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Cell Biology of Prokaryotic

Dorothee Murat, Meghan Byrne, and Arash Komeili

Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720-3102 Correspondence: [email protected]

Mounting evidence in recent years has challenged the dogma that are simple and undefined cells devoid of an organized subcellular architecture. In fact, once thought to be the purely eukaryotic inventions, including relatives of actin and control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmental- ization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of -bounded and -bounded organelles. In this article we highlight some of these prokaryotic organelles and discuss the current knowledge on their ultrastructure and the molecular mechanisms of their biogenesis and maintenance.

he emergence of in a world Skeptical readers might wonder if a pro- Tdominated by prokaryotes is one of the karyotic structure can really be defined as an defining moments in the of modern . Here we categorize any compartment day organisms. Although it is clear that the cen- bounded by a biological membrane with a dedi- tral metabolic and information processing ma- cated biochemical function as an organelle. This chineries of eukaryotes and prokaryotes share a simple and broad definition presents cells, be common ancestry, the origins of the complex they eukaryotes or prokaryotes, with a similar eukaryotic cell plan remain mysterious. Eukary- set of challengesthat need to be addressed to suc- otic cells are typified by the presence of intracel- cessfully build an intracellular compartment. lular organelles that compartmentalize essential First, an organism needs to mold a cellular mem- biochemical reactions whereas their prokaryotic brane into a desired shape and size. Next, the counterparts generally lack such sophisticated compartment must be populated with the pro- subspecialization of the cytoplasmic space. In per set of proteins that carry out the activity of most cases, this textbook categorization of eu- the organelle. Finally, the cell must ensure the karyotes and prokaryotes holds true. However, proper localization, maintenance and segrega- decades of research have shown that a number tion of these compartments across the cell cycle. of unique and diverse organelles can be found Eukaryotic cells perform these difficult mecha- in the prokaryotic world raising the possibility nistic steps using dedicated molecular pathways. that the ability to form organelles may have Thus, if connections exist between prokaryotic existed before the divergence of eukaryotes and eukaryotic organelles it seems likely that from prokaryotes (Shively 2006). relatives of these molecules may be involved in

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D. Murat, M. Byrne, and A. Komeili

the biogenesis and maintenance of prokaryotic in a variety of applied settings have made them organelles as well. center of most studies on magnetosomes (Bazy- Prokaryotic organelles can be generally div- linski and Frankel 2004). ided into two major groups based on the com- From a cell biological perspective, however, position of the membrane layer surrounding it is the often-neglected magnetosome mem- them. First are the cellular structures bounded brane that may hold the key to understanding by a nonunit membrane such a protein shell or fundamental properties of prokaryotic organ- a lipid monolayer (Shively 2006). Well-known elles. Detailed electron microscopic (EM) work examples of these compartments include lipid and biochemical studies have shown that the bodies, polyhydroxy butyrate granules, carbox- magnetosome membrane has the cytological ysomes, and gas vacuoles. The second class con- and chemical properties of a lipid bilayer mem- sists of those organelles that are surrounded by a brane (Gorby et al. 1988; Gru¨nberg et al. 2004). lipid-bilayer membrane, an arrangement that is Additionally, numerous proteomic studies have reminiscent of the canonical organelles of the shown that this compartment contains a unique eukaryotic . Therefore, mix of soluble and transmembrane domain- this article is dedicated to a detailed exploration containing proteins, implying the existence of of three prokaryotic lipid-bilayer bounded or- a dedicated protein sorting pathway (Okuda ganelle systems: the magnetosomes of magneto- et al. 1996; Gru¨nberg et al. 2001; Gru¨nberg tactic bacteria, photosynthetic membranes, and et al. 2004; Tanaka et al. 2006). The magneto- the internal membrane structures of the Planc- some membrane loaded with its protein cohort tomycetes. In each case, we present the most is present before crystal formation and serves as recent findings on the ultrastructure of these the site of biomineralization further confirming organelles and highlight the molecular mecha- that it is an independent organelle (Komeili nisms that control their formation, dynamics, et al. 2004). The organization of magnetosomes and segregation. We also highlight some pro- into one or multiple chains also suggests that tein-bounded compartments to present the mechanisms must exist for the proper localiza- reader with a more complete view of prokaryotic tion and division of this structure within the compartmentalization. cell. This already detailed view of the magneto- some has been pushed to the next level with two recent imaging studies that describe the use of Magnetosomes: Bacterial Compasses cryo-electron tomography (CET) to obtain The magnetosomes of magnetotactic bacteria high resolution three-dimensional images of (MB) are one of the most fascinating prokary- MB (Komeili et al. 2006; Scheffel et al. 2006). otic compartments (Fig. 1). MB are a phyloge- In CET a series of two-dimensional images of netically diverse group of microorganisms with a specimen, taken by tilting the stage of an elec- the ability to use geomagnetic field lines as tron microscope at various angles relative to the guides in their search for their preferred redox electron beam, is translated into a three-dimen- conditions (Bazylinski and Frankel 2004; Kom- sional image using a specific algorithm. This eili 2007). This behavior is achieved through the technique provides such a detailed view of a use of a unique magnetic organelle termed the cell that disruptive fixing and staining treat- magnetosome. A magnetosome consists of a ments common in other EM techniques are lipid bilayer membrane that houses an approx- not needed. As a result one can prepare a sample imately 50-nanometer crystal of the magnetic by a simple rapid freezing method and sub- mineral magnetite (Fe3O4) or greigite (Fe3S4). sequently image a cell at high resolution in a Individual magnetosomes are arranged into one near-native state (Milne and Subramaniam or more chains within the cell where they act 2009). This combination of rapid preservation, passively to orient the bacterium within a mag- minimal disruption of cellular features, and netic field. The unusual properties of these mag- nanometer scale resolution revealed features of netic minerals and their potential to be exploited magnetosomes that had not been visualized in

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Cell Biology of Prokaryotic Organelles

Figure 1. Magnetosomes can be easily visualized with various forms of electron microscopy. The electron-dense magnetite crystals are seen as a chain running through the cell in (A). Cryo-electron tomography was instrumen- tal in demonstrating that the magnetosome membrane is an invagination of the inner cell membrane (B) and cytoskeletal filaments surround the magnetosome chain (C). (A, Reprinted, with permission from Komeili et al. 2004 [# National Academy of Sciences]; B, reprinted with permission from Komeili et al. 2006 [# AAAS]; C, image courtesy of Zhuo Li and Grant Jensen.)

