Increasing complexity of the bacterial cytoskeleton

Increasing complexity of the bacterial cytoskeleton

Increasing complexity of the bacterial cytoskeleton Jakob Møller-Jensen and Jan Lo¨we Bacteria contain cytoskeletal elements involved in major cellula...

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Increasing complexity of the bacterial cytoskeleton Jakob Møller-Jensen and Jan Lo¨we Bacteria contain cytoskeletal elements involved in major cellular processes including DNA segregation and cell morphogenesis and division. Distant bacterial homologues of tubulin (FtsZ) and actin (MreB and ParM) not only resemble their eukaryotic counterparts structurally but also show similar functional characteristics, assembling into filamentous structures in a nucleotide-dependent fashion. Recent advances in fluorescence microscopic imaging have revealed that FtsZ and MreB form highly dynamic helical structures that encircle the cells along the inside of the cell membrane. With the discovery of crescentin, a cell-shape-determining protein that resembles eukaryotic intermediate filament proteins, the third major cytoskeletal element has now been identified in bacteria as well. Addresses MRC-Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK Corresponding authors: Møller-Jensen, J (e-mail: [email protected]) and Lo¨we, J (e-mail: [email protected])

Current Opinion in Cell Biology 2005, 17:75–81 This review comes from a themed issue on Cell structure and dynamics Edited by Anthony A Hyman and Jonathon Howard Available online 16th December 2004 0955-0674/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2004.11.002

Introduction Despite their minute and deceptively simple appearance, closer study of bacterial cells reveals considerable intricacy. In recent years the use of fluorescent imaging techniques has led the way in uncovering the surprising level of internal organisation in bacteria [1]. In particular, the discovery of true bacterial homologues of the three cytoskeletal elements, tubulin, actin and intermediate filaments, has challenged our previous perception of the cytoskeleton as a hallmark of eukaryotic cells. Essential processes of the bacterial cell, such as cell shape maintenance, DNA segregation and cell division, rely on the cytoskeleton. Just like microtubules and F-actin in eukaryotes, the bacterial cytoskeletal equivalents are highly dynamic, thus providing the cell with a flexible scaffold rather than a static ‘skeleton’ as such. www.sciencedirect.com

In addition to its three filamentous components, the eukaryotic cytoskeleton is associated with motor proteins, which actively transport cellular components along the polymer tracks or move the polymers relative to each other, and a myriad of accessory proteins that serve to regulate the turnover of polymer subunits. The activity of auxiliary protein modulators also seems to be important for the function of the bacterial cytoskeleton. However, so far none of the proteins found to be associated with the bacterial cytoskeleton share homology with eukaryotic proteins. Here, we review the recent discoveries of how bacterial cytoskeletal elements display highly dynamic localisation patterns in vivo and discuss their important roles in determining cellular infrastructure.

The bacterial tubulin homologue, FtsZ The notion of a bacterial cytoskeleton was first prompted a decade ago by the identification of the cell division protein FtsZ as a putative tubulin homologue [2–5]. FtsZ is the major bacterial cell division determinant and is found almost ubiquitously in eubacteria and archaea as well as in some intracellular organelles of eukaryotic cells [6]. FtsZ localises specifically to the midcell division site where it forms the cytokinetic Z-ring, which constricts the cell membrane during septation. In addition, FtsZ ring formation serves as a first cue in the assembly of the cell division machinery, which consists of at least seven different proteins depending on the species [6]. FtsZ binds and hydrolyses GTP in a manner coupled to the self-assembly of the protein. In vitro FtsZ forms single protofilaments that can be either straight or curved depending on the nucleotide state; polymers assembled in the presence of GTP are primarily straight, whereas GDP favours a more curved conformation (see Figure 1a). This transition between straight and curved conformations has been proposed as a means of transmitting energy from nucleotide hydrolysis into mechanical force for constriction of the FtsZ ring [7]. Although the amino acid sequence similarity between tubulin and FtsZ is low and confined to regions forming the active sites, their tertiary structures are surprisingly similar [8–10]. As in the case of tubulin, the active site of FtsZ forms by association of monomers such that the catalytic ‘T7-loop’ of one monomer inserts into the nucleotide-binding pocket of the adjacent monomer in the protofilament, thereby leading to polymerizationdependent activation of the GTPase activity [11]. Recent advances in fluorescent imaging have shown the intracellular localization pattern of FtsZ to be more complex and dynamic than thought previously. By Current Opinion in Cell Biology 2005, 17:75–81

