The bacterial cytoskeleton Joe Pogliano

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1 Available online at The bacterial cytoskeleton Joe Pogliano Bacteria contain a complex cytoskeleton that is more diverse than previously thought. Recent research provides insight into how bacterial actins, tubulins, and ParA proteins participate in a variety of cellular processes. Addresses Division of Biological Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA , United States Corresponding author: Pogliano, Joe (jpogliano@ucsd.edu) This review comes from a themed issue on Cell structure and dynamics Edited by Yixian Zheng and Karen Oegema /$ see front matter Published by Elsevier Ltd. DOI /j.ceb Introduction Bacterial cells have a complex subcellular organization that is established and maintained by a diverse set of polymerizing proteins that make up the bacterial cytoskeleton. At least three general classes of dynamic polymers have been identified: proteins with homology to the eukaryotic polymers actin and tubulin, and members of the ParA/MinD family. Among the bacterial actins, at least five different families have been characterized and shown to participate in many processes, including cell division, maintaining cell shape, positioning bacterial organelles, and catalyzing DNA segregation. Most known bacterial tubulins are closely related and are required for cell division, but recent work has identified additional divergent members that participate in plasmid DNA replication or segregation. The ParA/ MinD superfamily of ATPases form a large and diverse set of proteins that rely upon their dynamic assembly properties to mediate the localization of many types of protein complexes within the cell and for catalyzing the segregation of both plasmid and chromosomal DNA. Several in-depth reviews have recently focused on the bacterial cytoskeleton [1 4]. This review highlights recent progress on these three highly conserved classes of cytoskeletal proteins with an emphasis on new insights into how they function and on the identification of recently discovered family members. Bacterial tubulins FtsZ One of the first cytoskeletal proteins recognized in bacteria was the tubulin homolog FtsZ. The sequences of FtsZ from bacteria and archaea form a family of highly conserved proteins that are very divergent from eukaryotic tubulins, with only amino acids involved in GTP binding and hydrolysis conserved between the two families [5 8]. Despite this divergence the three-dimensional structures of FtsZ and tubulin are very similar, suggesting they evolved from a common ancestor [5 10]. Like tubulin, FtsZ polymerizes cooperatively and in a GTP-dependent manner in vitro [7 12]. FtsZ is an essential component of the cell division apparatus, assembling a cytokinetic ring at midcell required to recruit other members of the cell division complex [5,8 10,13 16]. The FtsZ ring constricts with septum invagination and reassembles at new division sites from spirals of FtsZ [17 20]. In addition to recruiting septal biogenesis enzymes to the cell midpoint, recent reports implicate FtsZ in affecting peptidoglycan synthesis along the side wall as well [21,22 ]. In vitro, purified FtsZ assembles protofilaments, tubes and sheets under a variety of different polymerization conditions, but how FtsZ polymers are arranged in vivo has been unclear. New techniques such as electron cryotomography that allow high-resolution imaging of cells in a near-native state [23,24,25 ] promise to reveal the in vivo structure of FtsZ and many other bacterial cytoskeletal filaments. The first high-resolution glimpse of the FtsZ ring of Caulobacter crescentus using electron cryotomography was recently provided by Li et al. [26 ]. FtsZ rings were observed to consist of multiple, short (100 nm) overlapping protofilaments approximately 5-nm wide (Figure 1a). Surprisingly, these filaments always occurred about 16 nm away from the cell membrane, suggesting the existence of an adaptor protein that links the filaments to the membrane. BtubA/BtubB At least eight families of tubulin have been described in eukaryotes, while in bacteria the only tubulin relative recognized for many years was FtsZ. The availability of genomic sequences recently led to the identification of several additional families of tubulin-like proteins encoded within bacterial and archaeal genomes [8,27,28 30]. A pair of tubulin homologs, BtubA and BtubB, characterized from Prosthebacter dejoneii were shown to be closely related to a and b tubulin and assemble as a heterodimer into GTP-dependent polymers in vitro [28 30]. BtubA/BtubB were probably acquired from a eukaryotic cell by horizontal gene transfer. The functions of the BtubA/BtubB polymers within Prosthebacter are currently unknown [28 30].