more than 30 years of work on MB. Most strik- crystals implying that this organelle is an invag- ing was the finding that in Magnetospirillum ination of the inner membrane at all times magneticum AMB-1, individual magnetosomes (Komeili et al. 2006). Although such an organ- are not separated into vesicles and are instead in- ization might seem puzzling at first it does make vaginations of the inner cell membrane (Fig. 1B). sense in the context of magnetosome function This state was observed in empty magnetosomes and magnetite biomineralization. Because the as well as those that contained fully formed primary job of the magnetosome chain is to

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D. Murat, M. Byrne, and A. Komeili

orient the cell in external magnetic fields the or- magnetosome island or MAI, carries signature ganelle must be attached to the rest of the features of other genomic islands found in bac- cell and by integrating the magnetosome into teria and encompasses a substantial portion of the cell membrane no additional machinery is the genome. For instance, in AMB-1 the MAI needed to achieve proper orientation in mag- is predicted to contain over one hundred genes netic fields. It has also been hypothesized that accounting for approximately 2% of the organ- the biomineralization of magnetite may involve ism’s gene content (Fukuda et al. 2006). From the formation of precursor minerals such as fer- an evolutionary perspective the organization rihydrite in the periplasmic space (Frankel et al. of core magnetosome genes into an unstable 1983). In such a case the small opening between genomic segment implies that the appearance the magnetosome lumen and the periplasm of this organelle in diverse bacterial species was would provide a simple path for the transport accomplished through lateral transfer of the of these precursor minerals. The CET imaging MAI (Jogler et al. 2009). What makes the MAI of Magnetospirillum gryphiswaldense MSR-1, intriguing to cell biologists is the possibility an organism closely related to AMB-1, did not that it contains the unique functions required specifically explore the existence of any connec- to build a magnetosome. Biochemical and ge- tions between the magnetosome membrane and netic studies have shown that a number of MAI the inner cell membrane (Scheffel et al. 2006). genes encode proteins that can influence the size However, in this organism the magnetosomes and morphology of magnetite crystals (Arakaki were found juxtaposed against the cell mem- et al. 2003; Scheffel et al. 2008; Murat et al. brane consistent with the possibility that they 2010). Other factors, such as the MamA protein, are also invaginations of the inner cell mem- appear to function in activating or priming pre- brane (Scheffel et al. 2006). These tremendous formed magnetosomes for biomineralization imaging studies have revealed the organization (Komeili et al. 2004). And, as described later, and ultrastructure of the magnetosome at nano- one core region of the MAI is essential for the meter scales and recent studies are beginning formation of the magnetosome membrane, to define the molecular basis of magnetosome protein sorting to this organelle and its specific formation and organization. localization within the cell (Komeili et al. 2006; MB are fastidious and slow growing or- Scheffel et al. 2006; Murat et al. 2010). ganisms but offer multiple advantages as model At the heart of the MAI is the mamABE systems for the molecular study of organelle for- operon, a gene cluster conserved in multiple mation in prokaryotes. Multiple MB genomes species of MB. A comprehensive genetic analysis have been sequenced in the past few years, mag- of the MAI showed that in the absence of the netosomes can be readily purified from cell ex- mamABE operon, AMB-1 is nonmagnetic and tracts using simple magnetic columns and most fails to even form empty magnetosome mem- importantly, magnetosomes are not essential branes (Murat et al. 2010). An analysis of indi- for cell survival under laboratory growth condi- vidual deletions of each of the 18 genes of this tions, opening the door to the use of genetics as cluster revealed a range of mutant phenotypes a tool for uncovering the steps involved in mag- with defects at every step of magnetosome for- netosome formation. A combination of these mation. Interestingly, four genes, mamI, mamL, approaches has led to the identification of a mamQ and mamB, seem to be essential for the large list of genes thought to be involved in the formation of the magnetosome membrane formation and function of magnetosomes. Sur- (Murat et al. 2010). None of these genes en- prisingly, most of these genes are organized into codes for proteins with homology to known a coherent and unstable genomic region whose membrane deformation factors found in eukar- core components are conserved across multi- yotes. However, they contain intriguing features ple species of magnetotactic bacteria (Ullrich that may hint at a potential mechanism for mag- et al. 2005; Fukuda et al. 2006; Richter et al. netosome formation. MamB and MamQ share 2007; Jogler et al. 2009). This region, termed the homology with large families of membrane

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Cell Biology of Prokaryotic Organelles

