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Provided for non-commercial research and educational use only. Not for reproduction, distribution or commercial use. This chapter was originally published in the Comprehensive Biophysics, the copy attached is provided by Elsevier for the author’s benefit and for the benefit of the author’s institution, for non-commercial research and educational use. This includes without limitation use in instruction at your institution, distribution to specific colleagues, and providing a copy to your institution’s administrator. All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier’s permissions site at: http://www.elsevier.com/locate/permissionusematerial From K. Kruse, Bacterial Organization in Space and Time. In: Edward H. Egelman, editor: Comprehensive Biophysics, Vol 7, Cell Biophysics, Denis Wirtz. Oxford: Academic Press, 2012. pp. 208-221. ISBN: 978-0-12-374920-8 © Copyright 2012 Elsevier B.V. Academic Press. Author's personal copy 7.13 Bacterial Organization in Space and Time K Kruse, Theoretical Physics, Saarland University, Germany r 2012 Elsevier B.V. All rights reserved. 7.13.1 7.13.2 7.13.2.1 7.13.3 7.13.3.1 7.13.3.2 7.13.3.3 7.13.3.4 7.13.4 7.13.4.1 7.13.4.1.1 7.13.4.1.2 7.13.4.1.3 7.13.4.2 7.13.5 7.13.6 7.13.6.1 7.13.6.2 7.13.7 7.13.7.1 7.13.7.2 7.13.8 References Introduction Organization Principles Mechanisms vs. Models The Bacterial Cytoskeleton Prokaryotic Cytoskeletal Proteins Helices and Rings Induced Assembly and Disassembly of Cytoskeletal Filaments Spatial Distribution of Magnetosomes and Carboxysomes Positioning The Z-Ring The Min Oscillations Biochemistry of the proteins MinD and MinE Min oscillations result from MinD-MinE self-organization in presence of a membrane Cooperativity is needed for spontaneous emergence of Min oscillations Polar Localization of MinD/DivIVA in B. subtilis Polar Protein Localization Chromosome segregation and protein distributions Entropic chromosome segregation Active Chromosome Segregation Temporal Organization Division Entry A three-protein Circadian clock Concluding remarks Glossary Divisome The molecular assembly that generates formation of a new cell wall to divide a bacterial cell. Meanfield models A variant of computational models used to describe the evolution of protein distributions that takes the form of partial differential equations. 7.13.1 Introduction Bacteria were long considered to be mere bags of proteins and DNA with the macromolecules they contained homogenously distributed. This view was based on several experimental observations: Diffusion constants for cytosolic macromolecules in bacteria have been measured to be on the order of 1–10 mm2s1,1–3 such that for cells of typical bacterial sizes of a few micrometers, diffusion homogenizes these molecules on time scales of a second. Furthermore, early optical and electron microscopy did not reveal clearly discernable compartments that would separate different parts within bacteria as is the case for eukaryotes. Finally, based on sequence comparisons, bacteria were thought to possess no cytoskeleton, which in eukaryotes is to a large extent responsible for spatial and temporal organization. This view of unstructured bacteria has been shattered in the last 20 years or so, by the use of modern electron and 208 208 209 210 210 210 210 212 212 213 214 214 214 215 216 216 217 217 217 218 219 219 219 219 Nucleoid A region of the bacterial cell that contains the chromosomal DNA. Origin of replication A site on the chromosomal DNA where DNA replication starts. Walker ATPase An ATPase for which the nucleotidebinding pocket contains two conserved sequence motifs, namely the Walker A and Walker B motif. fluorescence microscopy as well as by the discovery of the bacterial cytoskeleton.4 Even prior to this time, it was, of course, clear that bacteria are to some extent internally organized: cell division does not occur randomly in space and the duplicated chromosome is evenly divided between the two daughter cells. Furthermore, some bacteria like Caulobacter crescentus, Bacillus subtilis, or Myxococcus xanthus were known to be at least temporarily polarized. The degree of bacterial organization we are aware of today, however, came as a complete surprise. We now know that bacteria can have a nose, they possess organelles, which are non-randomly distributed in the cells, and regular filamentous structures exist, either in form of rings or helices. In addition, a number of bacterial cytoskeletal proteins have been discovered and by now it is clear that bacteria contain homologs of the eukaryotic cytoskeletal proteins actin and tubulin. Also intermediate filaments have found prokaryotic counterparts. Comprehensive Biophysics, Volume 7 doi:10.1016/B978-0-12-374920-8.00717-7 Author's personal copy Bacterial Organization in Space and Time Biophysics has played an important role in discovering bacterial organization, and in revealing the mechanisms underlying their formation. Think of the major advances in optical microscopy that not only allow us to locate proteins within bacterial cells with an extension of only a few microns, but also to track individual proteins and quantify their mobility. Theoretical analysis is indispensible if one aims at a quantitative understanding of spatiotemporal protein patterns, and this can often be used to distinguish between qualitatively different mechanisms. Furthermore, the reconstitution of bacterial systems in vitro plays an increasingly important role.5 In the following chapter, a number of prominent bacterial structures will be discussed. Instead of trying to be comprehensive, the systems are chosen to illustrate various general organizational principles. A major part is devoted to the bacterial cytoskeleton, because similar to its eukaryotic counterpart it plays an essential role in bacterial organization. Before discussing specific examples of bacterial organization, some general principles underlying the generation of spatiotemporal order will first be presented. 