2 20 Cell structure and dynamics Figure 1 Progress in understanding the bacterial cytoskeleton is revealed in a collection of cell biology images from the last year. (a) A reconstruction of FtsZ protofilaments (red) near the inner membrane (blue) based on electron cryotomography of C. crescentus. The outer membrane is shown in green. The panel on the right shows the localization of FtsZ-GFP at the division site of C. crescentus. Reprinted from [26 ] with permission from the publisher. (b) TubZ-GFP assembles polymers required to stably maintain plasmid pbtoxis in Bacillus thuringiensis [27]. (c) Fluorescently labeled ParM (green) polymerizes between two beads (yellow) coated with parc DNA bound with ParR, pushing the beads apart over time (s). The right two panels show electron microscopy images of ParM filaments attached to the beads. Reprinted from [77 ] with permission from the publisher. (d) A phylogenetic tree showing the relationship of several of the known families of bacterial actins. The bottom panel shows that the B. subtilis plasmid segregation protein AlfA assembles polymers (green) extending throughout the cell (red membranes). FRAP experiments (right two panels) show that AlfA-GFP filaments dynamically exchange subunits. Reprinted from [80 ] with permission from the publisher. (e) C. crescentus MipZ interacts with ParB at the cell poles and assembles a protein gradient (graph) that prevents FtsZ from assembling near the poles, thereby favoring FtsZ assembly at midcell. Reprinted from [103 ] with permission from the publisher. (f) V. cholerae ParA1-GFP (red) migrates in front of the separating YFP-ParB-labeled origins (green), suggesting a mitotic mechanism in which ParA pulls the origins apart. Panels I through VI show different cells at various stages of the cell cycle. Reprinted from [114] with permission from the publisher. TubZ and RepX Many bacteria and archaea encode relatives of tubulin and FtsZ that are so vastly divergent that they do not fit into either family [8,27 ]. All of the divergent bacterial tubulins identified thus far are encoded by large plasmids in various species of Bacillus [27 ]. Recent work demonstrates that some of these proteins comprise a previously unrecognized tubulin-based bacterial cytoskeleton. The

3 The bacterial cytoskeleton Pogliano 21 first member of this family shown to polymerize was TubZ from Bacillus thuringiensis [27 ]. TubZ is encoded by pbtoxis, a virulence plasmid that carries several of the insecticidal crystal toxins for which B. thuringiensis is well known [31]. TubZ-GFP fusions assemble dynamic polymers in B. thuringiensis that span the length of the cell [27 ](Figure 1b). In time-lapse microscopy and FRAP experiments, TubZ-GFP polymers are polarized with plus and minus ends and translocate through the cell by a treadmilling-type mechanism. TubZ can assemble by itself in either B. thuringiensis or Escherichia coli, and appears to have a critical concentration for assembly in vivo. TubZ appears to play an important role in stably maintaining plasmid pbtoxis. A mutant TubZ protein (TubZD269A) predicted to be defective in GTP hydrolysis assembles static rather than dynamic polymers. When the mutant protein is expressed in trans from a compatible plasmid, it coassembles with wild-type TubZ, trapping it in a nonfunctional form, and this leads to loss of pbtoxis from the cell [27 ]. TubZ is encoded in an operon together with TubR, a DNA-binding protein that regulates TubZ expression. It therefore seems likely that TubZ and TubR are essential components of a plasmid maintenance machinery, but their precise roles are still not understood. Given TubZ s dynamic assembly properties, one possibility is that TubZ plays a role in plasmid DNA segregation, potentially representing a very simple and ancient tubulin-based mitotic apparatus. However, as discussed below, these proteins might be also involved in DNA replication. How conserved are the polymerization properties of TubZ? At least four