proteins whereas MamI and MamL are unique structures similar to the magnetosome-specific to MB. These two latter proteins are small cytoskeleton can still be seen by CET but they (70 ) polypeptides with two pre- are no longer associated with magnetosomes. dicted transmembrane domains. MamI does Given that MamJ can associate with MamK in not possess any distinguishing structural fea- a bacterial two-hybrid system a simple and tures but MamL contains a cytoplasmic tail attractive model has been proposed whereby that is rich in positively charged residues. One MamJ can anchor MamK to the magnetosome potential model for membrane deformation is membrane allowing it to organize individual that this tail interacts with one leaflet of the organelles into a chain (Scheffel et al. 2006; inner cell membrane creating an asymmetry Scheffel and Schu¨ler 2007). Taken together that favors the bending of the membrane. An- these results suggest that similar to eukaryotic other finding of this work is that membrane for- cells, prokaryotes can take advantage of cytoske- mation can be decoupled from the sorting of at letal elements to position and organize subcel- least a subset of magnetosome proteins. When lular compartments. the putative protease, MamE, is absent, empty As can be seen, progress in the study of magnetosome membranes are still formed al- magnetosomes has been rapid in the last few though a number of magnetosome proteins are years and the incredible gains made from the mislocalized arguing for a step-wise assembly of ultrastructural characterization of this organelle this organelle (Murat et al. 2010). are beginning to be matched with molecular One of the central genes of the mamABE studies. Yet, much work remains to be per- operon, mamK, is homologous to the large and formed. Although the discovery and genetic diverse family of bacterial actin-like proteins analysis of the MAI provides a potential “parts discovered in the last decade (Carballido-Lo´pez list” for the magnetosome formation machi- 2006). When mamK is deleted in AMB-1, the nery, the specific mechanisms that control resulting mutants are not defective in magneto- membrane biogenesis and protein sorting have some membrane formation or biomineraliza- yet to be defined. Moreover, even though the tion of magnetite. Instead, magnetosomes are discovery of the MamK/MamJ system for chain no longer organized into chains and are spread formation is a breakthrough advance in this out across the cell membrane (Komeili et al. field, its mechanism of action remains elusive. 2006). The CET imaging studies of AMB-1 A resolution of these key issues is necessary and MSR-1 had also discovered that the magne- before evolutionary comparisons can be drawn tosome chain is surrounded by a network of between eukaryotic organelles and magneto- cytoskeletal filaments with dimensions similar somes. The elucidation of these key cellular to bacterial actin-like filaments (Komeili et al. mechanisms will also provide new modes for 2006; Scheffel et al. 2006) (Fig. 1C). Interest- exploitation of magnetosomes in a variety of ingly, these filaments are no longer present applied settings. when mamK is deleted (Komeili et al. 2006). Together with recent observations that MamK Photosynthetic Membranes: Variations can form filaments in heterologous systems on a Theme and in vitro, these results suggest that this actin- like protein constitutes the structural compo- Photosynthetic membranes are perhaps the nent of the magnetosome-specific cytoskeleton most thoroughly studied of all prokaryotic (Pradel et al. 2006; Taoka et al. 2007). MamJ, a organelles (Fig. 2). They fall into three general highly acidic protein encoded by a gene directly categories and each has unique and intriguing upstream of mamK, seems to play a crucial role characteristics that make it ideal for the study in the organization of magnetosome chain as of membrane dynamics and intracellular organ- well. When mamJ is deleted in MSR-1, the ization in prokaryotes. The first category, histor- magnetosome chain collapses into a ball within ically referred to as chromatophores, contains the cell (Scheffel et al. 2006). In this mutant, the various intracytoplasmic membrane (ICM)

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D. Murat, M. Byrne, and A. Komeili

Figure 2. Photosynthetic membranes were the first of bacterial organelles to be imaged with electron microscopy. (A) is an image from a 1967 imaging study of Rhodopseudomonas palustris. The photosynthetic membranes (Th) are arranged as ribbon-like structures that are clearly continuous with the inner cell membrane (CM) at the point indicated by the arrow. These features are revealed in three dimensions in a surface rendered reconstruction of Rhodopseudomonas viridis in (B). Thylakoid membranes of (C) are arranged in several circular layers and display species-specific morphologies. In contrast with photosynthetic membranes of purple bacteria, thylakoids appear to be fully separated from the inner cell membrane. (A, Reprinted, with permission, from Tau- schel and Drews 1967 [# Springer]; B, reprinted, with permission, from Konorty et al. 2008 [# Elsevier]; C, reprinted, with permission, from Nevo et al. 2007 [# Nature Publishing Group].)

structures that house the photosynthetic pro- the efficiency of photosynthesis by increasing tein complexes of the purple photosynthetic the number of available photosynthetic protein bacteria. The second category consists of the complexes, maximizing the size of the light- numerous examples of thylakoid membrane exposed membrane surface and by providing compartments found in cyanobacteria. The an idealized subcellular environment for this chlorosome compartments of green photosyn- vital reaction. However, despite their functional thetic bacteria constitute the third major cate- relatedness these organelles differ in fundamen- gory of bacterial photosynthetic compartment. tal ways that impact the mechanisms by which All of these organelle systems act to maximize they are formed and maintained.

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Cell Biology of Prokaryotic Organelles

Chromatophores were first studied bio- Donohue 2006). These protein complexes in- chemically where it was shown that a defined clude the light harvesting 2 (LH2) protein com- fraction of cellular extract was capable of carry- plex and the “core” complex consisting of the ing out certain light-dependent reactions in multimeric light harvesting 1 (LH1) and reac- vitro. Elegant EM imaging of different species tion center (RC) polypeptides. In some species, of purple phototrophic bacteria showed the dimers of core complexes are formed through a presence of extensive intracellular membranes linkage with the PufX protein. These protein with distinct and species-specific morpholog- complexes are first assembled in the inner cell ical characteristics (Oelze and Drews 1972). membrane at sites that will invaginate to form For instance, the photosynthetic membranes chromatophores (Tavano and Donohue 2006). of Rhodopseudomonas palustris appear as neatly Genetic studies with R. sphaeroides have shown folded membrane stacks that are continuous that in the absence of LH2 the chromatophore with the cell membrane (Tauschel and Drews membranes lose their characteristic stacked 1967) (Fig. 2A). In contrast, in the Rhodobac- spherical shape and instead turn into long tericiae species these structures are spherical tubules within the cell (Tavano and Donohue invaginations of the inner membrane where 2006). Interestingly, when pufX is deleted in multiple bubbles are connected to one another. these LH2-deficient strains the membrane tu- These early ultrastructural studies have recently bules disappear and instead large spherical been augmented with the CET imaging of internal membranes are observed (Tavano and Rhodopseudomonas viridis, an organism that Donohue 2006). Thus, the major protein com- forms membranes that are similar in morphol- ponents of chromatophores play a decisive role ogy to those observed in R. palustris (Konorty in determining the morphology of the mem- et al. 2008) (Fig. 2B). The near-native state pre- brane. Recent structural and biophysical mod- served by cryofixation reveals much of the same eling studies of photosynthetic membrane features observed in traditional electron mi- proteins have built on these functional studies croscopic imaging of the same organism. Mem- to provide a mechanistic basis for the remodel- branes are folded in an accordion-like structure ing of the cell membrane into chromatophores. and they are invaginations of the inner mem- When chromatophores are imaged by atomic brane with a distinct 128 nm wide opening to force microscopy (AFM) ordered arrays of the periplasmic space (Konorty et al. 2008). In photosynthetic protein complexes are seen to many organisms, including R. viridis, chroma- densely pack the membrane surface (Sturgis tophores are produced only under photosyn- et al. 2009). Based on the known structures of thetic growth conditions. The CET imaging of these complexes distinct domains containing R. viridis at early time points after switch to dimers of the RC-LH1-PufX complex as well photosynthetic growth reveals the presence of as rings of LH2 can be placed in the AFM images small vesicular structures adjacent to the cell (Sturgis et al. 2009). Interestingly, three-dimen- membrane (Konorty et al. 2008). Presumably sional electron microscopic reconstruction of these early compartments eventually mature negatively stained single particles reveals that into the membrane stacks seen in cells grown the dimers of RC-LH1-PufX form a complex under continuous photosynthetic conditions. that is bent toward the lumen of the chromato- How are these exquisite and species-specific phore (Qian et al. 2008). An in silico model of membrane morphologies generated? The chromatophores based on this bent structure answer appears to be a simple and elegant of the core complex predicts the formation of mechanism in which the inherent properties long membrane tubules with dimensions simi- of photosynthetic protein complexes determine lar to that observed in mutants lacking LH2 the resulting shape of the membrane. The (Qian et al. 2008). Using these functional and chromatophores of purple bacteria such as structural results as a guide, other modeling R. sphaeroides house the major components studies have also supported the hypothesis that of the photosynthetic machinery (Tavano and the biophysical properties of photosynthetic

