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Functional Taxonomy of Bacterial Hyperstructures

Department of Science, University of Rouen, 76821 Mont Saint Aignan Cedex, and Epigenomics Project, Genopole, 91000 Evry, France,¹ Swammerdam Institute for Life Sciences, University of Amsterdam,1098 SM Amsterdam, The Netherlands,² Molecular and Cellular Biology, University of California, Davis, California 95616,³ Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712,⁴ Laboratoire de Génétique Microbienne, INRA, 78352 Jouy en Josas, France,⁵ Department of Genetics, Faculty of Sciences, Universidad de Extremadura, E06080-Badajoz, Spain,⁶ Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute-Frederick, NIH, Frederick, Maryland 21702,⁷ Department of Biology, Washington University, St. Louis, Missouri 63130,⁸ Department of Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, Texas 77030,⁹ Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel,¹⁰ Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093-0116,¹¹ Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Oslo, Norway,¹²

SUMMARY

INTRODUCTION

PRINCIPLES

CANDIDATE HYPERSTRUCTURES

Ribosomal or Nucleolar Hyperstructures?

A lac Hyperstructure as a Paradigm for Transertion Hyperstructures?

Flagellar Hyperstructures

Chemosignaling Hyperstructure

Cellulosomal Hyperstructures

PTS-Glycolysis Hyperstructures

Cytoskeletal Hyperstructures

DNA Repair Hyperstructures

Competence Hyperstructures

DNA Replication Hyperstructures

Segregation Hyperstructures

Compaction Hyperstructure

Cell Division Hyperstructures

CANDIDATE PROCESSES IN HYPERSTRUCTURE ASSEMBLY, DISASSEMBLY, AND INTERACTIONS

Supercoiling

Transcription and Translation

Chromosome Compaction

Local Concentrations

Distribution of Sequences on Nucleic Acids

Chromosome Replication

Membrane Domain Formation

Phospholipid Turnover, RNA Degradation, and Proteolysis

Intracellular Streaming

Ions and Ion Condensation

Gel/Sol Transitions

Tensegrity

Water Structures

INTERACTIONS BETWEEN HYPERSTRUCTURES

Hyperstructures Send and Receive Messages via Their Constituents

Synergistic Interactions between Processes Lead to the Assembly and Disassembly of Hyperstructures

Coupled Oscillations Contribute to the Interactions between Hyperstructures

The Assembly and Disassembly of Different Hyperstructures Are Coupled

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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A great deal of effort is currently being put into studying the connections between the constituents of cells as revealed in the transcriptome, proteome, metabolome, and even interactome. Intriguing results have been obtained in terms of scale-free networks and small worlds (107, 219, 276). An indelicate question is on what concept of a cell are such analyses based. It is not certain that biologists understand the essence of even the "simple," well-studied bacterium Escherichia coli. Hence, one of the first priorities for students of biological complexity should be to understand what bacterial cells are. A full understanding would entail answering three questions: what are they doing, how are they doing it, and why are they doing it?

Here, we limit ourselves essentially to addressing the second question—how are they doing it—in structural terms, since this is actually easiest insofar as a remarkable degree of structure in bacteria has been revealed over the last decade. This advance in our understanding has led to the proposal that a level of organization exists midway between genes/proteins and whole cells. This is the level of hyperstructures (197). A hyperstructure is more than what is usually meant by a "supramolecular assembly" or a "molecular machine" or even a "module" (2, 96). In our hypothesis, hyperstructures are spatially extended assemblies of molecules and macromolecules that come in nonequilibrium and equilibrium flavors, that command signaling molecules and macromolecules, that interact with one another, and that determine the phenotype of the cell. Hence, in our terminology, the entire eukaryotic nucleolus is a hyperstructure while in that of Hartwell et al., even a single ribosome can be a module (96). Here we summarize the evidence in favor of hyperstructures in bacteria and discuss the possibilities they offer.

PRINCIPLES

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The controversy over the importation into biology from physics of concepts such as dissipative structures and Turing structures is far from over. More generally, nonequilibrium structures are proposed to exist that are maintained by a dissipation of free energy, most of the time in the form of a sustained irreversible chemical reaction. Examples of nonequilibrium structures include at the level of the weather, a tornado, and at the level of simple chemicals, the spatiotemporal patterns produced in the Belousov-Zhabotinsky reaction (89). It has also been argued that dissipative structures do not occur in biology but instead that biological structures are close to thermodynamic equilibrium even though there is a flux through them. There is little controversy, though, over the existence of equilibrium structures, such as a fully grown crystal in a saturated solution, that are generally stable for long periods (i.e., their size and composition do not change) in the absence of a source of energy in the environments usually encountered by the bacterium. These structures, which minimize the total free energy of the system, can nevertheless be formed from very mobile constituents. An equilibrium structure may be shifted out of equilibrium if the thermodynamic potentials of its components are also shifted from their equilibrium values. These changes in the values of the chemical potentials may result, for example, from changes in the environment of the cell or from the global nonequilibrium activity of the cell itself. In what follows, we try not to put the cart before the horse. We use these concepts with caution, aware that other, more specifically biological, terms may need to be devised. We shall try to focus first on those extended structures that have been observed to exist or that can reasonably be expected to exist. In doing this, we shall try to class these putative hyperstructures according both to the above concepts and to other, more biologically oriented, concepts.

CANDIDATE HYPERSTRUCTURES

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There is also an equilibrium aspect to ribosomes (271). During the stationary growth phase of E. coli, 70S ribosomes are converted to translationally inactive, 100S forms of ribosomal dimers by the binding of the ribosome modulation factor. Under energy depletion conditions in a variety of eukaryotes, ribosomes actually form crystal arrays in which they are protected (for references see reference 179); to our knowledge, equivalent hyperstructures of ribosomes in a crystalline state have not been observed in bacteria. As in the case of ribosomes, RNA polymerase is also stored in an inactive but reusable form in the stationary phase: the core enzyme forms complexes with polyphosphate (129), while the sigma70 subunit forms a complex with an anti-sigma factor, RSD (regulator of sigma D) (for references, see reference 271).