7.13.2 Organization Principles The mechanisms underlying the spatiotemporal organization of proteins in cells can generally be separated into two classes, depending on whether or not they exploit external cues.6 In this context, an external cue does not necessarily refer to a signal coming from outside of the bacterium, but rather to any entity that determines the localization of the protein under investigation. Such entities could be other proteins. For example, a ring built from the protein FtsZ, called the Z-ring, forms the scaffold for all proteins that will assemble into the divisome, the structure that drives cell division by generating the formation of a new cell wall, which ultimately leads to two daughter cells. As another example, the dynamic distribution of MinC in Escherichia coli is imposed by the distribution of MinD. As a different kind of cue, membrane curvature has long been thought to play a role in bacterial organization. Membrane curvature could be used to localize proteins, for example, to the poles of rod-shaped cells. Indeed, there is now compelling evidence that some proteins assemble preferentially on membranes with a specific curvature.7 Also the lipid composition of the cytoplasmic membrane, which in turn might depend on membrane curvature,8,9 could be exploited as an external cue to localize proteins. As a final example, let us mention active directed transport, which might play a minor role in prokaryotes, but is heavily used in eukaryotes, where it relies on external cues provided by the microtubule network (see Chapter 4.15). With exception of the last example, the protein distributions result in the presence of external cues from a diffuse-andcapture mechanism; proteins move randomly in the cytoplasm or on the membrane until they hit an appropriate target to which they adhere. Due to the comparatively large diffusion constants of molecules in the bacterial cytoplasm,2,3 which makes them explore the whole cell volume within seconds or faster, this mechanism is very efficient. However, it relies on pre-existing structures and the obvious following question is: 209 How do these structures themselves form in the right places at the right time? Mechanisms underlying the emergence of structures in absence of external cues can again be divided into two classes. Self-assembled structures are equilibrium structures, while self-organized structures require a constant flux of energy and/or matter to persist. As a non-biological example of self-assembly, consider the formation of salt crystals. As a contrasting example, the spontaneous emergence of convection rolls in water heated from below is due to self-organization; the rolls vanish if the heating is switched off and no energy is put into the system.10 Both self-assembly and selforganization are also present in bacteria. For example, receptor clusters on membranes can result from self-assembly (In spite of the article’s title, the mechanism underlying formation of membrane clusters of chemotactic receptors proposed in Greenfield1 is really based on self-assembly of the receptors: No energy or matter flux is required for maintaining the clusters.), whereas the Min oscillations (see below) are clearly self-organized patterns.11 Physics has developed an impressive armada of tools and concepts to analyze self-assembled and self-organized patterns,10 which can all be employed to understand bacterial organization. The distinction between self-organization and -assembly on one hand, and organization due to external cues on the other may not always be as clear cut as the previous paragraphs might suggest. This is due to numerous feedbacks that exist between various components. We consider selforganization to be a powerful and adequate concept if the structure at hand results from the interaction of a small number of components. This is the case for the Min oscillations in E. coli. Other examples exist, and for eukaryotes this concept has also been successfully applied to explain cellular structures.12,13 As soon as the number of components becomes too large, though, the insight gained by classifying a pattern as self-organized might be limited, even if correct. Think, for example, of the Z-ring that is influenced by a rather large number of regulatory proteins, which themselves are localized to the Z-ring by diffusion and capture.14 In such a case, one can either focus on a subsystem consisting of few different proteins, or it might pay to ‘‘coarse grain’’ the system and to consider interactions between a small number functional entities rather than between individual proteins. Such entities could capture essential features of single proteins or of protein complexes. Depending on the context, it is thus sometimes useful to emphasize the aspect of self-organization or -assembly, and sometimes to point out external cues. There are a few common structural ‘‘motifs’’ that result from these mechanisms. Protein gradients are widely employed to spatially structure multicellular organisms15 and eukaryotic cells, for example, during cell division,16 but are also present in bacteria. In developing organisms, they often emerge from localized protein synthesis, diffusion away from their source, and degradation. In single cells, gradients are rather formed by proteins in a specific phosphorylation state. Gradients provide a nice example of structures that can form by external cues (although they do not have to) and that themselves serve in turn as external cues in the formation of other structures. In rod-shaped bacteria, such gradients can take a special form with the proteins being accumulated at the Author's personal copy 210 Bacterial Organization in Space and Time cell poles. Another common motif is given by filamentous structures. These can either be straight, helical, or they can form a closed ring around the cytoplasm. The spectrum of temporal structural motives is much smaller and in essence limited to oscillations. In the following, these general motifs will be illustrated by particular examples. As mentioned before, the focus will be on protein structures and how they organize bacterial DNA, organelles, and other proteins. However, also the distribution and organization of the chromosomal DNA contributes to the internal organization of bacteria. By presenting specific binding sites and possibly through a process termed ‘‘transertion’’17,18 it can strongly influence the distribution of proteins. On several occasions, we will briefly touch on this subject. The mechanical aspects of bacterial organization will only play a minor role in the following discussion, even though they might be very important. They are discussed in more detail in (see Chapter 7.6) on bacterial morphology. Before discussing these examples, a few comments on the role of theory in our endeavor to reveal the mechanisms of bacterial structure formation seem in order. 