other Bacillus plasmids encode tubulin-like proteins, each very distantly related to the other and to TubZ [27 ]. One of these, RepX encoded by plasmid px01 of Bacillus anthracis, was identified as an important component of plasmid replication [32 ]. A mini-replicon constructed from px01 could only be introduced into B. anthracis by electroporation if an intact copy of RepX was present. A parallel finding was also made for pbtoxis, where a mini-replicon containing TubZ and TubR was constructed [33]. RepX was shown by electron microscopy and dynamic light scattering to undergo dynamic, GTP-dependent polymerization in vitro [34]. RepX has a GTPase activity that is required to establish the plasmid in vivo during transformation experiments [32 ]. Taken together, it is now clear that these divergent tubulins (TubZ and RepX) are important for plasmid stability, functioning in replication, segregation, or possibly both. It seems likely that these functions are conserved among the TubZ-like proteins encoded by plasmids in B. megaterium and B. cereus. A function in DNA stability might also be conserved among the archaeal TubZ-like proteins, many of which are also plasmid-encoded. However, given the extreme divergence of the archaeal proteins, they might have alternative functions, raising the possibility that divergent tubulin homologs, like divergent bacterial actins, assemble a variety of different types of polymers that participate in many different aspects of cellular physiology. Bacterial actins MreB Bacteria contain many proteins distantly related to eukaryotic actins. FtsA, MreB, and ParM were long ago recognized to contain key amino acid motifs conserved within the larger actin/hsp70/hexokinase superfamily [35]. Elucidation of the crystal structure of MreB and the discovery that it assembles filaments in vitro and in vivo demonstrated that these divergent actins are part of an essential bacterial cytoskeleton that probably arose billions of years ago [9,36 39]. MreB and closely related proteins (such as B. sutbilis Mbl and MreBH) assemble dynamic polymers that move rapidly in a tight spiral pattern beneath the cell membrane in many different organisms [37,38,40 44]. The mechanism of movement could be via treadmilling, as reported for MreB-YFP in C. crescentus [45]. Purified MreB from Thermotoga maritima assembles actin-like polymers in the presence of either GTP or ATP [36,39,46 ]. Proteins of the MreB family have several important functions, the most conserved being a direct role in maintaining cell shape by influencing the position of peptidoglycan synthesis [37,38,42,47 52]. Cell shape control requires the concerted actions of MreB with several other proteins, including MreC, MreD, and Pbp2 [53 59]. Many bacteria contain only a single MreB protein, but in B. subtilis, cell growth depends upon three closely related proteins, MreB, MreBH, and Mbl, all of which colocalize within the cell [37,48,60 ]. MreBH interacts with a cell wall hydrolyase (LytE) and directs its localization in a helical pattern, providing a potential mechanism by which MreBH can directly influence peptidoglycan structure [60 ]. In addition to maintaining cell shape, MreB participates in a number of other functions within the cell including protein localization and chromosome segregation (reviewed in [1,4]) [41,51,56,61 69]. The extent to which MreB directly functions in chromosome segregation is still being investigated in some organisms [66,68], but in an elegant study of C. crescentus, a direct role for MreB in chromosomal origin separation was established [67]. Using a small molecule inhibitor (A22) that allowed the rapid inhibition of MreB function in synchronized cell cultures [67], inactivation of MreB prevented segregation of GFP-tagged chromosomal origins without affecting DNA replication. Future studies using small molecules to inhibit MreB and other cytoskeletal proteins will probably be instrumental in deciphering the many functions of these dynamic proteins.