The story does not stop here. LacY is a permease that is inserted into the membrane. If a gene encoding an abundant membrane protein was similarly transcribed, translated, and inserted, there would be hundreds of nascent proteins anchoring the entire hyperstructure to the membrane via "transertion." Transertion is defined as the coupled transcription, translation, and insertion of proteins into and through membranes (27). Transertion in bacteria has been calculated to occupy most of the cytoplasmic membrane and has been established by membrane fractionation and electron microscopy studies (for references, see reference 282) and by supercoiling studies (153). A gene that is transcribed frequently to yield mRNAs that are translated often is, of course, more likely to generate a transertion hyperstructure than a gene that encodes a product that is rare. Given the abundance of complexes of ATP synthase in the cytoplasmic membrane of E. coli, it might therefore be expected that transertion would also create a nonequilibrium ATP synthase hyperstructure comprising the atp genes, the mRNA, and the nascent proteins. Such transertion hyperstructures might also comprise lipids if the nascent proteins (or the export apparatus that handles them) have preferences for particular phospholipids, which seems to be the case for certain subunits of the ATP synthase (8, 126). It should be noted that the correct folding of the lac permease, like that of many other permeases, requires phosphatidylethanolamine (68), and if association with a specific phospholipid can determine the conformation of a protein, reciprocally, the conformation of a protein may determine its association with a phospholipid. Hence, we might expect transertion hyperstructures to play a major role in the structuring of the membrane. This does indeed seem to be the case (27).

The secretory system may also be important in the formation of transertion hyperstructures, although we can see no simple explanation for the different locations of secretory components in different bacteria. In B. subtilis, for example, the translocases SecA and SecY are organized in clusters that spiral around the cell (40). This location of SecA is dynamic and depends on both active transcription and translation; it also depends on the presence of anionic phospholipids such as phosphatidylglycerol and cardiolipin. However, SpoIIIJ and YqjG, which are believed to be involved in the transfer of membrane proteins from the Sec apparatus into the membrane, are randomly located in B. subtilis, while their homologue in E. coli is located mainly at the poles (264). This can be contrasted again with the concentration of Sec translocons at the single export site which is often associated with the division site in the gram-positive coccoid Streptococcus pyogenes (232).

There is another sort of multimolecule assembly involving the lac operon that is different in both nature and size. In the absence of an inducer such as lactose (or in the presence of the preferred sugar, glucose), the lac operon is not transcribed. This is because some of the 10 copies per cell of the tetrameric LacI repressor bind with their dimers to the operator O1 and to two auxiliary operators, O2 and O3, nearby on the DNA; this on-off binding (which is an equilibrium process) increases the local concentration of LacI at these operators if they are close enough and brings them closer still to increase further the local concentration of LacI at O1 (188). This structure, albeit small, might be considered a hyperstructure, especially since it serves as a model for much larger ones. In a sense, it is an equilibrium hyperstructure because it is stable in the absence of a flow of energy through that part of the system. We are tempted to term it a "repression" or "site-binding" hyperstructure, and we point out that this equilibrium, site-binding hyperstructure can give way to a nonequilibrium, transertion hyperstructure and vice versa.

There is, however, another aspect to flagella, and that concerns the possible existence of a transertion hyperstructure. As with the lac operon and the atp genes, the transertion of the 20,000 or so FliC subunits of the bacterial flagellum might help create a flagellar hyperstructure bringing together the fliC gene and neighboring flagellar genes, nascent mRNA, and nascent proteins (38). Flagellar transcripts are organized into three classes, which are synthesized successively. The class 2 genes are under the control of the flhDC operon, which encodes transcriptional activators for class 2 promoters; class 2 genes encode the proteins of the hook/basal body and the regulatory proteins FlgM and ²⁸, and class 3 genes require ²⁸ for their expression (which is inhibited by the anti-²⁸ FlgM) and encode extracellular proteins such as FliC. A coupling between translation and secretion is achieved via Flk, a membrane-anchored homologue of ribosomal protein S1, which recruits the class 3 mRNA of flgM (note that there is also a class 2 mRNA of flgM that is not recruited) to allow the translated protein to be exported through the hook/basal body of the flagellum with the aid of FlgN, a specific chaperone (114). The logic is that when the construction of the hook and basal body of the flagellum is sufficiently advanced, FlgM is exported, its inhibition of transcription of the class 3 genes ends, and the synthesis of abundant proteins such as FliC begins. Recently, the flagellar transertion hyperstructure was proposed to act as a sensor of external hydration in Salmonella enterica serovar Typhimurium (273). It seems likely that lack of hydration leads to interference of filament subunit polymerization at the growing end of the flagellum, which in turn leads to a backup of nascent subunits of FlgM in the hollow channel, preventing FlgM levels from dropping and transcription of the class 3 genes from occurring. Hence, the growing flagellum itself acts as the sensor. In the model in which hyperstructures determine intracellular events, it should be noted that the feedback signals from the flagellum that switch off the class 3 genes also down-regulate nine virulence genes (273).