76 Cell structure and dynamics

Figure 1

Electron micrographs

Fluorescence micrographs

(a)

(b)

(c)

(d)

(e)

(f)

Bacterial tubulin, FtsZ

Bacterial actins, MreB and Mbl

Intermediate crescentin filaments

Protein polymers visualised by electron microscopy (a, c, and e) and their intracellular localisation as shown by fluorescence microscopy (b, d, and f). (a) Straight and curved FtsZ protofilaments assembled in the presence of GDP (adapted with permission from [7]). (b) Immunofluorescent localization of FtsZ in E. coli cells. The arrow indicates a constricting Z-ring. (c) MreB filaments and sheets assembled with ATP (adapted with permission from [43]). (d) Helical filaments formed by the MreB-like protein Mbl fused to GFP in Bacillus subtilis (from [48]). (e) Intermediate filaments formed by crescentin (adapted from [60]). (f) Crescentin–GFP localisation to the inner cell curvature of Caulobacter crescentus. The cell membrane is stained in red [60]. Size bars are 100nm in (a, c, e) and 2 mm in (b, d, f).

measuring FtsZ–GFP fluorescence recovery after photobleaching (FRAP), the Z-ring structure was shown to be extremely dynamic, continuously remodelling itself before and during constriction with a rate comparable to that of microtubules. The dynamic turnover of FtsZ proteins relies on GTP hydrolysis, as mutant proteins with reduced enzymatic activity displayed slower recovery [12,13]. Thus, the FtsZ protein is recycled in and out of the ring at the expense of GTP hydrolysis. Only 30–40% of all FtsZ in the cell is present in the ring structure at a given time. Time-lapse microscopic analysis of Escherichia coli cells expressing FtsZ–GFP has revealed that, in addition to forming the division ring, FtsZ moves rapidly in less bright helical patterns around the cytoplasm (not visible in Figure 1b because of the phase contrast imaging) [14]. The helical pattern was visible even in newborn cells where the Z-ring was not yet assembled. Similar helical structures of FtsZ serve as intermediates in ring formation at the onset of sporulation in Bacillus subtilis, when the division site is shifted from midcell to asymmetric positions [15]. Whether FtsZ rings and helices are part of the same structure is not known and the significance of dynamic FtsZ helices during vegetative growth remains to be established. As will be discussed below, FtsZ is not the only bacterial protein known to generate dynamic helical patterns. Current Opinion in Cell Biology 2005, 17:75–81

Consistent with its key role in cell division, FtsZ serves as the target for a number of auxiliary proteins (see Table 1). ZipA and FtsA, both essential for cell division in E. coli, are recruited independently to the division site through interaction with the FtsZ C-terminus region. Either ZipA or FtsA is required for stable Z-ring formation and both proteins are required for recruitment of subsequent components of the division machinery [16,17]. Moreover, ZipA is capable of promoting FtsZ bundling in vitro [18]. Another positive modulator of FtsZ assembly, ZapA, colocalises with FtsZ in vivo and promotes bundling of FtsZ protofilaments in vitro [19,20]. Deletion of the zapA gene has no immediate effect on cell division in B. subtilis. However, removal of ZapA in conjunction with other modulators, EzrA and DivIVA (described below), results in a synthetic lethal phenotype and, at abnormally low FtsZ concentrations, ZapA becomes essential for cell survival [19]. ZapA forms homodimeric and tetrameric complexes, which are presumed to stabilise the polymer by crosslinking individual FtsZ protofilaments within the ring [20]. Expression of the cell-division inhibitor SulA is induced as part of the SOS response to DNA damage in E. coli. SulA binds to the FtsZ T7-loop, thereby preventing assembly of the Z-ring until DNA has been repaired [21,22]. Interestingly, SulA homologues have been www.sciencedirect.com

Increasing complexity of the bacterial cytoskeleton Møller-Jensen and Lo¨ we 77

Table 1 Components of the bacterial cytoskeleton. Name

Homology

Function(s)

Intracellular localization

Structure solved?