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D. Murat, M. Byrne, and A. Komeili

proteins and their long-range interactions with (Nevo et al. 2007). CET studies as well as other each other would be sufficient to produce curv- attempts to reconstruct the cellular arrange- ed membrane structures in vivo (Chandler et al. ment of thylakoids have also revealed that, in 2008). Despite these convincing arguments in contrast with the chromatophore membrane, favor of a self-assembly model for chromato- the inner cell membrane and the thylakoid phore formation, it is possible that other factors membrane are not continuous with each other may be involved in the process. For instance, a (Liberton et al. 2006; van de Meene et al. recent proteomic analysis of R. sphaeroides 2006; Nevo et al. 2007; Ting et al. 2007). The has shown that a number of proteins outside lack of connections between thylakoids and the the major photosynthetic complexes may also cell membranes has also been shown through be present within chromatophores raising the the use of various fluorescent membrane dyes prospects that novel factors may have roles in (Schneider et al. 2007). FM1-43 is a hydropho- the development of this organelle (Zeng et al. bic dye that fluoresces once incorporated into 2007). membranes and is thought to be incapable of The thylakoid membranes of cyanobacteria diffusing past the inner cell membrane. When are the evolutionary precursors of chloroplasts. it is used to stain the cyanobacterium Synecho- As with chromatophores these organelles are cystis sp. PCC 6803 the inner membrane and responsible for some of the central light-de- outer membrane are labeled but the thylakoids pendent reactions of photosynthesis. However, do not incorporate the dye indicating that these the morphology and subcellular arrangement membrane systems are separate entities or that of thylakoids is markedly different than that of a physical barrier prevents the migration of chromatophores (Fig. 2C). Several recent CET the dye to the thylakoids. In contrast when studies have corroborated earlier EM studies Mitotracker, a membrane dye that can diffuse of thylakoids and provided additional insights past cellular membranes, is used as a marker into the organization and species-specific diver- all membranes including the thylakoids are sity of this fascinating organelle (van de Meene stained in this organism. Long incubations et al. 2006; Nevo et al. 2007; Ting et al. 2007). In with FM1-43 initially stain intracellular struc- most cases thylakoids appear as several flattened tures resembling vesicles and eventually high- and stacked layers of lipid-bilayer membrane light the thylakoid membranes indicating a that encircle the cell. The number of layers mode for transfer of and proteins from and the spacing between them follows a spe- the cell membrane to this organelle (Schneider cies-specific arrangement (Nevo et al. 2007). et al. 2007). Thus, similar to eukaryotes, cyano- Although these layers cover much of the cyto- bacteria form membrane structures that are dis- plasmic space there is still substantial flow of continuous from the cell membrane implying cellular components in between the thylakoid the presence of mechanisms for bending and stacks. This is because of the presence of fission of cellular membranes. numerous perforations within the thylakoid Given their evolutionary connections, one membrane and in CET images a number of clue to the mechanisms of thylakoid membrane macromolecules such as ribosomes and storage formation has come from examining the path- granules are seen within these openings (Nevo ways of chloroplast biogenesis in plants. The et al. 2007). The three-dimensional images pro- vesicular inducing protein in plastid 1, Vipp1, vided by CETalso reveal numerous bridges and is a protein implicated in membrane remodel- fusions formed by membranes that traverse the ing and vesicular trafficking in chloroplasts in different stacks of thylakoids (Nevo et al. 2007). Arabidopsis (Kroll et al. 2001). Cyanobacteria Finally, large cytoplasmic vesicles are seen near contain homologs of Vipp1 and its absence and at times fused to the thylakoids. The highly in Synechocystis results in the loss of stacks of networked nature of this membrane system sug- thylakoid membranes (Westphal et al. 2001). gests that long-range communication and trans- These findings had suggested a possible role port may occur throughout the whole organelle for Vipp1 in the biogenesis of thylakoid

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Cell Biology of Prokaryotic Organelles

