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Bacterial Cells Have Cytoskeletons, Too Bacterial cells contain cytoskeletal structures that impart long-range order within the cell Lawrence Rothfield, Aziz Taghbalout, and Purva Vats lthough eukaryotic cells contain a complex internal cytoskeleton, until recently microbiologists believed that bacteria contained no comparable elements except for the murein exoskeleton located outside the cytoplasmic membrane. Indeed, the absence of a cytoskeleton was one of the hallmarks used to distinguish bacteria from eukaryotic cells. That view changed dramatically in 2001 when Laura Jones, Rut Carballido-López, and Jeff Errington of Oxford University, Oxford, United Kingdom, described the cytoskeletal nature of the actinlike MreB and Mbl proteins of Bacillus subtilis. Within these rod-shaped gram-positive cells, the two proteins form structures that extend between the two poles and help to regulate cell shape. This finding prompted a period of rapid progress that changed our view of bacterial cellular organization. The eukaryotic cytoplasm has long been known to contain several types of extended cytoskeletal networks, composed of microtubules, intermediate filaments, and actin filaments, that communicate with other intracellular and membrane-associated components. It is now clear that bacteria contain proteins resembling both the actin and nonactin cytoskeletal proteins of eukaryotic cells as well as proteins unrelated to the eukaryotic cytoskeletal proteins, organized into extended membrane-associated structures. These structures provide long-range order to the cell—far more than microbiologists once recognized. A Lawrence Rothfield is a Professor, Aziz Taghbalout is an Instructor, and Purva Vats is a Postdoctoral fellow in the Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington. Homologs of Actin Help To Shape Bacterial Cells Cells of B. subtilis contain three actin homologs, designated MreB, Mbl (Fig. 1B), 582 Y ASM News / Volume 71, Number 12, 2005 and MreBH. Further, most other types of rodshaped bacteria, including E. coli, contain actinlike proteins homologous to MreB (Fig. 1C). In contrast, MreB-related proteins are absent from those coccal species whose genomic sequences are known, supporting the idea that a major role of MreB is to support establishment of a rod shape. The three-dimensional structures of MreB and actin are very similar, as shown by F. van den Ent, Linda Amos, and Jan Löwe of the MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. Within the cell, MreB is organized into two helical strands composed of actin-like protofilaments, running along the inner surface of the cytoplasmic membrane and usually coiling around the rod-shaped cell along its long axis (Fig. 1C). The coiled structure changes its appearance and position during the cell cycle, according to work from the laboratories of James Gober of the University of California, Los Angeles (UCLA) and Lucy Shapiro of Stanford University, Stanford, Calif. At a specific stage of the C. crescentus cell cycle, MreB rearranges to form a ring-like structure near the division site. • Bacteria, long thought to lack a cytoskeleton, contain proteins resembling both the actin and nonactin cytoskeletal proteins of eukaryotic cells as well as additional proteins that play cytoskeletal roles. • Actin homologs perform a variety of functions, helping to determine cell shape, segregate chromosomes, and localize proteins within bacterial cells. • Nonactin homologs and unique bacterial cytoskeletal proteins are involved in determining cell shape and in regulation of cell division, chromosome segregation, and probably other cellular functions. This resembles the behavior of FIGURE 1 eukaryotic actin, which assembles into a cytokinetic ring at the division site early in the division process. Other bacterial actin-like structures do not show this degree of plasticity. The overall coiled structure of Mbl , for example, appears not to vary although particular parts of the structure turn over at a rapid rate, according to fluorescence photobleaching analysis. The different behavior of the MreB and Mbl structures emphasizes that bacterial cells contain several cytoskeletal structures, with different properties. The actin-like cytoskeletal structures perform several important cellular functions in bacteria. One such function is to regulate the shape of rodshaped bacteria by regulating the pattern of synthesis of the cell wall peptidoglycan. Thus, loss of MreB in all species that have been examined, and of eiCytoskeletal structures in bacterial cells. (A) Crescentin in Caulobacter crescentus. The crescentin ther MreB or Mbl in B. subtilis, filamentous structure (red) is located along the concave edge of the comma-shaped cells, in which causes cells to lose their rod DNA (blue) has been co-labeled with DAPI. (Reproduced with permission from Ausmees, Kuhn and shape. The link between the acJacobs-Wagner, 2003, Cell 115: p. 707.) (B) Mbl protein of B. subtilis- The helical Mbl structure, visualized by immunofluorescence, is visible along the length of the