Within the cellulosome, the proteins are organized in a highly ordered chain-like array (164). It is believed that the cellulose-bound cellulosome clusters are the sites of active cellulolysis and that the products are channeled down fibrous structures to the cell (60). Over 95% of the endoglucanase activity of Clostridium thermocellum is associated with the cellulosome (25), which is consistent with there being advantages to the enzyme colocalization. This concentration of enzymes acting on related substrates provides the synergy that has been found between cellulases, between cellulases and hemicellulases, and between a cellulosomal enzyme and noncellulosomal enzymes (60, 67). The idea is that the synergy between the enzymes in cellulosomes makes the cellulosome structure more effective in attacking the substrate, as might be the case of the family 9 endoglucanases, which not only cleave cellulose molecules internally but also proceed in a processive manner along the chain from the cleavage site. Moreover, the synergy observed between cellulosomes and noncellulosomal enzymes would also be consistent with direct interactions between them.

Is the cellulosome (or polycellulosome) an equilibrium hyperstructure or a nonequilibrium hyperstructure? Clearly, it can be induced by substrate: cellulosomal genes are expressed as a function of the presence of different substrates, resulting in a population of cellulosomes with activities directed towards the available substrate. Indeed, the growth medium affects both the subunit structure and function of the cellulosome, and when cells are grown on different substrates, such as glucose, cellobiose, xylan, mannan, or pectin, chromatographic fractions of cellulosomes that differ in subunit compositions and enzymatic activities can be obtained (60, 94). In general, protuberances are not produced when the bacteria are grown under cellulase-repressing conditions. Protuberances form in about 4 h when Clostridium cellulovorans is grown on cellulose. This does not mean that the hyperstructure is a nonequilibrium one. However, within 5 min of the addition of the soluble sugar glucose, cellobiose or methylglucoside, the protuberances can no longer be detected; in other words, the protuberances dissociate rapidly when no longer needed. Similarly, when Clostridium thermocellum is grown on cellobiose, the polycellulosomes are compact and quiescent, while when it is grown on cellulose, the polycellulosomes change morphology radically to form what has been termed "contact corridors" (16). In this case, one interpretation is that the hyperstructure can exist in both equilibrium and nonequilibrium states. It would be interesting to learn whether there is a relationship between the transertion hyperstructure responsible for producing the cellulosomal proteins and the dynamic nature of the polycellulosomal structure.

In the case of glycolysis, there has been controversy over the existence of glycolytic metabolons in prokaryotes and eukaryotes, to which we do not intend to contribute much (48, 168, 211). Rather, we propose to explore the idea of a metabolic hyperstructure in which large numbers of each of the different species of enzymes bind dynamically to one another to increase their local concentration and that of the intermediates. Consider first the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which is responsible for the sensing and uptake of a large number of extracellular sugars and for feeding their products, cytoplasmic sugar phosphates, directly to the enzymes that constitute the glycolytic cycle (233). In E. coli, for example, there are many sugar-specific PTS permeases or enzyme II complexes, and each consists of three or four proteins or protein domains, i.e., IIA, IIB, IIC, and sometimes IID. The IIC and IID components are always integral membrane constituents, while the IIA and IIB components are localized to the cytoplasmic surface of the membrane. Glucose transport, for example, depends on a membrane-bound IICBGlc which interacts with a cytoplasmic IIAGlc, and IIAGlc-P is in turn phosphorylated by another cytoplasmic protein, P-HPr. P-HPr derives its phosphoryl group from phosphoenolpyruvate in a reaction catalyzed by enzyme I. Fluorescence studies in vivo of the distribution of enzyme I revealed three patterns of distribution, i.e., polar, punctate, and diffuse, depending, for example, on carbon source, cell density, and growth phase (213). This does not, of course, demonstrate a PTS hyperstructure, since the locations of the other enzymes were not determined. Nevertheless, it does show that the distribution can be nonrandom and that it can change. There are other reasons to suspect the existence of a PTS hyperstructure. For example, it has been proven that IIC is dimeric, and it is likely that the enzymes II form multiprotein complexes with the PTS energy-coupling enzymes, enzyme I, and HPr (for references, see reference 233).

Glucose-6-phosphate, released from the enzyme II complex of the PTS, enters the glycolytic pathway. Evidence also exists for an extensive glycolytic metabolon (245). In eukaryotic cells, interactions between sequential pairs of glycolytic enzymes have been demonstrated, with glycolytic enzymes being partitioned reversibly between cytoplasmic and cytomatrix-bound states depending on physiological conditions (for references, see reference 278) or indeed confined to an organelle, the glycosome, in trypanosomes (117). In E. coli, the glycolytic pathway has been isolated as an equimolar multienzyme complex in which compartmentation of substrates was demonstrated. One such complex was reported to have a molecular mass of 1.65 MDa, similar to that calculated for an equimolar complex of the enzymes of glycolysis, and had a particle diameter of 30 to 40 nm (90, 187). Finally, the full enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, and enolase (all glycolytic enzymes) results from their homo-oligomeric association, supporting the idea that a single species of enzyme can be activated to oligomerize by substrate (259); such an association could help nucleate and stabilize a hyperstructure.

The potential advantages of an enzymatic or metabolic hyperstructure include (i) reduction in the size of the pools of intermediates, since enzymes pass substrates to their partners with minimal delay and, even if the substrate were passed loosely from one enzyme to the next in the pathway so that it sometimes escaped, the presence of identical adjacent enzymes would increase the chance of recapture and the resumption of processing; (ii) protection of unstable or scarce intermediates by maintaining them within the hyperstructure away from other cellular factors that might act on them; (iii) avoidance of an "underground" metabolism in which intermediates become the substrates of other enzymes; and (iv) protection of the cell from toxic or very reactive intermediates that can be transformed rapidly and effectively within the hyperstructure. For example, colocalization within a joint hyperstructure of PTS enzymes actively engaged in sugar transport with glycolytic enzymes engaged in sugar metabolism not only would facilitate the processing of substrates but also could provide enzyme I of the PTS with a high local concentration of the phosphoryl donor for sugar uptake, phosphoenolpyruvate, the product of glycolysis.