4 22 Cell structure and dynamics ParM ParM is a bacterial actin required for segregation of the E. coli plasmid R1 [70 72]. MreB and ParM have threedimensional structures similar to actin, though all three share an incredibly low level of sequence similarity (<12%) [36,73]. ParM assembles polymers that act together with the ParR DNA-binding protein and its cognate DNA recognition sites (clustered within a centromere-like region of DNA, parc) to segregate plasmid DNA [74,75]. The polymerization dynamics of ParM are very different from actin [76,77 ]. Purified ParM displays dynamic instability in vitro in which spontaneously nucleated filaments extend bidirectionally at equal rates and rapidly decay if not stabilized by interactions with ParR/parC nucleoprotein complexes [76]. Remarkably, this entire system was reconstituted in vitro with only these three components [77 ]. Fluorescently labeled ParM assembled a radial array of dynamically unstable polymers when added to beads coated with parc DNA and ParR. When both ends of a dynamic polymer became captured by parc/parr complexes, polymerization continued by incorporation of new subunits adjacent to the nucleoprotein complexes, driving the beads rapidly apart [77 ](Figure 1c). A refined model for ParM filaments recently demonstrated that they have a left-handed twist rather than a right-handed twist like actin [78 ]. ParM, like other actins, contains a central nucleotide-binding cleft formed by two domains of the protein. The degree of opening between the two domains of a single monomer is proposed to contribute to the degree of filament twist observed in vitro [78 ]. The model generated suggests that the subunit subunit contacts within the ParM filaments are different from that of F-actin. These new results lead to the idea that divergent actin relatives may assemble a variety of different structures. Given the significant sequence divergence of many bacterial actins, even within the MreB family, the implication is that there may in fact be a large number of ways actin-like proteins have evolved to assemble a polymer. Evidence for this last point awaits the structural characterization of additional divergent members of the bacterial actin superfamily. How do ParM filaments attach to the plasmid DNA? An elegant structure provided by Moller-Jensen [79 ] now provide a possible mechanism by which ParR may provide a link to the filament. The DNA binding N-terminus of ParR forms a ribbon helix helix structure that binds cooperatively to 10 sites within parc [75,79 ]. Surprisingly, ParR dimers assemble into a gently curved helix with 12 dimers required to make one full turn. The DNA recognition domains are positioned with a spacing of 3.5 nm on the outside of the ParR helix to make sitespecific contacts as the DNA wraps around. This helical structure, which appears as a large donut when viewed on end in the electron microscope, predicts that several amino acids on the outer helix should be important for DNA binding. Mutations in these amino acids eliminate both DNA binding and segregation functions, providing additional support for the model. Another surprising aspect of the structure is the presence of a small (6 nm diameter) hole just large enough to accommodate a ParM filament if amino acids of the flexible C-terminal helix of ParR give way. These in vitro studies of ParM and ParR suggest a detailed model for the architecture of the plasmid partition complex in which ParM filaments interact with the internal C-terminus of the ParR helix bound to parc DNA. Two ends of spontaneously nucleated ParM filaments become stabilized by ParR/parC, and as ParM-ATP monomers add to both growing ends of a filament, multiple sites of interaction with the ParR helix increase processivity, driving plasmids apart without letting go. An elegant feature of the ParR helix structure is its complete symmetry, allowing ParM filaments to interact with plasmids from either side. AlfA Another highly divergent actin relative, AlfA, was recently shown to play a role in segregating plasmid DNA during both vegetative growth and sporulation in B. subtilis [80 ]. The process of sporulation in B. subtilis poses a unique challenge for plasmid inheritance, because plasmids must localize to one extreme end of the cell before polar septation to be inherited by the spore [80 ]. Actin-like polymers that assemble between plasmids and push them toward the cell pole would be ideally suited for this function. AlfA assembles polymers that can extend the entire length of the cell (Figure 1d). Fluorescence recovery after photobleaching (FRAP) experiments demonstrate that these polymers are highly dynamic, rapidly exchanging subunits with a cytoplasmic pool. Like many other plasmid segregation systems, a DNAbinding protein, AlfB, is also required for segregation and probably serves as an adaptor protein connecting the filament to the DNA. Unlike ParM, dynamic instability has yet to be observed for AlfA, raising the question of how similar the mechanism of AlfA-mediated segregation is to that described for ParM. MamK Magnetotactic bacteria synthesize unique organelles called magnetosomes that they use to align themselves in a geomagnetic field. In Magnetospirillum species, magnetosome formation depends upon a cytoskeletal network composed at least in part of MamJ and MamK [81,82,83,84,85]. As visualized by electron cryotomography, the actin homolog MamK assembles a series of 6-nm wide linear filaments that extend over much of the length of the cell and provide a scaffold for the assembly of membrane invaginations containing magnetite (magnetosomes). MamJ is thought to be required to attach magnetosomes to the polymer [84 ]. MamJ and MamK