cell and appears to be tin-like proteins and the pepticomposed of two intertwined helices. (Reproduced with permission from Jones, Carballido-Lopez doglycan synthetic machinery and Errington, 2001, Cell, 104, p. 916.) (C) MreB protein of E. coli- The double helical organization is exemplified by the observaof the E. coli Yfp-MreB protein is visualized in a three-dimensional reconstruction from a series of optical sections of fluorescence micrographs. (Reproduced with permission from Shih, Le and tion that the sites of peptiRothfield, 2003, Proc. Natl. Acad. Sci. U.S. 100: p.7868.) (D) FtsZ protein in sporulating B. subtilisdoglycan synthesis are distribThe FtsZ spiral structure represents FtsZ-Gfp in a fluorescence micrograph of a sporulating cell. uted in a coiled pattern that (Reproduced with permission from Ben-Yehuda and Losick, 2002, Cell 109: p. 258.) (E) Representation of panel D. The FtsZ spiral structure is indicated in red. (F) MinD protein in E. coli. The helical extends along the length of the structure of Gfp-MinD is visualized in an E. coli cell that is coexpressing Gfp-MinD and MinE. The cell in a helical arrangement high level fluorescence in the coils in the upper portion of the cell represents the high concentration similar to that of Mbl, and this of MinD within the polar coils (“polar zone”) of the MinD helical array that runs the entire length of the cell. The MinD in the polar zone oscillates between the two ends of the cell with a periodicity distribution depends on the of 1–2 minutes. . (Reproduced with permission from Shih, Le and Rothfield, 2003, Proc. Natl. Acad. presence of Mbl, as shown by Sci. U.S. 100: p.7867). (G) Representation of panel F. The high concentration of MinD in the polar Richard Daniel and Jeff Errzone is indicated in red. The lower concentration of MinD in the remainder of the pole-to-pole helical array is indicated in blue. ington. During the process of cell shape regulation in B. subtilis, Mbl apparently controls longitudinal growth along the cell cylinder helical pattern in C. crescentus, according to whereas MreB controls cell width, according to Rainer Figge and his collaborators at UCLA. Daniel and Errington. Similarly, penicillin-bindA second important function of the MreB ing protein 2 (PBP2), which is involved in pepcytoskeleton is to participate in movement of the tidoglycan synthesis and is required to maintain two newly replicated daughter chromosomes to rod shape, also depends on MreB to form a the opposite poles of a cell. Depletion of MreB Volume 71, Number 12, 2005 / ASM News Y 583 Several Forces Helped Steer Rothfield from Clinical onto Research Career Path When Lawrence Rothfield entered Cornell University before his 16th birthday, his head was filled with images of physicians and scientists from books. “I wanted to be a doctor from my early teens because I wanted to make great discoveries about the causes and cures of diseases,” he says. He assumed, as did many researchers like him, that he needed to be a physician before he could make medical discoveries. After graduating from Cornell, Rothfield studied medicine at New York University (NYU) and soon became a practicing internist on Park Avenue in Manhattan. Because he also conducted research, supported by his first research grant for a modest $6,500 from the National Institutes of Health, “it meant 80-hour weeks, and eating a sandwich for lunch in the car, while driving from my medical office to the medical school,” he recalls. “Although I think I was a pretty good doctor,” Rothfield says, the lifestyle was too frantic. Thus, after about four years, he stopped doing clinical work and turned his full attention to research. After applying for a more expansive grant, Rothfield experienced what he calls a “life-changing” event—a visit from NIH reviewers who told him that “my ideas were good, but that I was totally unqualified to do the work.” They advised him to apply for a fellowship to do biochemical research with Bernard Horecker, who was then head of microbiol- ogy at NYU, where he was assigned to work with Mary Jane Osborn, then a junior assistant professor in Horecker’s group, and still a friend and colleague. “After two postdoctoral years, when I was scheduled to return to medicine, it became clear to Bernie Horecker that I was doing so reluctantly, and he offered me a faculty position in his new department of molecular biology at the Albert Einstein College of Medicine,” Rothfield says. “I didn’t hesitate to accept.” He never again practiced medicine. Today Rothfield is professor of molecular, microbial, and structural biology at the University of Connecticut Health Center, where he served as department chair from 1968 to 1980. His current research focuses on the mechanism of cell division, and the long-range organization of the bacterial cell. “Beyond the obvious biological interest in understanding how cells divide, and the potential application of this knowledge in developing antibacterial agents that block bacterial cell division, I see our work as an example of a much broader biological question,” he says. “How do cells