Reference

FtsZ

Tubulin

Principal cell-division determinant

Dynamic Z-ring and cytoplasmic helices

Yes

[2,8,12,14,15]

FtsA

Actin

Stabilization of the Z-ring; recruitment of proteins to the division site

Z-ring associated

Yes

[16,17,44]

Stabilization of the Z-ring; recruitment of additional proteins to the division site

Z-ring associated

ZipA

[16–18]

ZapA

Cross-linking of FtsZ protofilaments

Z-ring associated

Yes

[19,20]

SulA

Stress-induced inhibitor of FtsZ polymer assembly; required for cell division in cyanobacteria and plastids

Z-ring associated

Yes

[21,22,23,24,63]

EzrA

Inhibitor of FtsZ polymerisation involved in division-site placement

Membrane associated

MinC

Inhibitor of FtsZ polymerisation involved in division-site placement

Oscillating (E. coli) or polar (B. subtilis).

Yes

[25,29]

Membrane-associated ATPase; binds MinC to determine division-site placement

Oscillating (E. coli) or polar (B. subtilis).

Yes

[25,29,64,65]

MinE

Regulator of MinCD localisation; involved in division-site placement

Oscillating

Yes

[25,29]

DivVIA

Polar anchor of MinCD (during vegetative growth) and chromosomal oriC (during sporulation); mediates polar cell wall synthesis in Streptomyces coelicolor

Polar

[31–33,34,35]

Noc

Mediator of nucleoid occlusion in B. subtilis

Co-localized with the nucleoid

[39]

MinD

Nitrogenase iron protein, NifH

[40,41]

MreB/Mbl

Actin

Cell shape determination in non-spherical bacteria; chromosome segregation; establishment of cell polarity in Caulobacter

Helical, dynamic

Yes

[48,49,50,51,52, 53,54,55]

ParM

Actin

Plasmid segregation

Dynamic filaments

Yes

[45,58]

MreB-associated; chromosome segregation; sugar efflux transport

Helical, membrane-localized

[56]

Cell shape determination in certain curved bacteria

Helical. Probably static.

[60]

SetB Crescentin

Lamin, keratin

reported recently to be required for cell division in cyanobacteria as well as in Arabidobsis thaliana plastids, suggesting a role for SulA in cell division under normal growth conditions [23,24]. Other mechanisms act during vegetative growth to negatively regulate FtsZ polymerisation. Together they impose spatial restrictions upon Z-ring formation to ensure that cell division takes place only at the proper position [25]. One such regulator is the Min system, which in E. coli is composed of three proteins, MinC, MinD and MinE. The negative effector MinC prevents assembly of the Z-ring by interacting directly with FtsZ www.sciencedirect.com

and the spatial regulation of MinC activity is controlled in spectacular fashion by MinD and MinE. MinC is recruited to the membrane by interaction with the membrane-associated ATPase MinD, the localization of which is in turn influenced by MinE. As a result, the Min proteins oscillate back and forth along the inner cell membrane, thereby inhibiting FtsZ assembly at polar positions. The self-organising oscillating behaviour of the Min proteins has been simulated by computer models based on theory of biological pattern formation [26–28]. Recent fluorescent imaging revealed that the MinD ATPase polymerises into helical filaments that may serve as a track for the oscillatory behaviour [29]. The ability to Current Opinion in Cell Biology 2005, 17:75–81

78 Cell structure and dynamics

oscillate along helical tracks is shared by other MinD-like ATPases, which play a role in the segregation of DNA [30]. In B. subtilis the MinCD proteins prevent polar cell division without requiring a protein related to MinE. In this case the MinCD complex is recruited to the cell poles by the DivIVA protein [31–33]. DivIVA is also found at the poles of the mycelial bacterium Streptomyces coelicolor, where it plays an essential part in the regulation of hyphal growth and morphogenesis [34]. Interestingly, DivIVA purified from B. subtilis has been found to oligomerise in vitro into oligomers shaped like ‘doggy-bones’, which are capable of further assembly into fibres and twodimensional lattices [35]. A second mechanism for the negative regulation of septum placement, termed nucleoid occlusion [36], relates cell division to the position of the DNA in the cell, with the result that FtsZ rings are unable to form in areas of the cell occupied by the nucleoid [37,38]. A non-specific DNA binding protein, Noc, has been shown recently to be responsible for nucleoid occlusion in B. subtilis by an as-yet-unresolved mechanism [39]. EzrA is a third negative regulator of FtsZ ring formation in B. subtilis and is found generally in Gram-positive bacteria with a low proportion of G/C nucleotides in their genomes [40]. It is a transmembrane protein present in high numbers and is homogeneously distributed on the cell membrane, where it interacts directly with FtsZ to inhibit aberrant Z-ring formation [41].