5 The bacterial cytoskeleton Pogliano 23 directly interact and mutations in the genes encoding either protein disrupt the linear arrangement of magnetosomes [81,83,84 ]. Purified MamK assembles into bundles of filaments in vitro [85]. It will be interesting to see a comparison of the biochemical and structural properties of MamK with ParM, MreB, and AlfA. FtsA Sequence and structural data show that the cell division protein FtsA forms a separate and divergent family of bacterial actins [35,86]. FtsA localizes to the future site of cell division by interacting with the C-terminus of FtsZ and contributes to the recruitment of other members of the cell division complex [10,13 15]. For many other bacterial actins such as ParM, MreB, and AlfA, the ability to assemble dynamic polymers is central to their function, but for FtsA, the role of polymerization, if any, is currently unclear. Recent studies have shown that S. pneumoniae FtsA assembles polymers in vitro [87], suggesting that polymerization might also play an important role in FtsA function, but this function remains poorly defined. Dynamic localization of proteins belonging to the ParA/MinD superfamily The ParA/MinD family of P-loop ATPases [88] are highly conserved in bacteria and are key components of the bacterial cytoskeleton. Two different subfamilies, MinD and ParA, play a multitude of roles in bacterial subcellular organization. MinD and related proteins were recognized long ago as spatial regulators of the site of cell division, but only more recently were they found to assemble polymers that oscillate rapidly within the cell. ParA proteins form dynamic polymers that catalyze the segregation of plasmid and chromosomal DNA, and more recently have been shown to determine the position of other protein complexes within the cell. MinD In E. coli, MinD works together with MinC and MinE to determine the position of cell division by specifying the assembly of FtsZ at the cell midpoint. MinD is a polymerizing ATPase [89 91] that associates with the membrane via its C-terminus [92 94] and also with MinC to form a complex (MinC/MinD) that inhibits FtsZ polymerization. MinC and MinD cycle from pole to pole along the cell membrane, generating a temporal protein gradient in which the highest time-averaged concentration occurs near the cell pole thereby preventing polar FtsZ assembly [95 98]. Assembly of FtsZ over the nucleoid is inhibited by SlmA in E. coli [99] and Noc in B. subtilis [100]. Although MinC and MinD are conserved in many bacteria, surprisingly, oscillation is not. In B. subtilis, MinC and MinD localize statically to the poles by interactions with DivIVA [101,102], making it unclear whether dynamic polymerization is an evolutionarily conserved feature of MinD function. C. crescentus MipZ An elegant strategy for determining the position of FtsZ assembly independently of the Min system was recently elucidated in C. crescentus, where the ParA family member MipZ was shown to couple chromosome segregation to cell division [103 ]. At the beginning of the cell cycle, MipZ localizes to the chromosomal origin of replication (oric) via the DNA-binding protein ParB. After DNA replication, one of the daughter origins migrates to the opposite cell pole simultaneously with MipZ, which is also an inhibitor of FtsZ polymerization. The arrival of MipZ and oric at the opposite pole stimulates the release of a pool of FtsZ from this location, thereby coupling FtsZ assembly to DNA segregation. MipZ localizes to both poles after oric separation and establishes a protein gradient within the cell that extends inward from each pole (Figure 1e), providing a mechanism for directing the assembly of FtsZ in the center of the cell. Plasmid DNA segregation by ParA ParA ATPases are important for the efficient segregation of both plasmid and chromosomal DNA in many bacteria [3,71]. ParA proteins usually occur in an operon together with a DNA-binding protein, ParB, which interacts with a set of specific DNA-binding sites that form the equivalent of a simple centromere parc [3,71]. Plasmids with ParA partitioning systems such as F, P1, and RK2, are positioned in the middle of the cell, where they remain as the cell continues to grow. After replication, daughter plasmids are separated slightly and generate two distinct plasmid complexes that later rapidly separate from each other at approximately 0.2 mm/min. During the process of separation, daughter plasmids are repositioned to the quarter-cell positions, that is, to what will be the midcell positions of the future daughter cells. The ATPase activities of ParA proteins are presumed to play a central role in targeting plasmids to midcell and in driving plasmid separation, but the underlying mechanisms are still unclear. Recent studies from several different systems show that plasmid ParA proteins assemble polymers in vivo and in vitro [104,105,106,107,108]. In many instances, ParA-GFP fusions oscillate within the cell and the cognate ParB/parC is required for this dynamic localization [104, ]. These results have led to a model in which ParA proteins assemble polymers that cycle back and forth both between plasmids and on either side of them, such that when the forces generated by the polymers are balanced in all directions, a single plasmid is held near the cell midpoint, while two plasmids are held near the quarter cell positions [107,108]. This model is strikingly different from the ParM model, in which a single, linear filamentous structure (or bundles of filaments) mediates plasmid separation [74 76,77 ].