determine spatial relationships between different parts of the cell and use this information to undergo specific differentiation events at predetermined locations? “For example, in the case of cell division proteins, the site is usually at the midpoint of the cell, whereas other cellular proteins or certain mutational changes in MreB impairs chromosome segregation, according to work by Herve Soufo and Peter Graumann at the University of Marburg, Marburg, Germany, and from 584 Y ASM News / Volume 71, Number 12, 2005 will be located at one or both cell poles,” he continues. “How does the cell distinguish between these locations? This problem of topological recognition is a general biological problem that is encountered in all types of cells.” Rothfield grew up in New York City, where his father—a “man of total honesty and integrity” — worked as a pharmacist. His mother, who became a full-time housewife, dropped out of law school to support her husband during pharmacy school. Rothfield’s wife Naomi, a physician and rheumatology expert, is a professor of medicine at Connecticut. He credits her—along with the NIH grant review group—with propelling him along a research career path. “She encouraged me despite the significant blow to our not-too-robust financial situation,” he says. They have four grown children—not a scientist in the group—two involved with music professionally, the third a computer programmer, and the fourth a faculty member in a university English department. Rothfield vividly recalls Ludwig Eichna, professor of medicine at NYU, who influenced him greatly. “Eichna never accepted sloppy thinking and always demanded of students ‘Show me the evidence!’” Rothfield adds, “I never forgot that lesson.” Marlene Cimons Marlene Cimons is a freelance writer in Bethesda, Md. the laboratories of Lucy Shapiro at Stanford University and of Kenn Gerdes at the University of Southern Denmark, Odense, Denmark. Zemer Gitai and his collaborators at Princeton University and Stanford University also showed that MreB molecules associate, directly or indirectly, with the Caulobacter chromosome near its origin of replication. Thus, the MreB cytoskeleton is likely to be associated with the bacterial equivalent of a centromere, working cooperatively with other bacterial proteins at this site to move the oriC region of each daughter chromosome to a cell pole, perhaps by moving it along the MreB filament toward a polar binding site. Yet a third MreB function is to deliver or maintain certain proteins at the cell poles, according to work from the laboratories of Shapiro, Marcia Goldberg at Harvard University, and Yu-Ling Shih and her collaborators at the University of Connecticut (UC). This process also could involve movement of the proteins along the MreB cytoskeletal track. Bacteria Encode Homologs to Other Eukaryotic Cytoskeletal Proteins In addition to cytoskeletal elements composed of actin homologs, some bacteria contain cytoskeletal structures formed by homologs of the two other main groups of eukaryotic cytoskeletal proteins—intermediate filament proteins and tubulins. For instance, the comma-shaped bacterium C. crescentus contains a homolog of intermediate filament proteins, called crescentin. In C. crescentus cells, crescentin forms a curved filamentous structure adjacent to the inner surface of the cytoplasmic membrane (Fig. 1A), according to Nora Ausmees, Jeffrey Kuhn, and Christine Jacobs-Wagner at Yale University. Crescentin is required for the cells to maintain their crescent shape and, in its absence, the cells grow as rods. Moreover, when MreB is removed, the cells also lose their normal crescent shape, but become spherical. Therefore, this species contains two shape-determining cytoskeletal proteins, one (the actin homolog MreB) that is responsible for the underlying rod shape of the cell and the other (the intermediate filament protein homolog crescentin) responsible for imparting curvature to the rod. Additionally, almost all bacterial species encode FtsZ, a close structural homolog of the tubulin proteins that are the major components of eukaryotic microtubules. FtsZ is required for cell division and forms a ring structure at the division site during the first step of assembling the bacterial cell division machinery. The FtsZ ring acts as a scaffold for assembly of the large number of other proteins of the division apparatus. However, FtsZ can also assume a coiled structure that winds around the length of rodshaped cells, and it is likely that what appears to be a FtsZ-containing ring at the division site is a tightly compressed coiled structure. Thus, in E. coli cells, a portion of FtsZ is present in a dynamic helical structure that resembles the structures formed by the cytoskeletal MreB and Min proteins, according to Swapna Thanedar and William Margolin at the University of Texas Medical School in Houston. Furthermore, in sporulating B. subtilis cells, a coiled FtsZ structure extending from midcell toward the cell poles appears to represent an intermediate stage in redistributing FtsZ from the ring structure at midcell to a new FtsZ ring that is