membranes but a recent study suggests that this were discovered in an acidobacterium isolated defect may have less to do with membrane bio- from a microbial mat community in Yellow- genesis than it does with the assembly of photo- stone National Park making it the first photo- synthetic complexes (Gao and Xu 2009). Using synthetic bacterium that has been identified in a repressible promoter, Vipp1 was depleted to the phylum Acidobacteria (Bryant et al. 2007). levels in which cells could no longer perform Chlorosomes are flattened, ellipsoidal struc- photosynthesis. Under these conditions the thy- tures that are connected to the cytoplasmic lakoid membranes had a wild-type appearance membranes by a relatively thick baseplate (Fig. suggesting that Vipp1 may function at a step 4A). The chlorosome envelope is 3–5 nm thick downstream of membrane biogenesis (Gao and and electron opaque, as seen by thin-layer trans- Xu 2009). Another fascinating possibility has mission electron microscopy (Cohen-Bazire come from the observation that homologs of et al. 1964; Staehelin et al. 1980). This layer is eukaryotic dynamin can be found in several thinner than the cytoplasmic membrane species of cyanobacteria (Low and Lowe 2006). (8 nm), indicating it is not a lipid bilayer. In eukaryotes dynamin and dynamin-like pro- However, lipids have been identified in purified teins are important for membrane fission and chlorosomes, and the chlorosome envelope tubulation in processes ranging from endo- fractures in freeze-fracture electron microscopy cytosis to cytokinesis (Praefcke and McMahon in a manner characteristic of lipids, suggesting 2004). As with eukaryotic dynamins, the puta- that the envelope is a lipid monolayer (Staehelin tive dynamin homolog found in cyanobacteria et al. 1980; Frigaard and Bryant 2006). is also aGTPase, can bind liposomes invitro,and Chlorosomes primarily contain bacterio- localizes to cellular membranes in vivo (Low chorophyll (BChl) c, d, or e, which can number and Lowe 2006). More strikingly, the three- 150,000–300,000 molecules in a single organ- dimensional structure of prokaryotic dynamin elle. Tenproteins have been purified from Chlor- is remarkably similar to that of eukaryotic dy- obium tepidum chlorosomes, and all of them namin (Low and Lowe 2006). Given these sim- have been shown to be susceptible to cleavage ilarities in structure and biochemical activity it by proteases, suggesting they are surface ex- has been postulated that cyanobacterial dyna- posed. Antisera to these proteins can precipi- mins may play a role in establishing the complex tate chlorosomes, further supporting the model assemblies of thylakoid membranes (Low and that these proteins are in the chlorosome enve- Lowe 2006). However, dynamin-like proteins lope (Chung and Bryant 1996; Vassilieva et al. are not found in all cyanobacteria and in the 2002). A number of these envelope proteins strains where they do exist, no functional data show similarity with each other leading to the exists to suggest that they have a dedicated role hypothesis that they perform redundant func- in thylakoid membrane biogenesis. tions. This idea is supported by genetic studies Chlorosomes are the largest light-harvesting in which individual deletions of 9 of the 10 systems found thus far in photosynthetic organ- chlorosome genes had virtually no effect on isms, and they have been shown to allow cells to chlorosome structure or function (Frigaard harvest light energy at extremely low light in- et al. 2004). However, when double, triple and tensities (Frigaard and Bryant 2006). A striking quadruple mutants were created in which com- example of this is a chlorosome-containing ob- binations of genes predicted to be in the same ligate phototroph that was found 2391 meters family were deleted, dramatic phenotypes in below the surface of the Pacific Ocean and the size and morphology of chlorosomes were thought to extract the energy necessary for uncovered suggesting that the protein content growth from the infrared radiation of a geother- of the organelle determines its ultrastructural mal vent (Beatty et al. 2005). Chlorosomes are properties (Li and Bryant 2009). The 10th gene, found in all Chlorobi or csmA, has been proposed to act in the flow of and some Chloroflexi or green filamentous energy from the antenna to the reaction center. anoxygenic phototrophs. Recently, chlorosomes Interestingly, in the aforementioned study csmA

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D. Murat, M. Byrne, and A. Komeili

could not be deleted, suggesting that it is essen- (Fuerst 2005). The simplest configuration is tial to the cells (Frigaard et al. 2004). found in organisms such as those of the genus The discovery of chlorosome proteins and Pirellula in which a large lipid-bilayer bounded the directed functional studies detailed earlier compartment contains and separates the chro- are important steps in understanding the mech- mosome and ribosomes from other cellular anism of chlorosome formation. The unique components. This organelle, termed the pirel- arrangement of lipids and envelope proteins lulosome, is surrounded by a small area of cyto- suggests that this mechanism will be different plasmic space known as the paryphoplasm than the one used to form other lipid-bounded (Fig. 3B). Unlike the periplasmic space of organelles. Toaccount for their architecture and Gram-negative bacteria macromolecules such composition a recent hypothesis suggests that a as RNA can be found in the paryphoplasm self-assembly process is responsible for the for- (Lindsay et al. 1997). mation of chlorosomes (Hohmann-Marriott In some Planctomycetes, more complicated and Blankenship 2007). According to this forms of compartmentalization have been ob- model, and other pigment served in which the pirellulosome is further molecules accumulate in between the two leaf- subdivided into smaller and more specialized lets of the inner membrane creating a growing compartments. The most dramatic example is bubble surrounded by a single lipid layer. In found in species such as Gemmata obscuriglo- fact, when the gene encoding for bacteriochlor- bus in which a compacted is sur- ophyll synthase c was deleted in tep- rounded by a double lipid-bilayer membrane idum normal chlorosomes were not formed and to form a nuclear body (Lindsay et al. 2001) instead smaller deflated structures containing (Fig. 3A). Ribosomes are found both within other pigments were seen within the cell (Frig- the nuclear body and throughout the rest of aard et al. 2002). Within this monolayer, glyco- the pirellulosome indicating that some transla- syl diacylglycerides are enriched because of their tional activity may be separated from transcrip- preferred interactions with the accumulated tion. The unusual membrane architecture and pigments. Finally, chlorosome proteins are re- the partial separation of transcription from cruited because of their preference for these translation are reminiscent of the eukaryotic chlorosome components. A combination of ge- nucleus thus raising the possibility that the netic and biochemical studies are now needed Planctomycetes may represent the early forms to directly test this simple self-assembly model of compartmentalization that has come to for chlorosome biogenesis. define the eukaryotes. This arrangement also implies that communication and transport of macromolecules must occur between the vari- Planctomycete Membrane Compartments: ous compartments of G. obscuriglobus. Al- True Ancestors of Eukaryotic Organelles? though molecular pathways and evidence for The examples discussed thus far represent the such transport have not been found, micro- broad spectrum of intracellular compartmen- scopic examination has revealed that the fold- talization that can be found in the prokaryotic ing of the lipid bilayer membrane surrounding world. These structures, however, do not resem- the nuclear body creates a small opening that ble the characteristic organelles that define the may be a portal for transport of macromole- endomembrane system of eukaryotes making cules (Lindsay et al. 2001). Time-lapse mi- it difficult to draw any evolutionary parallels. croscopy experiments have also helped to The members of the Planctomycetes, a deep elucidate the steps involved in the segregation branching phylum of the Bacteria, however, may of nuclei and biogenesis of organelles during contain the bacterial ancestors of eukaryotic cell division. Many of the Planctomycetes, organelles. Most species of this phylum are including G. obscuriglobus, divide by budding characterized by extensive and truly unique com- rather than the binary fission mechanism often partmentalization of their cytoplasmic space seen in bacteria (Lee et al. 2009). During early

