contacts along the left-handed genetic helix.

[1] We have tested the additivity of partial anhysteretic remanent magnetization (pARM) for suites of eight synthetic magnetites with mean grain sizes from 65 nm to 18 μm and 18 natural samples including lake sediments, oceanic and continental volcanic rocks, gabbros, and granites. In both synthetic and natural sample suites, domain states inferred from hysteresis and other magnetic properties vary from single-domain (SD) through pseudosingle-domain (PSD) to multidomain (MD). For each sample, total ARM intensity was compared with sums of partial ARMs of three different types: four conjugate pairs of parallel pARMs; four pairs of perpendicular pARMs; and one set of five neighboring parallel pARMs. In each case, the intervals of alternating field (AF) over which a steady field H was applied to produce the partial ARMs are nonoverlapping and cover the entire AF range (0–100 mT) used to produce the total ARM. Additivity of partial ARMs was verified to better than ±3% for all the samples, whatever the domain state (SD, PSD, and MD) or composition (ranging from pure magnetite to x = 0.6 titanomagnetite). The universality of pARM additivity is unexpected because its analog, partial thermoremanent magnetization (pTRM), deviates from ideal behavior as the grain size increases and the domain structure becomes MD. The different behaviors probably result from the fact that pARM is produced at ordinary temperatures over short times, whereas the most intense pTRM is produced at temperatures approaching the Curie point with significant dwell times, promoting such processes as isothermal remanence acquisition, domain nucleation, and domain wall reequilibration. Verification of the law of additivity of pARMs is an encouraging first step toward validating pseudo-Thellier and other methods of paleointensity determination that use ARM in place of, or in addition to, TRM.

The distribution of remanence coercivities in a rock sample can be obtained by differentiating alternating field (AF) demagnetization curves of anhysteretic remanent magnetization (ARM); however, significant errors can be introduced by grain interactions and by the noise inherent in differentiation. We have found that 1) this error can be significantly reduced by measuring partial ARM's (pARM's) and 2) pARM patterns show a strong empirical correlation with grain-size. pARM's are imparted by exposing a sample to a small direct magnetic field in the presence of a large, decreasing AF; the direct field is switched on over a specified range of AF's, thus magnetizing a fraction of the magnetite grains in the corresponding coercivity range. We have further found in both synthetic and natural sediments that measurement of the anisotropy of pARM enables us to detect the preferred orientations of distinct subpopulations of magnetite particles, related to the size and shape of the grains.

Fruit-bodies and mycelia of Byssomerulius corium (Fr.)Parm. contain a large quantity ( ca. 18 and 5–12% respectively of dry weight) of byssomeruliols, new phloracetophenone derivatives. Structures were elucidated by spectroscopic methods with emphasis on mass spectrometry. These compounds appear to be restricted so far to this species.

X-ray structure analysis of actin and of the NH2-terminal domain of the heat-shock cognate protein Hsc70 has revealed an unexpected extensive structural similarity between these two molecules. Despite the absence of significant similarity of their amino acid sequences, both proteins share the same core architecture and a common nucleotide binding site resembling the structure of hexokinase. All three are ATPases or kinases and bind ATP in association with Mg2+ or Ca2+. The common fold consists of two alpha/beta domains, which are connected by a putative hinge with an ATP-binding site situated between the domains. Each domain contains a five-stranded beta-sheet of identical topology, which suggests that the molecules may have evolved by gene duplication. From a comparison of the three aligned structures, a fingerprint sequence of the adenine nucleotide binding pocket was derived, which predicted that members of the glycerol kinase family should also have a similar fold of their nucleotide binding domain. This was later confirmed when the X-ray structure was published. Data base search with a refined consensus sequence has retrieved other sugar kinases, as well as the prokaryotic cell cycle proteins FtsA, MreB, and StbA, and two Escherichia coli phosphatases. These proteins are predicted to possess a structure similar to actin in the common core region. As exemplified for actin, Hsc70, and glycerol kinase, the diversity of biological function is provided by the polymorphism of the loops joining the beta-strands and helices in the core region and by inserted domains that show high variability.

ATP conversion to ADP is a central process in all living organisms and is catalyzed by a vast number of different enzymes. The energy generated can drive metabolic processes, directed transport, force-generation and movement as well as signal transduction and regulation. While ATPases generate ADP and free phosphate, kinases transfer the terminal phosphate of ATP to a wide variety of substrates, from metabolic intermediates to proteins, so controlling their activity. Hence, assays to monitor ADP concentrations have wide applications in biochemical and biomedical research, ranging from detailed understanding of mechanochemical coupling in motor proteins to screening for ATPase and kinase inhibitors.Fluorescent, protein-based biosensors have been reported for a number of biomolecules such as sugars, amino acids, metal ions and phosphate. This approach takes advantage of the highly specific interaction of a protein with the target molecule, which can be coupled to an optical signal by attaching fluorophores in suitable positions on the protein. Following this strategy, we have developed sensors for ADP based on fluorescently labelled mutants of the bacterial actin homologue ParM. We report two ADP sensors with distinct optical properties and ADP-binding characteristics, suitable for different types of in vitro assays. A coumarin-labeled variant binds ADP tightly and fast and can detect submicromolar concentrations of ADP. The sensor is particularly useful for mechanistic studies, where high sensitivity and high time resolution are required. The second variant is labeled with two rhodamine dyes, exploiting the stacking of rhodamines to generate a signal change. This variant has a more photostable fluorophore, higher wavelength excitation and lower ADP binding affinity, making it more suitable for high-throughput screening assays.