The bacterial actin homologues The first evidence of distant bacterial actin homologues came from a clever sequence homology search based on the catalytic core shared by actin, hsp70 proteins and hexokinase, which were known to have similar structures [42]. This approach identified the bacterial proteins FtsA, MreB and StbA (ParM) as putative actin-like proteins. Subsequent crystallographic analyses have revealed that, although these proteins share only very limited sequence similarity with actin, the structures of MreB and ParM are in fact very similar (FtsA less so), thus providing strong support for the existence of true actin homologues in bacteria [43–45]. MreB is an important bacterial cell shape determinant and its presence in bacteria is correlated with nonspherical cell morphology [46–48]. MreB protein (and paralogues like the B. subtilis Mbl protein) form helical filamentous structures close to the cytoplasmic face of the cell membrane, as shown in Figure 1d, and polymerise in a nucleotide-dependent manner in vitro (Figure 1c) [29,43,48,49,50]. The helical filaments have been proposed to affect the cell shape by serving as a scaffold for the peptidoglycan synthesis machinery [50,51]. In Caulobacter crescentus, one such enzyme has been shown to localise in an MreB-dependent helical pattern [50]. Moreover, using fluorescent vancomycin to probe the incorCurrent Opinion in Cell Biology 2005, 17:75–81

poration of nascent peptidoglycan, lateral cell wall synthesis in B. subtilis was shown to occur in a helical pattern. The specific pattern of cell wall synthesis was found to depend on the MreB-like Mbl protein, but not on MreB itself [52]. Mbl filaments were investigated in vivo using the FRAP method and found to be highly dynamic. GFP–Mbl was recycled within the filaments with a fluorescence half-life of about eight minutes. Surprisingly, Mbl fluorescence recovery occurred without apparent polarity, suggesting that the helical MreB and Mbl filaments may in fact consist of multiple short protofilaments arranged in a bundle [51]. Another recent time-lapse study of GFPfusions to MreB and Mbl in B. subtilis demonstrated that both proteins form multiple dynamic helical filaments that move along the long cell axis in actively growing cells [53]. Their dynamic localisation pattern displayed slight variations in that MreB seemed to overlap only with the nucleoid, whereas GFP–Mbl fluorescence was found throughout the entire cell length. Furthermore, in anucleate cells generated by an artificially induced block in chromosome segregation, only Mbl fluorescence was detectable; no MreB was present in cells devoid of DNA. Other reports have linked MreB with a function in chromosome segregation. Expression of mutant MreB protein with impaired ATPase activity in merodiploid E. coli produced rod-shaped cells with altered MreB filament morphology and severely impaired chromosome organisation, indicating that MreB filaments are important for active segregation of chromosomes [49]. Consistent with this, depletion of MreB in both Caulobacter and B. subtilis results in mislocalisation of chromosomal origins of replication, which are actively transported to opposite cell halves under normal conditions [54,55]. A third function of the MreB protein is related to generation of cell polarity in the asymmetric bacterium Caulobacter [54]. Upon depletion of Caulobacter MreB, specific proteins normally targeted to a specific pole displayed aberrant localisation. Remarkably, when MreB levels were replenished, these polar markers now localised to either one or the other cell pole, suggesting that the polar information provided by MreB was restored in random orientation. In Caulobacter, MreB helices rearrange during the cell cycle, compacting at the midcell in the predivisional cell and expanding to encircle the newborn cells along their entire length after cell division [50,54]. As a result, the MreB polymers always extend away from the new pole and this cyclic behaviour may serve to equip the Caulobacter cell with overall polarity [54]. Thus, it seems that the filaments formed by B. subtilis MreB and Mbl might be involved in chromosome segregation and cell wall synthesis, respectively, whereas in other organisms like E. coli and Caulobacter, which only contain a single mreB gene, diverse cellular functions might be attributed to a single filamentous structure. www.sciencedirect.com