7.13.2.1 Mechanisms vs. Models Patterns resulting from the presence of external cues are relatively easy to grasp and their understanding does usually not require an elaborate conceptual physical basis. While experimental biophysical methods (FRET, PALM etc.) might make essential contributions to elucidate such mechanisms, theoretical insights are expected to be minor in such a case. This is utterly different for mechanisms involving self-assembly or self-organization! These mechanisms rely on interactions between a large number of proteins.12,13 Consequently, a quantitative understanding cannot be achieved without the concepts of Statistical Mechanics and Non-linear Dynamics. Even on a qualitative level it is often difficult to see whether a proposed mechanism can result in the desired structure without invoking some kind of formal analysis. In this context, one should not confuse the computational models that are used to investigate mechanisms with the physical basis of these mechanisms. The models can take various appearances, depending on the aspects of the problem they are emphasizing. Sometimes so-called meanfield models in the form of partial differential equations will provide an appropriate framework, on other occasions stochastic models might be preferable.19 As a consequence it often makes no sense to quarrel about different models that represent the same mechanism. In the following, the focus will be on those mechanisms that are interesting from a biophysical point of view, although other mechanisms will be mentioned, too. 7.13.3 The Bacterial Cytoskeleton While the cytoskeleton was once thought to be a defining feature of eukaryotic cells, we now know that all eukaryotic cytoskeletal proteins have one or more bacterial analogs,4 see Figure 1. Similar to its role in the eukaryotic cell, the cytoskeleton is an important factor for determining bacterial organization. 7.13.3.1 Prokaryotic Cytoskeletal Proteins The first prokaryotic cytoskeletal protein to be identified was FtsZ,20,21 a homolog of the eukaryotic tubulin. In spite of their weak sequence similarity, FtsZ and tubulin bear a high structural similarity.21,22 Another, more recently discovered, similar protein is TubZ.23 Prokaryotic homologs of eukaryotic actin include MreB, FtsA, ParM, and MamK.4 Crescentin was the first prokaryotic intermediate filament to be discovered.24 It is a coiled coil rich protein and can form filaments. Other filament forming coiled coil rich proteins are FilP and AglZ. A further family of bacterial cytoskeletal proteins is formed by the Walker Box ATPases ParA and ParF, and the deviant Walker-type ATPase MinD.25 All the above proteins are capable of forming filamentous structures. These filaments can be straight like MamK,26 wind as helices around the cytoplasm like MreB,27–29 or form rings like FtsZ.20 These forms are not mutually exclusive. For example, FtsZ can switch between an arrangement in a ring and in a helix.30,31 With the exception of the intermediate filament-like proteins, all other prokaryotic cytoskeletal proteins are ATPases or GTPases. In a cellular environment, these filaments are thus kept out of equilibrium. Often they present a high turnover with rates up to 0.1/s. Some of these filaments (e.g., TubZ and MreB), have been reported to show treadmilling,23,32 that is, assembly at one and disassembly at the other end. This dynamic behavior requires a structural polarity of the filament. Other filaments show a behavior similar to the dynamic instability of microtubules as they present alternating phases of shrinkage and of growth at both ends.33 The bacterial cytoskeleton is employed in various processes. First of all, it determines cell morphology. For example, the rod shape of E. coli or of C. crescentus depends on the presence of MreB. Destruction of MreB filaments by the drug A22 results in aberrantly large round cells.34 In C. crescentus, MreB, furthermore, determines the polarity,35 while Cresentin accounts for the curved (crescent) shape of the bacterium.24 A second major function lies in the proper distribution and localization of cellular components: Magnetosomes that endow magnetotactic bacteria with the ability to sense external magnetic fields are aligned by MamK filaments,26 low copy number plasmids are distributed evenly on the daughter cells by ParA or ParM,36 and a ring formed by FtsZ recruits the division inery to the future site of cell division14 In Myxococcus xanthus, AglZ is involved in cell motility.37 Cytoskeletal proteins are thus of broad importance for bacteria, in determining their structural and dynamical properties. In contrast to eukaryotic tubulin or actin, most prokaryotic cytoskeletal proteins can assemble spontaneously into filaments.38 Still, their dynamics can be controlled by auxiliary proteins. In the following, the formation of cytoskeletal structures in bacteria will be discussed. 7.13.3.2 Helices and Rings As mentioned above, cytoskeletal filaments can form rings or helices; see Figure 2. In Bacillus subtilis, but also in Escherichia coli, Author's personal copy Bacterial Organization in Space and Time -tubulin GDP GTP + BtubA/B GDP empty -tubulin 211 FtsZ GTP GTP BtubA FtsZ FtsZ BtubB -tubulin (a) − F-actin MreB Protofilament axis ParM:ADP ParM filament ParM (b) Figure 1 Structure of bacterial cytoskeletal proteins and their eukaryotic homologs. (a) Structure of the a/b tubulin heterodimer (left) and the FtsZ dimer (right). (b) Structure of actin filaments (F-actin), MreB- and ParM-filaments. The arrow indicates the direction of the filament orientations. From Michie, K. A.; Lowe. J. Annu. Rev. Biochem. 2006, 75, 467–492. Copyright by Annual Review. FtsZ is observed to switch between annular and helical arrangements; helices can grow out from existing rings.30,31 In B. subtilis, under starvation conditions, the helix will collapse at another location and there induce spore formation. In C. crescentus, MreB forms a helix that determines cell polarity.35 Prior to cell division, the helix collapses into a ring and then reappears in the two future daughter cells. In E. coli, MreB rings and helices can coexist.39 A mechanism based on the growth of filaments states that the configuration of a filament is completely determined by the orientation of a filament nucleus from which it grows straight.40 However, this approach neglects any mechanical contributions, but turns the question into what determines the orientation of the nucleus and can thus not provide a completely satisfying answer. The arrangements of the filaments can be understood purely on mechanical grounds, apart from their assembly and disassembly dynamics. Accounting for a possible spontaneous curvature of the filaments, their bending stiffness, and possible anisotropies in the interaction between the filaments and the membrane, the filaments can form rings and helices on curved membranes.41 Which form is then adopted depends on the values of the spontaneous curvatures and the spontaneous twist. These values can be affected by the state of the nucleotide-binding site. For example, FtsZ curls when bound to GDP and is straight when bound to GTP. Author's personal copy 212 Bacterial Organization in Space and Time 2 1 3 4 (a) 1 2 3 4 5 (b) Figure 2 Examples of rings and helices formed by cytoskeletal proteins in rod-shaped bacteria. (a) FtsZ rings and helices in B. subtilis. From Ben-Yehuda, S.; Losick, R. Asymmetric cell division in B-subtilis involves a spiral-like intermediate of the cytokinetic protein FtsZ. Cell 2002, 109, 257–266. (b) MreB rings and helices in E. coli. From Shih, Y.; Le, T.; Rothfield, L. Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles. Proc. Natl. Acad. Sci. USA 2003, 100, 7865–7870. Copyright by PNAS. An alternative mechanism, motivated by the apparent lack of spontaneous curvature of MreB, is based on constrain forces resulting from filament-membrane interactions.42 Such forces could result from filament polymerization or from other forcegenerating entities. If these forces are located at the filament ends, rings and straight lines can become unstable and result in a helix. Which of these mechanisms – if any – is responsible for helix formation in rod-shaped bacteria is currently unknown. Further experimental input is certainly necessary to improve our understanding. on the actin-like protein ParM, a centromere-like sequence on the plasmid denoted parS, and the protein ParR binding to parS.36 ParM forms a filament extending from the parS locus on one copy of one plasmid to the corresponding locus on the other plasmid. ParR then induces hydrolysis of the ATP bound to ParM, which results in unbinding of the filament from the plasmid, thereby allowing a new ATP-bound ParM molecule to attach to the filament and the plasmid. In this way, ParR stimulates growth of ParM filaments such that the two plasmids are pushed to opposite ends of the cell. This mechanism has been shown to work by an in vitro reconstruction containing ParM, ParR, and parS containing plasmids.43 There is an alternative mechanism that assures an equal distribution of low copy number plasmids in the daughter cells: Throughout the whole cell cycle, some plasmids are evenly distributed along rod-shaped bacteria in a manner that depends on ParA.44 The Walker Box ATPase ParA is a bacterial cytoskeletal protein without known homolog in eukaryotes. It is contained in the type I partitioning system, which in addition encodes for ParB, a DNA binding protein, and parC, the binding site of ParB on the DNA. ParA dimers bind cooperatively and nonspecifically to DNA. ParA filaments polymerize until they contact ParB, which itself may be bound or not to parC, stimulating hydrolysis of the ATP bound to ParA.44 In striking contrast to ParM, the ParA filament will not transiently detach from the plasmid and grow further. Instead it will shrink and thereby pull on the plasmid. A theoretical analysis has shown that this process will lead to an even distribution of plasmids if the rate at which the plasmid detaches from the ParA filament depends on the current filament length such that it is smaller for longer than for shorter filaments.45 An alternative mechanism has been proposed that assigns an important role to the nucleoid in distributing ParA and thus the plasmids,46 see below. 7.13.3.4 7.13.3.3 Induced Assembly and Disassembly of Cytoskeletal Filaments It has already been mentioned that, in contrast to tubulin or actin, there is no kinetic barrier for forming bacterial cytoskeletal filaments. They can thus form anywhere in the cell. As in eukaryotes and as mentioned above, the assembly and disassembly of prokaryotic cytoskeletal filaments can nevertheless be regulated by other proteins. Such interactions can be used to position cytoskeletal structures or to control their effects on other cellular components. Some of these proteins are themselves distributed in structures independent of the cytoskeleton and act as proper external cues. Examples are the proteins MinC or SlmA that affect the position of the Z-ring (see below). In other cases, however, there is a feedback between their distribution and the cytoskeleton, opening the possibility for self-organization. The positioning of plasmids mediated by the bacterial cytoskeleton will serve to illustrate such processes. Plasmids are short circular strands of DNA. Some of them exist in very low copy numbers and active processes have evolved to guarantee an even distribution of such plasmids onto the daughter cells.36 The type II partitioning system relies Spatial Distribution of Magnetosomes and Carboxysomes Similar to the plasmids carrying the type I partition complex there are other macromolecular complexes that are evenly distributed along the long axis of rod-shaped bacteria. Two striking examples are provided by carboxysomes and magnetosomes; see Figure 3. Carboxysomes are organelles of cyanobacteria with a proteinaceous shell that sequester enzymes involved in carbon fixation.47 Within the rod-shaped bacterium Synechococcus elongatus, carboxysomes are evenly spaced in a ParA-dependent manner.48 Whether the underlying mechanism is the same as for the even distribution of plasmids carrying the type I partitioning systems is as yet unknown. Magnetososmes are membraneous organelles of magnetotactic bacteria.49 They contain magnetite, and are surrounded by a membrane that is continuous with the cytoplasmic membrane. In one cell, there are about 15–20 magnetosomes each containing a magnetite crystal of about 50 nm in size. Similar to carboxysomes, magnetosomes form chains that are necessary for efficient detection of an external magnetic field. Chain formation depends on another actin-like cytoskeletal protein, MamK, a homolog of MreB.26 As for carboxysomes, the mechanism behind the Author's personal copy Bacterial Organization in Space and Time 213 RbcL-CFP (a) (b) Figure 3 Alignment of bacterial organelles. (a) Linear arrangement of carboxysomes. From Savage, D. F.; Afonso, B.; Chen, A. H.; Silver, P. A. Spatially ordered dynamics of the bacterial carbon fixation machinery. Science 2010, 327, 1258–1261. Copyright by AAAS. (b) Localization of magnetosomes (A, B) and schematic representation of MamK filaments (C). From Komeili, A.; Li, Z.; Newman, D.; Jensen, G. Magnetosomes are cell membrane invaginations organized by the actin-like protein MamK. Science 2006, 311, 242–245. Copyright by AAAS. cytoskeleton-dependent regular distributions of magnetosomes is still unidentified. 7.13.4 Positioning The Z-Ring It was mentioned before that the Z-ring, a structure formed by FtsZ filaments, provides the scaffold for the proteins that make up the divisome. Correct positioning of the Z-ring is a vital task for bacteria, because it assures the even distribution of one copy of the chromosome onto each daughter cell. In contrast to animal cells, where the cytoskeleton plays an active part in positioning the contractile actin ring that will eventually cleave the cell, the position of the Z-ring is essentially determined by external cues. In many rod-shaped bacteria, two mechanisms cooperate to this end.14 One mechanism is called nucleoid occlusion and prevents formation of the Z-ring and thereby division over the nucleoids. A nucleoid is the bacterial equivalent of a eukaryote’s nucleus, that is, an intracellular region occupied by the bacterial chromosome (unlike the nucleus of eukaryotes, it is not delimited by a membrane). The physiological benefit is obvious; nucleoid occlusion prevents the chromosome being cut into pieces by the growing septum. A second mechanism is then necessary to select the region between the two segregated copies of the chromosome as the site of cell division. Otherwise, one daughter cell would remain without a chromosome. Different mechanisms have evolved to this