used to form the spore septum near the end of the cell (Fig. 1D, E), as shown by Sigal Ben-Yehuda and Richard Losick at Harvard University. Cytoskeletal-Like Organization of Other Bacterial Proteins Extended helical cytoskeletal-like structures are also formed in bacterial cells by proteins that are unrelated to eukaryotic cytoskeletal proteins. For instance, the E. coli MinCDE proteins that determine placement of the division site wind around the cell between the two poles in an arrangement similar to that of MreB (Fig. 1F, G), according to Shih and her collaborators at UC. This structure is distinct from the MreB cytoskeleton. The basic MinD coiled array may act as a lattice for the assembly and disassembly of the MinE and MinC site selection proteins and possibly other structures involved in division site placement. The E. coli SetB protein that affects chromosome segregation is also organized into a helical structure, according to Olivier Espeli and Kenneth Marian of Sloan-Kettering Institute, New York, N.Y. In this case, the SetB coiled array likely involves interactions with the MreB cytoskeleton. Additional bacterial proteins that form extended intracellular helical structures include the SecY protein, which is involved in exporting proteins across the cytoplasmic membrane; the ParA protein of plasmid pB171 that is involved in segregating plasmid DNA; and the Volume 71, Number 12, 2005 / ASM News Y 585 LamB protein of the E. coli outer membrane. The large and increasing number of proteins that adopt this long-range coiled organization is intriguing and implies that some common mechanism may be involved in their assembly. Future Challenges in Studying the Bacterial Cytoskeletons The discovery in 2001 of an actin-like cytoskeleton in B. subtilis quickly led to a new view of bacterial cells in which the cell is highly organized, with internal protein structures that show extensive long-range order. Based on this new knowledge, researchers studying the bacterial cytoskeleton face several major questions: Are there a limited number of cytoskeletal structures, with several functional elements associated with each one, or are there a large number of independent structures, each specialized to carry out a single function? Is there an underlying membrane-associated scaffold that is responsible for organizing all these coiled cytoskeletal structures? Are there additional structures that traverse the bacterial cytosol—perhaps facilitating the intracellular movement of specific proteins and other molecules—in addition to those thus far identified, which extend along the undersurface of the cytoplasmic membrane? Are there novel bacterial cytoskeleton-associated proteins within the structures that have thus far been identified? For example, in eukaryotic cells, several low-abundance proteins play key roles in the dynamic behavior of the tubulin-based microtubules, and the submembranous cytoskeleton of eukaryotic cells often contains proteins that communicate with cytosolic structures or with integral membrane proteins that in turn communicate with the outside world. Are there comparable proteins in bacterial cytoskeletal networks? One thing is certain. We are in the early stages of defining the nature of the bacterial cytoskeleton and are likely to encounter many surprises over the next few years that will further change our views of the bacterial cell. ACKNOWLEDGMENT Our research is supported by USPHS grant GM37– 60632. SUGGESTED READING Ausmees, N., J. R. Kuhn, and C. Jacobs-Wagner. 2003. The bacterial cytoskeleton: an intermediate filament-like function in cell shape. Cell 115:705–713. Ben-Yehuda, S., and R. Losick. 2002. Asymmetric cell division in B. subtilis involves a spiral-like intermediate of the cytokinetic protein FtsZ. Cell 109:257–266. Daniel, R.A., and J. Errington. 2003. Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell. Cell 113:767–776. Figge, R. M., A. V. Divakaruni, and J. W. Gober. 2004. MreB, the cell shape-determining bacterial actin homologue, coordinates cell wall morphogenesis in Caulobacter crescentus. Mol. Microbiol. 51:1321–1332. Gitai, Z., N. Dye, and L. Shapiro. 2004. An actin-like gene can determine cell polarity in bacteria. Proc. Natl. Acad. Sci. USA 101:8643– 8648. Jones, L., R. Carballido-Lopez, and J. Errington. 2001. Control of cell shape in bacteria: helical actin-like filaments in Bacillus subtilis. Cell 104:913–922. Kruse, T., J. Moller-Jensen, A. Lobner-Olesen, and K. Gerdes. 2003. Dysfunctional MreB inhibits chromosome segregation in Escherichia coli. EMBO J 22:5283–5292. Shih, Y.-L., T. Le, and L. Rothfield. 2003. 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 100:7865–7870. Soufo, H. J., and P. L. Graumann. 2003. Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins. Curr. Biol. 13:1916 –1920. Thanedar, S., and W. Margolin. 2004. FtsZ exhibits rapid movement and oscillation waves in helix-like patterns in Escherichia coli. Curr. Biol. 14:1167–1173. Van den Ent, F., L. Amos, and J. Lowe. 2001. Prokaryotic origin of the actin cytoskeleton. Nature 413:39 – 44. 586 Y ASM News / Volume 71, Number 12, 2005