There is another important aspect to consider, and that is the extent to which enzymatic hyperstructures are assembled in response to the cell's need for them. Certain transient, dynamic multimolecular assemblies form only in an activity-dependent manner (201, 209, 281), due, for example, to an association between enzymes that occurs only when they are engaged in transporting or transforming substrates or transducing a signal. We have proposed to term these assemblies "functioning-dependent structures" (FDSs) (256). In other words, an FDS assembles when functioning and disassembles when no longer functioning and thus is created and maintained by the very fact that it is in the process of accomplishing a task. This is, in its most useful form, a scale-free definition, and an FDS might describe a group of cells or a hyperstructure of molecules, providing that their performing a task is intrinsic to their assembly. This would lead to advantages for the hyperstructure in addition to those described above, and a functioning-dependent PTS-glycolytic hyperstructure might be expected to have its metabolic activity maximized by active, enzyme-promoted association of membrane and cytoplasmic constituents because (i) the multiple interactions involved in hyperstructure formation would help maintain the hyperstructure during fluctuations in substrate supply; (ii) enzyme association due to substrate-induced binding might select the appropriate transporter from a competing population, during, for example, diauxic growth of a bacterium on two substrate sugars; and (iii) the dissipative nature of the structure would imply that when the substrate was completely exhausted, the membrane domain would disperse, and the cytoplasmic structure would dissociate to free the space for other structures. In eukaryotes, numerous examples of FDSs exist, while in E. coli, there is the example of the promotion by substrate binding of the assembly of the three components of the protein-mediated transporter responsible for protein secretion (139).

So what does all this mean? There are equilibrium enzymatic hyperstructures such as the cellulosomes and some large complexes, such as those with the tricarboxylic acid cycle enzymes (182), that might be constituents in hyperstructures, but are there really substrate-induced enzymatic hyperstructures in bacteria? Evidence one way or the other is still insufficient, but since the synthesis of many of these abundant enzymes almost necessarily entails the formation of a synthesis hyperstructure, it is conceivable that the high concentration of enzymes newly released from a synthesis hyperstructure might drive their assembly into an adjoining enzymatic hyperstructure (see below).

A range of actin-like proteins also exist in bacteria (for references, see reference 143). After many suspicions that there might be a bacterial actin (for references, see reference 199), which were reinforced by sequence analysis revealing many candidate proteins (32), actin-like filaments were discovered in B. subtilis (109), and the crystal structure of MreB was subsequently shown to resemble that of actin (266); filaments formed by MreB have a short pitch (0.73 ± 0.12 mm) and assemble around the middle of the cell, while those formed by Mbl have a longer pitch (1.7 ± 0.28 mm) and cross the entire cell (109). The filamentation of MreB is ATP dependent (266), and the rate of extension of the growing end of filaments is similar to that of actin (0.1 µm/s), generating a potential poleward or centerward pushing velocity at 0.24 µm/min for MreB or Mbl, respectively (59). MreB is an important determinant of cell shape in the rod-shaped E. coli and B. subtilis as well as in the crescent-shaped C. crescentus, where it is reported to organize a PBP 2 complex involved in peptidoglycan synthesis and cell elongation into a band-like structure (76); immunoprecipitation data suggest that this complex contains PBP 1a, PBP 2a, PBP 2b, PBP 3a, and possibly other enzymes responsible for peptidoglycan synthesis (which would be consistent with other evidence for the existence of such a complex) (236). It should be noted that MreB does not appear to play such a role in B. subtilis (235). One idea is that MreB filaments not only act as a tracking device for the PBP 2-peptidoglycan biosynthesis complex but also are involved in switching peptidoglycan synthesis from an elongation mode to an invagination mode required for septation; this is because MreB is localized along the length of the cell during elongation but becomes concentrated at midcell early in division. Indeed, in Rhodobacter sphaeroides, MreB is localized to midcell in early septation, where it is also believed to be involved in the control of peptidoglycan synthesis (241). A possible dialogue between cytoskeletal hyperstructures was recently revealed. Separate and independent spirals of MreB and MreC occur in C. crescentus, with PBP 2 located both on the MreC spiral and at the division site (66, 70); the pattern of peptidoglycan synthesis depended on the existence of both spirals as well as on the level of FtsZ (70).

MreB may, however, have other cytoskeletal roles, since it appears to form part of a kinetochore-like complex that specifically segregates the replication origin region of the C. crescentus chromosome (87). However, the claim that the positioning of the replication hyperstructure in B. subtilis is partly dependent on MreB (58) has to be set against evidence that segregation does not depend on MreB in this organism (79). Finally, the motility of Spiroplasma melliferum, one of the helical Mollicutes which lack cell walls, depends on changes in the length and tension of cytoskeletal structures formed from two proteins, one of which is MreB (128).

In B. subtilis, another actin homologue, Mbl, is involved in maintenance of the cell wall. A banded pattern is made along the long axis of the cell by labeled vancomycin binding to sites of peptidoglycan assembly, and this pattern depends on Mbl (54). Turnover occurs along the length of the helical Mbl filaments, which have no obvious polarity and which appear to draw on a cytoplasmic pool that contains oligomers; the filaments are very dynamic, and when labeled and photobleached, they have a recovery half-time of about 8 min (41). The helical pitch of the filaments in cells of various sizes and at different growth rates remains relatively constant. Drawing on earlier ideas (167), it has been proposed that as the cell grows, the newly inserted helical strands of peptidoglycan stretch along the long axis of the cell to generate torsional stress in the direction of helix unwinding, and thus a helical rotation in the cell envelope, in the opposite (left-handed) direction to the growth of the Mbl helical filaments (41). This would enable the filaments to scan the inside surface of the cell membrane and so help generate a uniform new layer of wall material; moreover, the changing pitch of the older peptidoglycan strands would combine with the constant pitch of the newly inserted material via the Mbl filaments to generate a wall resistant to shearing (41). Insofar as peptidoglycan synthesis in B. subtilis is dependent on the helical structure of Mbl filaments (rather than simply on a collection of Mbl proteins), the Mbl filaments can be considered a hyperstructure. Similarly, if the segregation of the origins of replication in C. crescentus is dependent on the cytoskeletal structures formed by MreB (rather than on some aspect of these proteins that does not involve these structures), they might also be considered hyperstructures. Both are examples of how proteins act at the level of a hyperstructure to perform a cellular task and, arguably, of how a cytoskeletal hyperstructure might interact with other hyperstructures.