end, however, all those that we know of act through inhibition of Z-ring formation at unwanted sites rather than promoting it at the future division site. In E. coli and Bacillus subtilis, the negative regulator of Z-ring assembly is MinC. In B. subtilis it is localized in a stationary manner to the cell poles, while in E. coli its localization alternates between the two poles. In both species, though, the localization mechanism relies on the protein MinD. A rather different mechanism seems to operate in C. crescentus that apparently lacks nucleoid occlusion. There, the protein MipZ forms a static gradient with maxima at the two cell poles. MipZ directly inhibits Z-ring formation and thus directs formation of the divisome to the cell center. From a biophysical perspective, the mechanisms underlying the appropriate distribution of MinC are probably the most interesting ones. They will be discussed in detail below as they exemplify protein self-organization as well as the role of geometrical cues for bacterial organization. Before this, however, some words on nucleoid occlusion and the distribution of MipZ are in order. In E. coli and B. subtilis, nucleoid occlusion depends on DNA-binding proteins that are able to destabilize FtsZ filaments.50,51 Interestingly, these proteins, SlmA in E. coli and Noc in B. subtilis, seem to be unrelated although they ultimately act in very similar ways. SlmA interacts with specific binding sites on the bacterial chromosome and was suggested to sever FtsZ filaments by inducing hydrolysis of GTP bound to FtsZ.52 Its effect on FtsZ is enhanced many times by binding to DNA, such that SlmA floating freely in the cytoplasm does not destroy FtsZ structures. Similarly, for Noc, specific binding Author's personal copy 214 Bacterial Organization in Space and Time sites on the chromosome have been identified.53 How Noc mediates Z-ring disassembly is currently not known. Both SlmA and Noc have been suggested as playing a role in coordinating chromosome segregation and cell division (see below). Similarly to ParA and MinD, MipZ is a Walker-type ATPase which associates via ParB to the chromosome of C. crescentus.54 Binding occurs only in the vicinity of the replication origin of the chromosome. During segregation, the duplicated origin is localized in an MreB-dependent way to the cell poles. There, it is possible that MipZ filaments originate and thus establish a gradient. 7.13.4.1 The Min Oscillations In E. coli the Min system consists of three poteins, namely MinC, an inhibitor of FtsZ filament assembly, MinD, a deviant Walker-type ATPase, and MinE, a protein capable of enhancing the ATPase activity of MinD.55 Microscopy of fluorescently labeled Min proteins in living cells has revealed a remarkable spatiotemporal pattern of the Min-protein distribution: The proteins localize for about half a minute in one cell half, then switch rapidly to the opposite cell half, where they reside again for about half a minute, switch back and so forth, 56,57 see Figure 4(a). In this way, the Min system directs Z-ring assembly with an accuracy of a few percent to the middle of the bacterial long axis resulting in two equally sized daughter cells after division.58 Several lines of evidence suggest that these so-called Min oscillations emerge spontaneously from interactions of MinD and MinE with the cytoplasmic membrane and do not require any additional cue.59 Also, MinC is not involved in the generation of the oscillations. 7.13.4.1.1 Biochemistry of the proteins MinD and MinE Let us start by presenting some biochemical properties of the Min proteins,55 as illustrated in Figure 4(b). In absence of ATP, MinD is cytoplasmic. After binding ATP, it forms an amphipathic helix through which it can associate with the cytoplasmic membrane. Due to its low ATPase activity, in cells lacking MinE, most MinD will locate to the cytoplasmic membrane. On membranes, MinD can form aggregates, which take the form of tight helices on lipid vesicles.60 These helices have a pitch of about 6 nm (comparable to the long axis of a MinD molecule) and induce the formation of lipid tubes with a diameter of 50–100 nm. Their appearance requires the amount of MinD to exceed a critical concentration and most probably a conformational change of membrane-bound MinD that depends on ATP hydrolysis: In the presence of ATPgS, a non-hydrolysable analog of ATP, MinD still binds to lipid vesicles, but does not form helices. MinD has also been observed to form filaments in vitro, however, under unphysiological salt concentrations. The structure of membrane-bound MinD aggregates in vivo is currently not known. MinE can bind to membrane-bound MinD, where it competes with MinC for overlapping binding sites, and significantly increases its ATPase activity. Thereby, MinE can induce detachment of MinD from the membrane. It was shown in vitro that MinE can act processively, that is, it can remove several MinD molecules from the membrane before detaching itself.61 While the molecular mechanism for processive detachment is not yet understood, it likely involves transient MinE binding to the cytoplasmic membrane.62 In vivo it forms a marked structure at the boundary of a MinD aggregate, which is known as the MinE ring. 7.13.4.1.2 Min oscillations result from MinD-MinE selforganization in presence of a membrane A number of mechanisms have been proposed to explain the dynamic behavior of the Min-protein distribution in vivo as a consequence of Min-protein self-organization in the absence of external cues.11 Compelling evidence supporting the ideas have been provided by an in vitro assay consisting of a supported lipid bilayer and a buffer essentially containing MinD, MinE, and ATP,63 shown in Figure 4(c). In this setup, the Min proteins have been found to self-organize into traveling planar and spiral waves (see Figure 4(d)). Theoretical analysis has shown that the same mechanism underlying the patterns observed in vitro can also explain the patterns observed in vivo if one assumes a reduced diffusion constant of membrane-bound MinD in vivo compared to in vitro. While this indicates the possibility of generating Min oscillations by protein self-organization, other cues might still be important for producing the pattern in vivo. Several observations argue otherwise, though. First of all, the Minprotein pattern displays an intrinsic characteristic length: in filamentous cells the oscillating Min pattern has several nodes regularly separated from each other by a distance of about 3 mm.56 No structural unit has so far been associated with these nodes. Indeed, while the cells grow, transitions between standing waves as described above and traveling waves can be observed as the number of nodes is increased. Furthermore, in sufficiently short cells, the Min-protein distribution does not oscillate.64 Instead, the proteins accumulate in one cell half and switch only stochastically to the other half. Only