Increasing complexity of the bacterial cytoskeleton Møller-Jensen and Lo¨ we 79

SetB is the first protein reported to be associated with MreB [56]. Integrated in the inner membrane of E. coli, SetB appears to carry out multiple functions, serving in sugar transport across the membrane as well as having an apparently separate role in chromosome segregation [56,57]. Deletion of setB causes a delay in chromosome segregation, whereas overexpression of SetB appears to increase the pull on the segregating chromosomes, leading to nucleoid stretching and breakage. SetB localises into a helical pattern reminiscent of MreB and twohybrid analysis confirmed that SetB and MreB do in fact interact [56]. The exact role of SetB in chromosome segregation remains to be determined, though. The plasmid-encoded ParM protein is another prokaryotic actin homologue involved in DNA segregation. ParM assembles into dynamic polymers that act to push replicated plasmid molecules apart to opposite sites of the divisional plane, thereby ensuring their stable maintenance in the host cell [58,59]. Contrary to MreB, which assembles into straight protofilaments, polymers of ParM are strikingly similar to F-actin, assuming a double helix structure with an almost identical angular twist [43,45]. The dynamic behaviour of ParM is fuelled by ATP turnover and is subject to regulation by another protein, ParR, which is associated with the centromeric parC region on the plasmid DNA [58,59].

Bacterial intermediate filaments

each other or perhaps with some still unknown component? As mentioned above, the interaction of SetB with MreB has been established [56]. In Caulobacter, the condensation of MreB at the midcell is FtsZ-dependent, although no direct interaction appears to take place between FtsZ and MreB [50]. Moreover, in E. coli, the subcellular localisation of both FtsZ and MinD appears to be independent of MreB [14,29]. It is perhaps worth noting that proteins polymerising at any angle between 08 and 908 to the polar axis will assume a helical pattern because of the constraints posed by the rod-shaped cell; in other words, any filamentous shape ranging between a circle and a straight rod will be a helix. Thus, it is indeed possible that several helixshaped polymers can arise independently in the cell. As the resolution of light microscopy is limited to tenths of a micron, many questions concerning bacterial cell architecture may only be resolved by the use of whole-cell electron microscopy and tomographic image reconstruction. So far, this approach has been hampered by the very high protein density of the bacterial cytoplasm, which makes identification of individual filament structures in the tomographic sections difficult [61]. Prominent components of the eukaryotic cytoskeleton still remaining to be discovered in bacteria are motor proteins. Given the presence of prokaryotic tubulin-like and actinlike fibres, one would expect the likes of kinesins, dyneins and myosins to be present as well. Yet, so far, searches have been unsuccessful. One possible explanation for this is that they don’t exist; that bacterial cells manage with the ‘polymerization motor’, which is the result of ATP- or GTP-driven non-equilibrium polymerization of the cytoskeletal filaments [62]. Alternatively, as in the case of both FtsZ and MreB, the level of sequence identity between eukaryotic motor proteins and their putative prokaryotic homologues is too low for immediate identification by sequence search. In any case, it is likely that new exciting discoveries linking the dynamic cytoskeletal filaments with intracellular organisation are still to come.

With the recent discovery of the intermediate filament (IF)-like protein crescentin as an important morphological determinant in Caulobacter, it now appears that the IF proteins have distant relatives in bacteria [60]. Crescentin localises as a single cytoplasmic filament along the concave face of the crescent-shaped cell and deletion of the protein leads to the formation of straight rod-shaped cells (Figure 1f). As no structural information is yet available, it is unknown whether crescentin resembles the IF family of proteins beyond the extensive coiled-coil regions. However, crescentin shares the IF domain organisation and purified crescentin can be assembled into filaments of about 10nm thickness according to protocols developed for IF proteins (shown in Figure 1e) [60].

We thank Linda Amos for critical reading of the manuscript. J Møller-Jensen acknowledges support from the European Molecular Biology Organization.

Conclusions

References and recommended reading

With the discovery of bacterial cytoskeletal elements, one of the major central tenets in prokaryotic cell biology has been overturned. Even though the most extensively studied bacterial species lack distinct intracellular organelles, many enzymatic activities are executed at specific subcellular locations and at specific time-points in the cell cycle, thereby increasing the overall efficiency of growth. The dynamic cytoskeleton is at the very heart of this infrastructure.

Papers of particular interest, published within the annual period of review, have been highlighted as:

A number of bacterial proteins present helical patterns when studied in the microscope. Might they interact with www.sciencedirect.com

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Increasing complexity of the bacterial cytoskeleton Møller-Jensen and Lo¨ we 81