The translation elongation factor EF-Tu is a GTPase that delivers amino-acylated tRNAs to the ribosome during the elongation step of translation. EF-Tu/GDP is recycled by the guanine nucleotide exchange factor EF-Ts. The functional homologue of EF-Tu, EF-l, the eukaryotic aminoacyl-tRNA carrier, is also a major actin-bundling protein in eukaryotes. Bacterial EF-Tu has long been suspected of being an actin homologue. It forms filaments in vitro (18, 51, 98), it is more abundant (5 to 10% of soluble protein) than might be expected if its role were restricted to translation, it associates with the membrane, and its overproduction results in the loss of shape of E. coli. Remarkably, it has now been shown to form protofilaments and networks in vivo (163). The nature of the controls over its assembly and disassembly within cells is likely to be interesting, since ribosomes/polysomes were seen to be attached to protofilaments of EF-Tu; in terms of nonequilibrium structures, polymerized EF-Tu exchanges nucleotide rapidly and interacts with the other elongation factor, EF-Ts (18). However, both EF-Tu/GTP and EF-Tu/GDP can polymerize equally well in vitro (see below) (98). Below, we consider the significance of this in terms of ion condensation stabilizing inactive filaments and translational activity destabilizing them. In other words, we consider whether an EF-Tu cytoskeleton might be interpreted as a hyperstructure with a role in the sensing of metabolic activity.

The first SOS proteins to be produced are UvrA, UvrB, and UvrD; these, along with the endonuclease UvrC, catalyze the NER pathway (for a review, see reference 171). In E. coli, NER was shown to entail a significant increase of DNA-membrane contact points. To these points, many proteins, including UvrA, UvrC, the four units of RNA polymerase, DNA gyrase, and RecA, are recruited (145). DNA lesions are specifically relocated towards this DNA-protein-membrane hyperstructure, the formation of which depends upon UvrA and RecA. It was proposed that NER, recombination repair, transcription, and replication may have to cooperate with each other (145, 226). The ordered yet fluid nature of the cell membrane is likely to provide a suitable matrix on which these processes can be finely coordinated in space and time.

As a second line of defense, the homologous recombination pathway is activated to repair double-stranded DNA breaks (DSBs) (171). Homologous recombination proceeds through several sequential phases (125). Initially, a presynaptic filament is formed in which RecA molecules coat a single-stranded DNA substrate. This filament then participates in a sequence-specific search for double-stranded DNA sites that are homologous to the RecA-coated segment; a joint species results in which DNA strand exchange and heteroduplex extension occur. It turns out that this elaborate repair pathway involves a large, ordered hyperstructure(s) composed of DNA, many proteins, and presumably the cell membrane (179). How does this repair hyperstructure mature and how does it function?

In E. coli, formation of the presynaptic filament depends on the activity of multiple proteins, including the RecBCD, RecN, RecF, RecO, and RecR proteins (31, 125). RecN is an ATPase, a single-stranded-DNA-binding protein, and a member of the structural maintenance of chromosomes (SMC) superfamily (like the eukaryotic Rad50 protein) (119). Following generation of DSBs in B. subtilis, RecN assembles at the site of the DSB, followed successively by RecO (believed to be equivalent to Rad52) and RecF, which facilitate the loading of RecA (the equivalent of eukaryotic Rad51) onto single-stranded DNA, while RecF is thought to limit the extent of RecA binding (31, 120, 186). Once formed, RecA nucleofilaments perform a whole-genome search for the homologous duplex. High-resolution electron microscopy studies of E. coli cells exposed to DNA-damaging agents indicated that this homologous search is associated with a dramatic reorganization of bacterial chromatin into a tightly packed filamentous structure that appears to be associated with the cellular membrane (141). It has been argued that the tight, striated morphology of the assembly promotes homologous search by attenuating both the sampling volume and the dimensionality of the process (178, 179). Notably, DNA-RecA-membrane association has been proposed to be related to the activation of RecA (85) and to promote the coordination of repair processes (145). A RecA-dependent chromatin reorganization into a highly compacted structure has subsequently been observed to occur in B. subtilis cells exposed to DNA-damaging agents (242), implying that the formation of repair hyperstructures is essential and widespread in bacteria (228). The highly dynamic nature of the multiprotein-DNA repairosome or repair hyperstructure(s) was recently highlighted by observations which indicated that the nucleoprotein filaments extend and shrink on a time scale of a minute, a process interpreted as representing the search for the homologous duplex by the nucleofilaments (118). The notion that the repairosome indeed corresponds to a highly regulated hyperstructure is further buttressed by the recent finding that the SOS network exhibits precise temporal modulations (80), indicating the presence of an elaborate signaling network.

Repair of multiple double-stranded DNA breaks also appears to occur in eukaryotic cells in a large DSB repair center or hyperstructure, in which the enzymes in the Mre11-Rad50-Xrs2 complex come to the break sites to control end processing and signaling, followed by proteins that bind to single-stranded DNA as well as other signaling and repair proteins such as Rad51 and Rad52 (147, 148). As in eukaryotes, repair hyperstructures in bacteria are believed to be capable of containing several DSBs, (9), since increasing their number (up to the five or so that a bacterium can handle) does not lead to comparable increases in the number of hyperstructures per cell (120). Correspondingly, the size of this highly dynamic hyperstructure is dependent on the extent of the initial or ongoing DNA damage, and when the SOS response ends, the hyperstructure disappears.