after a certain critical length has been exceeded will the Min oscillations start. The critical length depends on the expression level of the Min proteins such that this regime does presumably not exist in wild-type bacteria.65 While the oscillatory behavior might be unique to the Min proteins, there is a system in B. subtilis that shows similar stochastic switching. It consists of the proteins Spo0J and Soj, which are involved in chromosome segregation and transcriptional regulation. The Soj proteins switch stochastically between the two cell poles66 or jump from nucleoid to nucleoid.67 Similarly to the Min oscillations, this process might result from a dynamic instability where Soj plays a role similar to MinD while Spo0J takes the part analogous to MinE.68 However, rather than binding to Soj, Spo0J switches between a condensed and an uncondensed form, where condensation is triggered by the presence of Soj and where the inverse process occurs spontaneously. Condensed Spo0J stimulates Soj’s ATPase activity. Theoretical analysis of this mechanism has revealed stochastic switching, but also the possibility of regular oscillations.68 A thorough experimental test of this prediction has not been attempted so far. Author's personal copy Bacterial Organization in Space and Time 215 MinE ring MinD MinE +ATP/−ADP x 2 Polar cap of MinD and MinC t 1 3 4b 5 –P 4a MinD-ADP MinC MinD-ATP MinE (c) FtsZ ring 1 min Intensity (a.u.) 2 µm 0 MinC concentration gradient Nucleoid occlusion 1 (b) Intensity (a.u.) (a) 0.5 Cell length MinD MinE 0 (e) 20 40 60 80 Distance (µm) 50 µm (d) Figure 4 Min oscillations in E. coli. (a) Kymograph of fluorescently labeled MinD and time-averaged distribution. (b) Schematic illustration of Z-ring positioning by the Min proteins and nucleoid occlusion. (c) Illustration of the reaction kinetics of MinD and MinE. (d) Fluorescenc profiles of MinD (green) and MinE (red) on a supported lipid bilayer. The structure moves in the direction indicated by the arrow. (e) Fluorescence intensity profiles of MinD and MinE in a wave. From Loose, M.; Kruse, K.; Schwille, P. Protein self-organization: lessons from the min system. Annu. Rev. Biophys. 2011, 40, 315–336. Copyright by Annual Review. 7.13.4.1.3 Cooperativity is needed for spontaneous emergence of Min oscillations A crucial ingredient to all mechanisms that have been proposed to explain the Min oscillations without reference to external cues is a reduced diffusion of membrane bound proteins compared to cytoplasmic proteins. Different diffusion constants are indeed essential for pattern formation in chemical systems, as proposed by A. Turing in his seminal paper on the chemical basis of morphogenesis.69 The introduction of membranes that on one hand reduce the diffusion constant and on the other hand reduce the dimensionality of the space the molecules reside in presents a new feature for reaction-diffusion systems. Their consequences are by far not yet fully explored. Another essential ingredient for all proposed mechanisms underlying the Min oscillations is the existence of some sort of Author's personal copy 216 Bacterial Organization in Space and Time cooperativity among the MinD molecules. It could manifest itself in cooperative binding of MinD to the membrane, or in the formation of MinD aggregates on the membrane by a mechanism reminiscent of nucleation; small clusters grow by incorporating further molecules diffusing on the membrane.11 In spite of in vitro experiments suggesting a two-step mechanism for forming membrane tubes induced by MinD, whereby by first MinD attaches to the membrane and then aggregates,60 the former seems to have more explanatory power than the latter. A careful analysis of the wave profiles on supported lipid bilayers suggests further sources of cooperativity,61 see Figures 4(d) and 4(e). Attachment of MinE to membrane-bound MinD occurs initially at a constant rate – as is revealed by the linear increase of the MinE concentration as a function of time. As a critical concentration is passed, however, the MinE profile becomes highly non-linear with a sharp maximum at a wave’s trailing edge, which is probably the in vitro analogue of the MinE ring in vivo. This sharp increase could be due to cooperative binding of MinE. However, as mentioned before, MinE presumably removes MinD processively from the membrane. It has been argued that such an effect can explain the formation of the MinE ring.70,71 This is similar in spirit to the accumulation of the depolymerizing, microtubule-associated, molecular motor Kin-13.72 To end this section let us give an intuitive explanation of one possible mechanism underlying the spontaneous emergence of the Min oscillations. Assume that all MinD reside in one cell half. Therefore, MinE will also be recruited to this half and start to remove MinD from the membrane. Due to the overwhelming presence of MinD in that half, most of the detached MinD will quickly rebind here. There will be a leak, though, through which some MinD will escape to the adjacent cell half. At some point, the system will topple over; due to the presence of MinE in the original cell half, more MinD will be removed from the membrane than added, while it can freely attach in the adjacent half. At some point also MinE will detach and after some time bind to the MinD, which is now bound to the membrane in the cell half originally devoid of MinD. At this point the process starts anew. Let us emphasize again, that this intuitive explanation can only be stated after the fact. Without a detailed computational analysis it cannot be clear whether this mechanism will really bring the desired effect or not. 7.13.4.2 Polar Localization of MinD/DivIVA in B. subtilis As already mentioned above, the distribution of MinD and hence of MinC is stationary in B. subtilis. There, the concentration of MinD is maximal at both poles simultaneously and decays towards the cell center. Furthermore, MinD accumulates at the in-growing septum. The localization of MinD depends on the protein DivIVA, which, similarly to MinE, affects the stability of MinD on the membrane. These two proteins are unrelated, though, and the effect of DivIVA is opposite to that of MinE, since it stabilizes membrane-bound MinD. In principle, the distribution of MinD could again result from a dynamic instability of the homogenous distribution induced by DivIVA. In this case one would expect a characteristic wavelength to be associated with the MinD distribution. The existence of a characteristic wavelength is, however, incompatible with the accumulation of MinD at nascent cell poles and its absence from the cell center in filamentous cells.73 An early theoretical work explaining polar localization of MinD invoked geometrical cues in terms of membrane curvature.74 The idea was that MinD has a reduced affinity for binding to curved membranes and is thus depleted at the cell poles. According to this proposal, DivIVA