6 24 Cell structure and dynamics Chromosome segregation by ParA proteins A number of bacteria contain ParA proteins with a role in chromosome segregation [113]. One recent example is Vibrio cholerae, whose genome is divided into two chromosomes (large and small) that replicate and segregate separately from each other. Both chromosomes rely upon proteins related to ParA for efficient segregation [114,115,116]. ParA1 from V. cholerae mediates the segregation of the origin of replication region of the large chromosome from one cell pole to the other [114 ]. Cells depleted for ParA1 display chromosome I segregation defects. ParA1-GFP assembles into a cytoplasmic haze of fluorescence that, in time-lapse microscopy experiments, migrates in front of the moving origin of replication (Figure 1f). These results suggest a model for chromosome segregation in which ParA1 assembles a mitotic machinery that pulls the daughter origins apart [114 ]. The smaller chromosome relies upon its own ParA system (ParA2) to position the origin of replication near the mid- and quarter cell positions [115 ]. ParA and the localization of cytoplasmic protein complexes Members of the ParA family have probably evolved to participate in localizing many different types of protein complexes in bacteria [117,118 ]. In R. sphaeroides, for example, complexes of chemotaxis proteins localize to the midcell and quarter cell positions [118 ]. A ParA homolog (PpfA) encoded within the chemotaxis operon was found to be responsible for positioning these chemotaxis complexes. In para mutants, the complexes are mislocalized and the cells are nonmotile, demonstrating that positioning by PpfA is essential for their function. The similarities between the localization behavior of bacterial plasmids and the chemotaxis proteins suggest a common positioning mechanism is at work. Crescentin Crescentin is an intermediate filament protein identified in C. crescentus in a screen for cell shape mutants [119]. Crescentin assembles a slightly curved filamentous structure that contributes to Caulobacter s characteristic comma shape appearance. In the absence of crescentin, cells become rod-shaped but are otherwise viable. The dispensability of intermediate filaments for Caulobacter growth and their general absence from many bacteria suggests that it serves a specialized role in these organisms, perhaps contributing to formation of a cell shape that provides a selective advantage as Caulobacter propels itself through aqueous environments. Conclusions The recent identification of many new families of bacterial polymers (MreB, ParM, MamK, AlfA, TubZ, ParA, and crescentin) suggests that the bacterial cytoskeleton is more diverse and complex than previously thought. This point was made beautifully most recently by Briegel et al. [25 ]. Using electron cryotomography, they discovered at least four distinct types of filaments within C. crescentus that could be grouped based on their size and location within the cell. Surprisingly, most of these filaments were still present when known cytoskeletal proteins such as MreB and crescentin were inactivated, indicating that many more families of bacterial cytoskeletal proteins await discovery [25 ]. An emerging paradigm in bacterial cell biology is that divergent actin relatives evolved in many different ways to harness the mechanical properties of polymerization for a variety of different processes, including cell division, cell shape, and DNA segregation. This theme likely applies to other families of dynamic polymers, including members of the ParA/MinD family that use dynamic localization to position protein and DNA molecules within the cell, and possibly also applies to divergent bacterial tubulins, which assemble dynamic cytoskeletal polymers required for plasmid maintenance. Our knowledge of cytoskeletal proteins will continue to expand with the development of more powerful fluorescence and electron microscopy tools. Acknowledgement This work was supported by a grant from the NIH (R01GM073898). References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Carballido-Lopez R: The bacterial actin-like cytoskeleton. Microbiol Mol Biol Rev 2006, 70: Shih YL, Rothfield L: The bacterial cytoskeleton. 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