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Cell Biology of Prokaryotic Organelles

Figure 3. The nucleus-like organelle of Gemmata obscuriglobus is shown in (A). The nuclear envelope (E) is a double lipid-bilayer membrane containing the chromosome (N). The inset highlights the intracytoplsmic mem- brane (ICM) that separates the riboplasm from the paryphoplasm (P) compartment. A simpler organization is seen in organisms such as Pirellula marina in which the intracytoplsmic membrane (ICM) differentiates the pir- ellulosome (PI) from the paryphoplasm (P) (B). Many of the Planctomycetes contain another unique organelle called the anammoxosome (C). Here a CETreconstruction of Brocadia fulgida is shown. The anammoxosome is the central compartment of this cell and iron particles (red) are found within it. (A, B, Reprinted, with permis- sion, from Lindsay et al. 2001 [# Springer]; C, reprinted, with permission, from Niftrik et al. 2008a [# ASM].)

stages of the budding process the newly divided to create the new nuclear envelope (Lee et al. nucleoid unbound by any membranes can be 2009). At present little is known about the seen in a relatively young bud. As the bud grows molecular mechanisms of organelle formation a complex migration of the mother cell inner in these organisms and the studies of this fasci- membrane and the daughter cell inner mem- nating topic are hampered by a lack of robust brane are followed by membrane fusion events genetic tools. However, recent sequencing of

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D. Murat, M. Byrne, and A. Komeili

several Planctomycete genomes may help in nature (Sinninghe Damste´ et al. 2002). These identification of novel gene products with a molecules, termed ladderane lipids form a unique role in organelle assembly and dynamics denser and more impermeable barrier than reg- (Studholme et al. 2004; Staley et al. 2005). One ular biological membranes that may prevent such clue has emerged from the genome of the diffusion of the toxic intermediates pro- Gemmata Wa-1 in which a homolog of the duced during the anammox reaction. The diffu- eukaryotic Gle2 protein, a component of the sion barrier provided by this organelle is also nuclear pore complex, has been discovered (Sta- thought to help in retaining the intermediates ley et al. 2005). A recent study conducted a more of the slow anammox reaction within the cell directed search for bacterial proteins that con- (Sinninghe Damste´ et al. 2002). A recent study tain signatures of eukaryotic membrane coat of this organelle by CET has revealed that its proteins, which play key roles in vesicle traffick- membrane is highly curved leading to the pro- ing and organelle maintenance in eukaryotes posal that the curvature could optimize the (Santarella-Mellwig et al. 2010). These proteins membrane surface and thus the membrane- are typified by an unusual combination of associated metabolic processes that happen in structural domains where a specialized arrange- the anammoxosome (van Niftrik et al. 2008a; ment of b-sheets, called a b-propeller, is fol- van Niftrik et al. 2008b). Some anammox bacte- lowed by an a-helical structure termed an a ria are also distinguished by their unique mode solenoid. These proteins are ubiquitous among of cell and organelle division. In Kuenenia stutt- the eukaryotes but when the genomes of all gartiensis cell division follows the typical binary sequenced bacteria where queried only species fission mode observed in other bacteria (van within the Planctomycete--Chla- Niftrik et al. 2009). As a result, the anammoxo- mydiae phyla contained genes encoding for eu- some is divided in half during each division karyotic coatlike proteins. Interestingly one of cycle and segregated equally among the two these candidates found in G. obscuriglobus was daughter cells. EM and CET imaging reveal seen to localize to the organism’s internal mem- the presence of a distinct cytokinetic ring appa- branestructures(Santarella-Mellwigetal.2010). ratus in the outermost compartment of this These results provide molecularevidence for the organism. Most bacteria use the tubulin-like possible ancestral link between Planctomycete protein FtsZ to form a division ring but the compartments and eukaryotic organelles. genome of K. stuttgartiensis is devoid of any Other species of the Planctomycetes have an homolog to ftsZ. Instead, another GTPase, additional membrane-bound compartment named kustd1438, was found to specifically called the anammoxosome capable of anaero- localize to the cytokinetic ring of this organism bic ammonium oxidation (Strous et al. 1999) (van Niftrik et al. 2009). The observation that (Fig. 3C). For decades, this anammox reaction kust1438 homologs are not found outside of hadbeenhypothesized toexistbased onthermo- the anammox bacteria also hints at its unique dynamic calculations but had never been associ- and important function in this process. How- ated with a living organism (Broda 1977). The ever, further functional studies are required to anammoxosome is located within the pirellulo- determine a direct role for this protein in cell some and it is the only Planctomycete organelle division and organelle partitioning. Ultimately, that can be purified, which has facilitated its development of robust genetic systems will help study (Lindsay et al. 2001). Among the pro- to further define the molecular mechanisms of teins found in the anammoxosome membrane organelle formation in the Planctomycetes. is hydroxylamine oxidoreductase, a unique enzyme that catalyzes ammonium oxidation PROTEIN-BOUNDED COMPARTMENTS (Schalk et al. 2000). Analysis of the anammo- xosome composition has also revealed that Carboxysomes are one of the best-known ex- its membrane is enriched in an unusual type amples of protein-bounded organelles in of concatenated lipids, never before found in bacteria (Yeates et al. 2008). They occur in all