furthermore binds preferentially to the rim of MinD regions, such that it accumulates at the cell poles or mature septa. Consequently, MinD would be stabilized and accumulate in these regions in spite of its low affinity for curved membranes. More recent observations, however, indicate that DivIVA itself senses membrane curvature; simplifying the picture considerably.75,76 7.13.5 Polar Protein Localization It is common in rod-shaped cells that proteins accumulate at the cell poles, as illustrated in Figure 5. As we have just seen, one example is DivIVA in B. subtilis, but other examples are legion. The mechanism underlying DivIVA localization is particularly simple, in that this protein directly reads out a geometrical cue, namely, (negative) membrane curvature. Also SpoVM, a small protein that recruits other proteins to newly forming spores in B. subtilis, has been found to locate preferentially to regions of a specific curvature.77 In contrast to DivIVA, though, SpoVM recognizes positive curvature. A different but conceptually similarly simple mechanism relies on cues associated with remnants from previous divisomes that are left behind at newly created poles after division. An example of such a protein is provided by TipN in C. crescentus.78,79 Understanding these two mechanisms does not require biophysical analysis, but rather a detailed knowledge of the structural elements of these proteins that mediate curvature recognition. There is, however, a third mechanism for polar localization that requires cooperative effects. In E. coli, chemoreceptors cluster at the cell poles.80 These clusters help to increase the bacterial sensitivity to changes in chemo-attractant or -repellant concentrations. It is known that chemoreceptors can form clusters anywhere on the membrane. High-resolution fluorescence microscopy has shown that the spatial distribution of such clusters correlates with their size; the largest clusters are found at the poles, and their size decreases as their location becomes closer to the cell center.1 The generation of this distribution does not need external cues, but can result from a simple nucleation process: Proteins attach to the membrane on which they diffuse81. As receptors meet, attractive interactions glue them together. As soon as a cluster exceeds a critical size, it becomes stable and will grow by incorporation of further receptor proteins. In an unbound system, this mechanism can lead to a periodic arrangement of clusters. In the confined geometry of an E. coli cell of wild-type length, it can lead to the observed localization of the largest clusters at the cell poles. As the reader might have noticed, such a nucleation mechanism operating on MinD is also at the heart of one of the proposed mechanisms explaining the spontaneous emergence of the Min oscillations.70 Author's personal copy Bacterial Organization in Space and Time Finally, the distribution of DNA is able to mediate protein localization at the poles. We will now briefly touch upon some aspects of chromosomal organization in bacterial cells and then discuss this possibility. 7.13.6 Chromosome segregation and protein distributions As has already become clear, the distribution of certain binding sites on the bacterial chromosome can serve as an external cue to organize the distribution of proteins. The mechanism behind A 217 the segregation of the two copies of a chromosome can have a strong influence on the internal organization of the chromosome. In this section, we will discuss mechanism behind the segregation of the two copies of a chromosome can have a strong influence on the internal organization of the cell.82 At the time when the existence of a bacterial cytoskeleton was ignored, Jacob and coworkers proposed that for rod-shape bacteria, the replication origins of the duplicating chromosome attach to the cell poles.83 Elongation of the cell would then lead to forces on the chromosomes that would pull them apart. Experimentally this view has been challenged using fluorescence microscopy, revealing that the replication origins in B. subtilis and C. crescentus separate 10 times faster than these cells elongate.34 Alternative mechanisms invoke entropic effects, ascribe an important role to cytoskeletal filaments, or postulate that the chromosome is indeed linked to the growing cell wall. 7.13.6.1 Entropic chromosome segregation A purely passive mechanism was proposed by Jun and Mulder, based on the observation that highly confined polymers segregate due to entropy.84 In fact, the free energy of two overlapping polymers consisting of N monomers confined to a tube of diameter D scales as FBD1/nNkBT, where kBT is the thermal energy and n ¼ 3/5 is the Flory constant defined through RgBNn, where Rg is the radius of gyration. This indicates a very strong repulsion between the two polymers that increases linearly with chain length. Monte Carlo simulations of two overlapping circular polymers in a bacterial geometry confirm the entropic segregation suggested by the scaling analysis. Adding the dynamics of DNA replication, the positions of the replication origins can be tracked. The simulations are in qualitative agreement with experimental data. B (a) Time DIC 7.13.6.2 Active Chromosome Segregation TipN-GFP Similar to plasmid segregation, the cytoskeleton has also been proposed to be involved in chromosome segregation. Similar to the suggestion by Jacob et al. mentioned above, the majority of the mechanisms involve site-specific sequestration of DNA. In C. crescentus, segregation of the chromosomal region close to the replication origin involves MreB.34 The Old New TipN-GFP New Old (b) Old epi-PALM (c) Figure 5 Polar localization of proteins in rod-shaped bacteria. (a) Distribution of DivIVA in wild-type (A) and mutant (B) E. coli. The mutant is unable to fission daughter cells after septum closure. In addition to the poles, DivIVA localizes to the curved regions of the newly formed septa. From Lenarcic, R.; Halbedel, S.; Visser, L.; Shaw, M.; Wu, L. J.; Errington, J.; Marenduzzo, D.; Hamoen, L. W. Localisation of DivIVA by targeting to negatively curved membranes. Embo. J. 2009, 28, 2272–2282. Copyright by Nature. (b) Localization of TlpN in dividing C. crescentus. After initial localization to the (younger) pole opposite to the stalk, it shifts to the nascent pole. From Lam, H.; Schofield, W. B.; Jacobs-Wagner, C. Landmark protein essential for establishing and perpetuating the polarity of a bacterial cell. Cell 2006, 124, 1011–1023. (c) Distribution of chemotactic receptors in E. coli. From Greenfield, D.; McEvoy, A. L.; Shroff, H.; Crooks, G. E.; Wingreen, N. S.; Betzig, E.; Liphardt, J. Selforganization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. Plos. Biol. 2009, 7, e1000137. Copyright by PLoS One. Author's personal copy 218 Bacterial Organization in Space and Time molecular details of this mechanism remain to be elucidated. A possibility is that