The vectorial search performed by the repair hyperstructure depends on the continuous consumption and dissipation of large amounts of energy. Again, we conclude that it is a nonequilibrium hyperstructure. If exposure to UV irradiation or to other DNA-damaging agents continues such that the rate of damage exceeds that of repair, ATP levels fall, and the dynamic RecA nonequilibrium hyperstructure collapses into a highly ordered RecA-DNA cocrystal where the tight crystalline packaging is believed to protect the DNA by physically sequestering (141). RecA is indeed known to protect chromosomal DNA from degradation. It is worth pointing out that these hyperstructures can be converted into one another depending on conditions (179).

Representatives of each of the above classes of competence proteins (ComGA, ComFA, and YwpH) accumulate preferentially at one (but sometimes both) of the cell poles, as does ComEA, although less markedly (93). In addition to the polar locations, these proteins are also present in a small number of foci close to the membrane, where they appear to follow a helical path. Moreover, given the estimated number of ComEC uptake pores of around 200, these foci are believed to contain many uptake assemblies (93). The interpretation of these results is that the binding, processing, and internalization of foreign DNA occur via molecular machines located at a very few sites and, in the limit, at one site in a cell pole (93, 119).

The story does not end with the internalization of the single-stranded DNA by a large hyperstructure. This DNA is used for homologous recombination with the chromosome, a process that depends on the binding of the ATPase RecA to single-stranded DNA, and as described above, RecN forms repair centers, to which first RecO and later RecF are recruited, on the nucleoids when DNA double-strand breaks occur (120). Both RecN and RecA have now been found to be localized at one cell pole, albeit with interesting differences. In competent cells, RecA colocalized with ComGA at one cell pole while RecN oscillated between both poles on a time scale of a minute (119). The essence of these findings is that foreign DNA enters the cell at one pole in a competence hyperstructure that includes RecN (which may protect it) and possibly RecA. RecA then forms long dynamic filaments that extend from the competence hyperstructure far into the cell to scan for homologous sequences on the chromosome to allow recombination to occur.

This initiation hyperstructure matures into a full-blown replication hyperstructure which comprises a DnaE-DnaE or PolC-DnaE strand polymerization complex along with clamp loader, primase, helicase, and single-stranded binding proteins. Other enzymes associated with replication are probably also present. It seems likely that several leading- and lagging-strand polymerases are needed per replication fork and that additional polymerases are required to aid in recombination and repair DNA, as exemplified by the location of RarA in the replication hyperstructure in E. coli (134). Ongoing replication requires feeding the hyperstructure with the four deoxyribonucleotides (dNTPs) at the rate of about 3,000 nucleotides per second, yet despite this high rate, there are sufficient dNTPs for only half a minute of synthesis. It is therefore not surprising that there is evidence in both eukaryotes and prokaryotes for the presence of ribonucleoside diphosphate reductase, which catalyzes the synthesis of the dNTPs, in the hyperstructure (92, 161). Dingman originally proposed that in bidirectional replication, the two replication forks remain together in a relatively static complex while the DNA passes through this complex (65); a quarter of a century later, evidence was found in support of this proposal, and this complex was termed the "replication factory" (136). It now seems that the exact spatial and functional relationships of the two forks to one another are uncertain and that the interactions holding the forks within the factory/hyperstructure are dynamic rather than static (36, 173). This picture of a dynamic hyperstructure dependent on its functioning is consistent with the disruption of the hyperstructure that occurs when replication forks encounter the many barriers that result in the arrest or breakage of the fork before reaching the terminus (as in the case of DNA damage and blocking proteins that force a restart by the reassembly of the replication machinery with the help of recombination proteins) (50, 165), when the supply of nucleotides is limited, and when the thymidylate synthetase gene is mutated (183).

Several mechanisms exist in E. coli to ensure that once initiation has occurred it does not immediately recur (137). Newly replicated DNA is transiently hemimethylated (with the old strand methylated and the new strand still to be methylated); SeqA, an oligomeric protein, binds preferentially to hemimethylated GATC sites, of which there are 11 in the minimal origin, to sequester this and other regions (such as the dnaA gene) and prevent multiple reinitiations. Hundreds of copies of SeqA, along presumably with the DNA to which it binds, form foci (206). There are 19,130 GATC sequences in the E. coli chromosome that are clustered around genes implicated in the replication, repair, and structure of DNA as well as in oriC; these genes include dnaA, dnaC, dnaE, gyrA, topA, hepA, lhr, parE, mukB, recB, recD, and uvrA, as well as genes involved in the synthesis of the precursors of DNA, purines and pyrimidines, namely, nrdA, purA, purF, purL, pyrD, and pyrI (for references, see reference 200). This has led to the idea of a sequestration/replication hyperstructure based on SeqA that would comprise not just the enzymes responsible for synthesizing DNA but would comprise these enzymes, the enzymes responsible for synthesizing and supplying DNA precursors, enzymes responsible for repair and recombination, the genes encoding many of these enzymes, and a specific region of the membrane (200). It is not surprising, therefore, that SeqA mutants are also affected in supercoiling and in the segregation of the chromosomes. (It should be noted here that formation of a SeqA-based replication hyperstructure is coupled to growth. In an E. coli mutant defective in initiation in which DnaC is inactivated for a while and then reactivated by temperature shifts, the number of SeqA foci equals the number of replication forks [11], presumably because the cell has grown too large during the period of inactivation of DnaC for the forks to come together in replication hyperstructures.)