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Cell Biology of Prokaryotic Organelles

cyanobacteria as well as chemoautolithotrophs increase the efficiency of RuBisCO and the fixa- where they serve as the site for the first step of tion of carbon. the Calvin cycle. The major catalytic compo- The defined set of proteins found in these nents of carboxysomes are the enzymes Ribu- operons is likely to be the minimal components lose-1,5-bisphosphate carboxylase oxygenase required to build a carboxysome. However, (RuBisCO) and carbonic anhydrase. RuBisCO recent results show that the proper organization catalyzes the reaction of CO2 with ribulose and segregation of carboxysomes across the cell bisphosphate to two molecules of 3-phospho- cycle require it to interface with other cellular glyceric acid (3PGA) and carbonic anhydrase components (Savage et al. 2010). Fluorescent catalyzes the conversion of bicarbonate to protein fusions to either a shell protein or to a CO2. By increasing the local concentration of RuBisCO component revealed that carboxy- RuBisCO and the CO2 substrate, carboxysomes somes are linearly spaced throughout the cell. are likely increasing the efficiency of the produc- The most relevant consequence of this arrange- tive carbon fixation reaction (Yeates et al. 2008). ment is that during cell division approximately This idea is supported by recent electron cryo- equal numbers of carboxysomes will be parti- tomography studies, which show that each car- tioned to each daughter cell (Savage et al. 2010). boxysome (measuring 80 to 150 nm) contains This arrangement relies on cytoskeletal systems over 200 RuBisCO enzyme complexes arranged as disruptions of either mreB (a bacterial actin- in concentric layers (Schmid et al. 2006; Iancu like protein) or parA lead to a disorganization of et al. 2007) (Fig. 4B). carboxysomes within the cell. In the parA mu- Only a few genes, found in one or more tants, some daughter cells do not receive any operons, are involved in the formation of car- carboxysomes meaning that they have to build boxysomes. In Halothiobacillus neopolitans, the their carbon fixation machinery de novo, which carboxysome genes encode for the large and in turn causes a significant lengthening of their small RuBisCO subunits, three small shell pro- doubling times (Savage et al. 2010). This fasci- teins that share high homology, a large shell nating study establishes a clear link between protein, carbonic anhydrase, and two unknown the cytoskeleton and carboxysome organiza- proteins that seem to have a regulatory func- tion. However, the specific connections between tion. Other bacteria that form carboxysomes this organelle and ParA, as well as the mecha- have slightly different genes in their operons, nisms by which the proper spacing of carboxy- but all contain homologs of the small shell somes is achieved remain to be elucidated. protein genes and genes that encode for the Carboxysomes are actually part of a larger RuBisCO subunits. Recently, small shell pro- family of protein-bounded compartments, teins from both a cyanobacterium and a che- which are all related through homology be- molithoautotrophic bacterium have been crys- tween their shell proteins. One such organelle tallized, which has provided valuable insights is the 1,2-propanediol use (Pdu) compartment into how the protein shell of the carboxysomes found in Salmonella enterica. Similar to carbox- may assemble (Kerfeld et al. 2005; Tsai et al. ysomes, Pdu compartments house specific en- 2007). These crystal structures reveal that the zymes that are important for their cellular proteins, purified individually, self-assemble function. Interestingly, a recent report has into hexamers that bind edge-to-edge to form shown that these enzymes all share a 20 amino monolayer sheets. These sheets of protein have acid amino-terminal sequence that is necessary been proposed to make up the walls of the car- for their packaging within the Pdu compart- boxysome. The crystal structures also revealed a ment (Fan et al. 2010). Furthermore, these positively charged pore at the center of the hex- amino-terminal sequences are also sufficient amers. This pore could allow for the passage of to target heterologous proteins such as GFP negatively charged molecules such as bicarbon- to Pdu compartments. Such amino-terminal ate while blocking the entrance of O2 creating sequence extensions were also detected in another way in which the carboxysome could enzymes thought to be associated with other

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D. Murat, M. Byrne, and A. Komeili

Figure 4. Chlorosomes of Chlorobium tepidum appear as flattened ovals arranged around the cell periphery (A). A representation of a single carboxysome based on CET imaging. The interior of the carboxysome appears to be packed with RuBisCO based on similarities between the known crystal structure of the enzyme and electron-dense entities seen in CET reconstructions (B). A TEM image of ta cyanobacterial cell reveals that the cytoplasmic space is filled with gas vesicles sectioned in two different orientations (C). (A, Reprinted, with permission, from Frigaard et al. 2002 [# ASM]; B, reprinted, with permission, from Iancu et al. 2007 [# Elsevier]; C, reprinted, with permission, from Walsby 1994 [#ASM].)

microcompartments making it likely that this their exposure to light, salt, nutrients and other mode of protein localization is universal among environmental stimuli. Gas vesicles are cylindri- protein-bounded organelles (Fan et al. 2010). cal or spindle-shaped and the size of gas vesicles Beyond their relevance to understanding the varies between species. Cells that grow at greater cell biology of organelles, this finding also depths have gas vesicles that are narrower in provides a method for engineering protein width and are able to withstand greater hydro- compartments in bacteria through the specific static pressure. targeting of heterologous enzymes. Ten to fourteen gas vesicle protein (gvp) Another unique protein-bounded organelle genes, depending on the species, have been in bacteria is the gas vesicle (Fig. 4C). Gas identified as being involved in gas vesicle forma- vesicles are gas-filled, protein-bound organelles tion. In Halobacterium halobium, at least ten gvp that function to modulate the buoyancy of cells genes were found to be required for gas vesicle (Walsby 1994). They are found in a number of formation (DasSarma et al. 1994), and eight bacteria and including halophilic and gvp genes in the halophilic archaeon Halobacte- methanogenic archaea and phototrophic and rium salinarum are necessary and sufficient for heterotrophic bacteria. Most bacteria and arch- gas vesicle formation (Offner et al. 2000). One aea that have been shown to form gas vesicles are of the essential genes encodes GvpA, the main found in aqueous environments and are non- vesicle wall component and one of the most hy- motile. The proteinacious walls of gas vesicles drophobic proteins known. The crystal struc- are freely permeable to gas molecules. Water is ture of GvpA has not been solved, mainly also able to enter the gas vesicles but cannot because GvpA aggregates and cannot be dis- form droplets on the inner surface because of solved without denaturation. Nonetheless, the its highly hydrophobic nature. Thus, any water structure of gas vesicles has been investigated that enters the gas vesicles evaporates (Walsby by X-ray analysis and atomic force microscopy 1994), and the gas-filled vesicles decrease the (Blaurock and Walsby 1976; Blaurock and overall density of the cells, allowing them to Wober 1976; McMaster et al. 1996), which float upward. By controlling the formation of revealed that the proteins form very ordered the gas vesicles, these organisms can specify ribs and that the protein subunits align at an their position in the water column to regulate angle of 548 to the rib axis. Interestingly, 548 is