treadmilling MreB filaments that are associated with the cytoplasmic membrane push the chromosomes apart.85 An interesting variant of this mechanism is the following: When genes of membrane proteins are transcribed, the nascent mRNA can immediately be translated into the corresponding amino acid sequence, which in turn inserts itself immediately into the cytoplasmic membrane/cell wall and thereby links the DNA mechanically to the wall.17,18 This process is called transertion. The compacted nucleoid is elastically linked along its whole extension to the elongating cell wall, and thus a force is generated on the replicating nucleoid that might pull the two copies apart. On the other hand, it 20 24 28 32 36 generates stress in the cell wall. This stress has a minimum in the cell center. It has been speculated that this stress distribution might be read by mechanosensitive proteins, and help to select the cell center as the site of Z-ring assembly.86 One consequence of active segregation that relies on specific DNA binding sites is a particular distribution of these binding sites. This distribution can be employed as an external cue to organize the bacterium.6 7.13.7 Temporal Organization Many of the spatial patterns discussed above appear in connection with cell division. For a cell, it is a vital point to decide min 80 Total T-KaiC ST-KaiC S-KaiC ori % KaiC 60 MipZ-YFP 40 20 0 Overlay 0 20 (b) 40 60 Time (h) 80 (a) B U KaiA 80 ST 60 % KaiC T KaiB A Total T-KaiC ST-KaiC S-KaiC 40 S 0 kXY (S) = kXY + A A(S) kXY k 1/2 + A(S) A(S) = max {0, [KaiA] − 2S} (c) 20 0 0 20 40 60 Time (h) 80 Figure 6 Examples of temporal regulation in bacteria. (a) Entry into cell division in C. crescentus mediated by MipZ. MipZ co-localizes with the replication origins. The Z-ring can form and induce division as soon as the regions of high MipZ-concentration have sufficiently departed from each other. From Thanbichler, M.; Shapiro, L. Getting organized – how bacterial cells move proteins and DNA. Nat. Rev. Microbiol. 2008, 6, 28–40. Copyright by Nature. (b) Periodic change in the phosphorylation level of the protein KaiA from the circadian clock formed by KaiA-C. (c) Wiring diagram indicating the mutual effects of the Kai proteins on each other (A) and numerical solution of a corresponding computational model (B). (b) and (c) from Rust, M. J.; Markson, J. S.; Lane, W. S.; Fisher, D. S.; O’Shea, E. K. Ordered phosphorylation governs oscillation of a three-protein circadian clock. Science 2007, 318, 809–812. Copyright by AAAS. Author's personal copy Bacterial Organization in Space and Time on the time she enters into cytokinesis. Hardly surprisingly, a number of propositions have been made which link the spatial patterns to check points for entering cytokinesis.54 In addition to the cell cycle, some bacteria are also temporally organized in order to match the day-night cycle on Earth. Such a circadian oscillator can be encoded in gene expression networks – as is the case for eukaryotes. A few years ago the proteins KaiA, KaiB, and KaiC of the cyanobacterium Synechococcus elongatus were shown to constitute a biochemical oscillator in vitro. In contrast to genetic oscillators it does not require protein degradation and synthesis, but rather periodically changes the phosphorylation status of KaiC. 7.13.7.1 Division Entry The evolving distribution of FtsZ-regulating proteins allows for straightforward ways to determine the onset of cell division. An early theoretical suggestion was based on a putative role of FtsZ-disassembly for the initiation of cytokinesis:87 As E. coli cells grow longer, the oscillation pattern of the Min proteins changes. Instead of pole-to-pole oscillations, the Min proteins will organize into a pattern with high concentrations simultaneously present at both poles followed by a phase where the Min proteins accumulate in the cell center. At this time the Z-ring is positioned correctly in the cell center and the divisome might have assembled. The presence of MinC could then activate the divisome through FtsZ disassembly. More recent studies on the dynamics of the Min proteins in dividing cells, however, show that division sets in before the pattern changes.64,88 Experimental evidence has been obtained, however, for a mechanism of cell division entry that works similar in spirit.54 In C. crescentus, the assembly of FtsZ is regulated by MipZ, which in turn is coupled to the distribution of the replication origins on the chromosome. As mentioned above, MipZ binds to specific DNA sites that are enriched close to the replication origin. For this reason, MipZ accumulates at these regions. As the chromosomes segregate, these regions depart from each other and a region depleted of MipZ emerges between them. At some points, the MipZ maxima will be sufficiently far apart that the MipZ concentration in between will be low enough as to allow for Z-ring formation. In this way, assembly of the divisome occurs only after chromosome segregation and can be followed immediately by cell division. In essentially the same way, the distribution of ParA was proposed to be regulated by the structure of the nucleoid, such that low copy number plasmids would be segregated faithfully46 (Figure 6(a)). 7.13.7.2 A three-protein Circadian clock A fair number of different mechanisms have been proposed to explain the periodic change in the amount of phosphorylated KaiC.89 KaiC is a hexameric molecule that possesses two sites which it can autophosphorylate and autodephosphorylate. Rather than giving a comprehensive account of all the proposed mechanisms, we will focus on one that accounts for the differences in the two phosphorylation sites as indicated by experiment (see Figures 6(b) and 6(c)).90 Let T-KaiC and 219 S-KaiC denote the two respective species of singly phosphorylated KaiC, ST-KaiC doubly phosphorylated KaiC, and U-KaiC unphosphorylated KaiC. During a complete cycle starting with U-KaiC, first T-KaiC forms, then ST-KaiC followed by S-KaiC, and finally the protein returns to U-KaiC. While all steps of this linear reaction chain are reversible, the presence of KaiA promotes the transitions from U-KaiC to TKaiC and from T-KaiC to ST-KaiC. Furthermore it inhibits the transition from ST-KaiC to S-KaiC, while promoting the inverse reaction. In turn, S-KaiC inactivates KaiA with the help of KaiB. That is, starting from U-KaiC, the system will arrive in a state that is dominated by ST-KaiC. If the latter concentration is high enough, the concentration of S-KaiC will exceed a critical value, which in turn inactivates KaiA such that the system relaxes through S-KaiC into a state dominated by U-KaiC and the cycle starts over again. The KaiABC system of S. elongatus thus presents an example of spontaneous oscillations. 7.13.8 Concluding remarks The modern view of bacteria is that of cells that are highly organized in space and time. 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