In the hyperstructure approach to replication, the dynamics of the sequestration hyperstructure define the period for which oriC is protected from new initiations. E. coli contains more than one copy of its chromosome when growing rapidly, and initiation occurs at several copies of oriC simultaneously; under these conditions immunofluorescence studies of SeqA suggest that the replication hyperstructure contains up to 6 forks immediately after initiation and that as the cell grows these divide into smaller structures that go to separate locations (183). This division of the replication/sequestration hyperstructure probably corresponds to the moment when oriC becomes available again for initiation. But what explains it? One possibility is that the continuing production of new methylated and hemimethylated GATC sequences titrates away SeqA and so weakens the giant sequestration hyperstructure; in the case of E. coli synchronized for chromosome replication, there is evidence that the SeqA foci change in composition as replication proceeds (288). Another, complementary, possibility is related to the existence of a specific segregation machine that might help pull apart both origins and sequestration hyperstructure.

The above evidence demonstrates the existence of specific hyperstructures that operate on origin regions to either segregate them or maintain them in polar positions. This is not the end of the story. A two-step model has been proposed for C. crescentus in which first oriC is rapidly moved poleward via MreB and then the rest of the chromosome moves independently of MreB (87). More generally, a two-level model might be envisaged, in which one or more specific hyperstructures operate on the origins but the entire chromosomes are separated by hyperstructure dynamics. In the latter case, a centrally located replication hyperstructure could push out the newly replicated daughters in opposite directions (136), aided and abetted by transcription (69). These ideas have been taken further, and in the strand-specific segregation model it is the association of each parental strand with a particular set of hyperstructures, and the continued association once replication has occurred, that ensures the separation of the daughter chromosomes (227). This is in line with the correlation between the location of genes on the chromosome and their position along the long axis of the cell (193, 253, 269) and with evidence showing a highly asymmetric pattern of segregation of markers around the terminus (275). Recent evidence suggests a progressive separation of the chromosomes (191), but against this there is also good evidence consistent with sister chromosomes remaining linked for a "significant" time after replication and with separation occurring simultaneously throughout the genome (14). It may prove important in this context to distinguish between slowly growing cells, where segregation of daughter chromosomes appears to be straightforward, and rapidly growing cells, where segregation of daughter and granddaughter chromosomes is more complicated and may require grouping into hyperstructures (K. Skarstad, unpublished data). Finally, proteins that compact the chromosomes after replication are also implicated in segregation (see below).

A successful round of cell division requires the coordinated orchestration of numerous cellular processes, including synthesis of proteins and lipids, peptidoglycan synthesis and hydrolysis, and the transport of these newly synthesized materials to the septal site. It is therefore not surprising that the majority of phospholipid synthases in B. subtilis are localized to the septal membranes, which are rich in cardiolipin and phosphatidylethanolamine, in an FtsZ-dependent manner (194). During growth in rod-shaped cells, new peptidoglycan is inserted randomly into the expanding lateral envelope, whereas new cell poles are generated de novo as a consequence of cell division (63). Old cell poles are completely inert with respect to peptidoglycan synthesis. The new cell poles are made during a burst of midcell peptidoglycan-synthesizing activity (279). While the absence of functional FtsZ in rod-shaped cells results in filaments that contain lateral peptidoglycan, defective FtsI or FtsQ, enzymes involved in coordinating cross wall synthesis with septation, results in the formation of filamentous cells that have bands of newly synthesized (polar) peptidoglycan at regular positions separated by one average cell length (63). Based on these observations, it has been suggested that the group of cell division proteins that are assembled into the early form of the hyperstructure initiate septal peptidoglycan synthesis by recruiting factors required for cell wall biosynthesis to midcell (1). While at least one component of the divisome is a transpeptidase (FtsI/PBP 3), many are not directly involved in peptidoglycan synthesis and instead must coordinate their activity with those of the peptidoglycan precursor synthetases MurG and MraY (33, 34, 101, 169) as well as the as yet-unidentified precursor translocase. The early cell division proteins and the proteins involved in peptidoglycan metabolism have a common purpose that cannot be achieved individually. Thus, all components involved in the synthesis of septal peptidoglycan represent a single hyperstructure.

In addition to orchestrating the coordinated invagination of the cell membrane(s) and the cell wall, the divisome must also coordinate DNA replication and nucleoid segregation, which are believed to occur simultaneously in bacteria, where the FtsZ ring assembly and DNA replication termination more or less coincide (61). A number of mechanisms are thus present to ensure that the divisome does not bisect the segregating nucleoid. First, DNA-binding proteins (SlmA in E. coli [24] and Noc [for nucleoid occlusion] in B. subtilis [285]) inhibit FtsZ assembly over the nucleoid, helping to prevent aberrant FtsZ assembly along the length of the cell until chromosome segregation reveals a nucleoid-free space or a membrane domain at midcell (see below). Should disaster occur, two divisome-associated proteins act to ensure that each daughter cell receives a complete copy of genetic material. In E. coli, should the nucleoids be accidentally concatenated, FtsK binds near the terminus region of the chromosome and allows the Xer proteins to decatenate the nucleoids (106) prior to the final closure of the invaginating septum. In B. subtilis this role is filled by the DNA translocase SpoIIIE. SpoIIIE, a domain of which shares a significant degree of homology with FtsK, is responsible for pumping DNA that has been trapped in the wrong compartment into the appropriate cell prior to cell separation (21).