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Cell Biology of Prokaryotic Organelles

close to the angle at which transverse and longi- a limited group of bacteria and photosynthetic tudinal stresses are equal in the wall of a cylin- membranes are formed through a self-assembly drical structure (Walsby 1994). mechanism using the photosynthetic proteins. Much work has been performed to under- These findings may imply that among bacteria stand the physical properties of gas vesicles membrane-bounded organelles evolved multi- including their structure, their ability to with- ple times independently. Second, self-assembly stand hydrostatic pressure, their ability to may be a common mode of organelle biogenesis exclude water, and their permeability to gas. in both lipid-bounded organelles such as pho- However, how the gas vesicle proteins interact tosynthetic membranes and chlorosomes and to form the gas vesicles, and how gas vesicle protein-bounded compartments such as car- formation is regulated in response to environ- boxysomes. At the moment, the restrictions mental cues remains largely unknown. Finally, placed on the cell by this mode of organelle it is possible that gas vesicle-like structures are formation remain unknown. For instance, can found in other bacteria that exist in nonaqueous newly synthesized enzymes still be targeted to environments. Homologs of gas vesicle genes carboxysomes after the shell has closed? There have also been found in actinomycetes that are clear exceptions to this rule as well. In the live in the soil yet no gas vesicle-like structures case of magnetosomes, a large number of mag- and no buoyancy phenotype has been seen netosome proteins can be eliminated and yet (van Keulen et al. 2005). the initial stages of membrane formation can still occur. Finally, cytoskeletal elements are used in multiple divergent systems as a means CONCLUDING REMARKS to organize and divide organelles. The magne- In conclusion, compartmentalization is not a tosomes of magnetotactic bacteria and the feature limited to the eukaryotic world and protein-bounded carboxysomes both require numerous examples of highly complex and dy- cytoskeletal proteins for accurate placement in namic organelle systems can be found among the cell, which aids in proper function and seg- the prokaryotes. The limited knowledge of the regation of these organelles during division. molecular mechanisms that control the biogen- Beyond the establishment of model systems esis of these prokaryotic organelles does not and robust tools, a change in perspective may allow for a direct mechanistic and evolutionary also be needed to move the understanding of comparison to their well-studied eukaryotic these organelles to the next level. For decades counterparts. In many cases attempts to study the major focus of research in the study of pro- prokaryotic organelles are hampered by their karyotic organelles has been to uncover the small size and a lack of molecular and genetic enzymatic basis of their function and to take tools. With the advent of high-resolution imag- advantage of the biochemical products of these ing systems such as CET and the availability reactions for applied purposes. Wewould like to of numerous genome sequences, some of the suggest that a dedicated focus on the cell biology barriers to the study of prokaryotic organelle of these organelles is needed to move forward. biology are beginning to fade. Although the The approach should be similar to that taken molecular understanding of organelle forma- by cell biologists studying organelle formation tion in prokaryotes is still at a relatively imma- in eukaryotes in which experiments are focused ture stage some general rules can be seen in on understanding the mechanisms that allow the recent findings detailed in this article. First, for membrane bending, protein sorting, and it is clear that the proteins that can influence organelle division. By defining the cellular organelle formation are unique to each of the basis for organelle formation in prokaryotes organelle systems discussed here. MamI and we may then be able to directly tackle the evolu- MamL are found only within the magnetotactic tionary basis of compartmentalization across bacteria, the putative eukaryotic-like proteins the various domains of life. Furthermore, this found in the Planctomycetes are also unique to avenue of research will shed light on the general

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D. Murat, M. Byrne, and A. Komeili

mechanisms used by prokaryotes to build large Fan C, Cheng S, Liu Y, Escobar CM, Crowley CS, Jefferson macromolecular assemblies and organize their RE, Yeates TO, Bobik TA.2010. Short N-terminal sequen- ces package proteins into bacterial microcompartments. cytoplasmic space. Finally, understanding the Proc Natl Acad Sci 107: 7509–7514. cell biology of prokaryotic organelles will allow Frankel RB, Papaefthymiou GC, Blakemore RP, Obrien W. for a more rational approach to their re-engi- 1983. Fe3o4 precipitation in magnetotactic bacteria. Bio- neering in biotechnological and biomedical chim Biophys Acta 763: 147–159. applications. Frigaard NU, Bryant DA. 2006. Chlorosomes: antenna organelles in green photosynthetic bacteria. In Complex intracellular structures in prokaryotes (Microbiology Monographs) (ed. JM Shively), pp. 79–114. Springer. ACKNOWLEDGMENTS Frigaard NU, Voigt GD, Bryant DA. 2002. Chlorobium tep- idum mutant lacking c made by A. 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Cell Biology of Prokaryotic Organelles

Dorothee Murat, Meghan Byrne and Arash Komeili

Cold Spring Harb Perspect Biol 2010; doi: 10.1101/cshperspect.a000422 originally published online August 25, 2010

Subject Collection Cell Biology of Bacteria

Electron Cryotomography Cyanobacterial Heterocysts Elitza I. Tocheva, Zhuo Li and Grant J. Jensen Krithika Kumar, Rodrigo A. Mella-Herrera and James W. Golden Protein Subcellular Localization in Bacteria Synchronization of Chromosome Dynamics and David Z. Rudner and Richard Losick Cell Division in Bacteria Martin Thanbichler Poles Apart: Prokaryotic Polar Organelles and Automated Quantitative Live Cell Fluorescence Their Spatial Regulation Microscopy Clare L. Kirkpatrick and Patrick H. Viollier Michael Fero and Kit Pogliano Myxobacteria, Polarity, and Multicellular The Structure and Function of Bacterial Actin Morphogenesis Homologs Dale Kaiser, Mark Robinson and Lee Kroos Joshua W. Shaevitz and Zemer Gitai Membrane-associated DNA Transport Machines Biofilms Briana Burton and David Dubnau Daniel López, Hera Vlamakis and Roberto Kolter The Bacterial Cell Envelope Bacterial Nanomachines: The Flagellum and Type Thomas J. Silhavy, Daniel Kahne and Suzanne III Injectisome Walker Marc Erhardt, Keiichi Namba and Kelly T. Hughes Cell Biology of Prokaryotic Organelles Single-Molecule and Superresolution Imaging in Dorothee Murat, Meghan Byrne and Arash Komeili Live Bacteria Cells Julie S. Biteen and W.E. Moerner Bacterial Chromosome Organization and Segregation Esteban Toro and Lucy Shapiro

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