While the ultrastructure of the cytokinetic ring has yet to be determined, protein localization, mutant analysis, and bacterial two-hybrid studies (64, 113) indicate that it is held together by an intricate network of interactions between all of its constituents that ultimately serves to link the founder proteins (e.g., FtsZ and FtsA) with the late arrivals (e.g., FtsI). Some of these interactions are formed sequentially on the ring itself, whereas others take place in the cytoplasm prior to FtsZ localization and these preassembled complexes are then loaded onto the ring as a single unit (37). Since the majority of cell division proteins are not present in sufficient numbers to form a ring structure or to interact with FtsZ in a one-to-one ratio, the divisome is envisioned as a ring with protein subcomplexes at regular distances (189). This structure is not a permanent complex but is likely to be very dynamic like the Z ring itself (see above). Given their functional rather than structural role, we envision that the subcomplexes would consist predominantly of late-localizing proteins such as the transpeptidase FtsI and other factors involved in peptidoglycan synthesis and the redirection of growth from lateral to perpendicular. Again the subassemblies localize in three compartments of the cell (i.e., the cytosol, the cytoplasmic membrane, and the periplasm). Regardless of their location or activity, together the individual constituents of the divisome, like those of other hyperstructures (and like, at a lower level, the ribosome), form an integrated structure whose function is greater than the sum of its parts.

The constituents of the division hyperstructure (or the hyperstructure itself) have more than one function. FtsK acts as a DNA translocase to assist chromosome dimer resolution during division when the developing septum may damage dimeric or intercatenated chromosomes (134). Such dimers are produced by homologous recombination, and their resolution involves Xer site-specific recombination whereby XerC and XerD recombine pairs of dif sites, which are 28-bp-long sequences located in the 330-kb terminus region where replication terminates (49). This requires interaction between FtsK and XerCD/dif.

The concept of a division hyperstructure that comprises many diverse molecules and that matures may help in grappling with a central problem in division. What lies upstream of FtsZ assembly at the division site? What is really the first step in division? What triggers 30% of the FtsZ to go to midcell (249)? It has been observed that cardiolipin domains occur in vivo at the division sites in E. coli (122, 174, 175) and B. subtilis (115) (note for those who like to look across the phyla: lipid domains are involved in division in fission yeast [220]). One possibility is that the transcription, translation, and insertion of proteins into the membrane (i.e., transertion) structure the bacterial membrane around the chromosomes (27, 78) such that the segregation of the chromosomes creates a domain enriched in cardiolipin between them (196). This domain might then concentrate not only FtsZ (perhaps even in a monomeric form) but also GTP plus other division proteins such as FtsA and ZipA, promoting effective polymerization in the right place at the right time, as well perhaps as other enzymes such as MurG (265). A cardiolipin-rich domain might also concentrate ions such as calcium, which can promote FtsZ polymerization in vitro (150, 289). There is some evidence that calcium levels are higher near the membrane (83) and that they can vary cyclically (43). Intriguingly, production in E. coli of S100B, a human protein that undergoes a calcium-dependent conformational change to bind to tubulin, results in it colocalizing with FtsZ and inhibiting division (75).

Does the division hyperstructure also include the genes encoding the enzymes involved in division? Several of these genes are located and transcribed together in the dcw cluster at the 2-min position on the E. coli chromosome. The 16 genes in this cluster include many involved in division or peptidoglycan synthesis, such as ftsZ, ftsQ, and ftsA. Polymeric proteins such as FtsZ are abundant, with estimates ranging from 3,000 to 15,000 monomers per cell (151), so it is conceivable that this cluster could be attached dynamically to the developing division hyperstructure for at least part of its existence. It may also contain, transiently, the terminus region or at least certain of the repeated motifs believed to direct the translocation activity of FtsK to the dif sites (26, 138).

Preventing FtsZ assembly at aberrant locations is critical to maintaining the fidelity of cell division. The Min proteins are one means by which bacteria prevent nonproductive division events at cell poles (140). In E. coli, the Min system consists of three proteins, MinC, MinD, and MinE (57). MinC is believed to inhibit the assembly of Z rings by binding to FtsZ polymers and inducing displacement of FtsA or possibly by preventing FtsA binding to FtsZ polymers (for references, see reference 6). MinC is recruited by MinD, which binds cooperatively to membranes in the presence of ATP and which forms a long, helical polymer that assembles from the pole (238). The MinE protein disassembles this structure, and the dynamics are such that the MinCD polymer is assembled first from one pole and then from the other with a period of 30 to 50 seconds. MinD interacts with lipids (251) and, in particular, with anionic phospholipids in vitro (where it forms oligomers) and in vivo (where its distribution depends on these phospholipids) (176); MinD has also been reported to convert phospholipid vesicles into tubes (102). Given the similarity of MinD to dynamin and the role of the latter in membrane insertion during division in eukaryotes, one speculation is that the MinCD polymer contains a tubule of fresh membrane to be incorporated into the membrane when released by MinE (6). Alternatively, the phospholipid tubes may be important constituents of the FtsZ assembly site and the Min system acts to remove them, thereby preventing aberrant Z-ring formation (204).

It should be noted that the Min system is not completely conserved in all bacterial species and is not therefore a universal system for preventing polar septation. In contrast to E. coli, B. subtilis does not have a MinE homologue and MinCD does not oscillate but remains at the poles through interactions with the DivIVA protein (95). In C. crescentus there are no Min homologues. Instead, MipZ, which interacts directly with both the chromosome partitioning apparatus and FtsZ, prevents aberrant FtsZ assembly at cell poles (254). Finally, while it has been suggested that the Min genes play a role in selecting the medial division site, (221), data indicating that the majority of division events take place at midcell even in min mutants, along with evidence that the nucleoid plays an important role in the spatial regulation of division, suggest that the primary function of the Min proteins is to prevent aberrant FtsZ assembly at cell poles (140, 204).

CANDIDATE PROCESSES IN HYPERSTRUCTURE ASSEMBLY, DISASSEMBLY, AND INTERACTIONS

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