Reports the identification of Noc, a protein which is responsible for nucleoid occlusion in B. subtilis. Noc binds non-specifically to the nucleoid and helps to prevent assembly of the division machinery at positions overlapping the DNA, thereby providing a link between key cell-cycle events. 40. Levin PA, Kurtser IG, Grossman AD: Identification and characterization of a negative regulator of FtsZ ring formation in Bacillus subtilis. Proc Natl Acad Sci USA 1999, 96:9642-9647. 41. Haeusser DP, Schwartz RL, Smith AM, Oates ME, Levin PA: EzrA prevents aberrant cell division by modulating assembly of the cytoskeletal protein FtsZ. Mol Microbiol 2004, 52:801-814. 42. Bork P, Sander C, Valencia A: An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc Natl Acad Sci USA 1992, 89:7290-7294. 43. van den Ent F, Amos LA, Lowe J: Prokaryotic origin of the actin cytoskeleton. Nature 2001, 413:39-44. 44. van den Ent F, Lowe J: Crystal structure of the cell division protein FtsA from Thermotoga maritime. EMBO J 2000, 19:5300-5307. 45. van den Ent F, Moller-Jensen J, Amos LA, Gerdes K, Lowe J: F-actin-like filaments formed by plasmid segregation protein ParM. EMBO J 2002, 21:6935-6943. 46. Doi M, Wachi M, Ishino F, Tomioka S, Ito M, Sakagami Y, Suzuki A, Matsuhashi M: Determinations of the DNA sequence of the mreB gene and of the gene products of the mre region that function in formation of the rod shape of Escherichia coli cells. J Bacteriol 1988, 170:4619-4624. 47. Wachi M, Matsuhashi M: Negative control of cell division by mreB, a gene that functions in determining the rod shape of Escherichia coli cells. J Bacteriol 1989, 171:3123-3127. 48. Jones LJ, Carballido-Lopez R, Errington J: Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell 2001, 104:913-922. 49. Kruse T, Moller-Jensen J, Lobner-Olesen A, Gerdes K:  Dysfunctional MreB inhibits chromosome segregation in Escherichia coli. EMBO J 2003, 22:5283-5292. The first evidence that MreB plays a role in bacterial chromosome segregation is presented. Flow-cytometric analysis shows that chromosomes segregate in pairs in MreB-depleted E. coli cells. Fluorescencemicroscopic imaging further reveals impaired segregation of chromosomal DNA as well as mislocalised chromosomal origin and terminus regions in cells where the MreB cytoskeleton is hampered by expression of mutant MreB forms. 50. Figge RM, Divakaruni AV, Gober JW: MreB, the cell-shapedetermining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus. Mol Microbiol 2004, 51:1321-1332. 51. Carballido-Lopez R, Errington J: The bacterial cytoskeleton: in  vivo dynamics of the actin-like protein Mbl of Bacillus subtilis. Dev Cell 2003, 4:19-28. Provides the first evidence of cytoskeletal dynamics in bacteria. The MreB-like Mbl protein, found previously to form helical structures in B. subtilis (see [48]), was localized in live cells using a functional GFP–Mbl fusion. Measuring FRAP, the authors determine the fluorescence half-life of Mbl filament turnover to be eight minutes. Mbl filaments remodel with no apparent polarity, suggesting that the fluorescent helix structure may in fact consist of a bundle of shorter protofilaments. 52. Daniel RA, Errington J: Control of cell morphogenesis in  bacteria: two distinct ways to make a rod-shaped cell. Cell 2003, 113:767-776. The innovative use of a fluorescent vancomycin derivative as a marker of nascent cell wall synthesis yields surprising insights into the different strategies employed by different bacteria to control their cell shape during growth. In B. subtilis, new cell wall material is inserted in a helical pattern by a mechanism that is dependent on Mbl but not on MreB. Conversely, in Corynebacterium and Streptomyces, non-septal cell wall material is inserted at the poles.

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53. Defeu Soufo HJ, Graumann PL: Dynamic movement of actin-like  proteins within bacterial cells. EMBO Rep 2004, 5:789-794. The three actin-like proteins in Bacillus subtilis (MreB, Mbl and MreBH) are studied using functional GFP fusions and found to localize into distinct helical patterns. Time-lapse microscopy reveals that the filaments are highly dynamic, moving along the inside of the cell membrane. By contrast to Mbl and MreBH, MreB is found to colocalise with the nucleoid. 54. Gitai Z, Dye N, Shapiro L: An actin-like gene can determine  cell polarity in bacteria. Proc Natl Acad Sci USA 2004, 101:8643-8648. MreB is reported to be involved in determination of cell polarity in the asymmetric bacterium Caulobacter. Depletion of MreB leads to mislocalisation of chromosomal origins as well as of specific polar-localized proteins. Upon restoration of the MreB levels the polar marker proteins localize to either cell pole, suggesting that MreB-dependent cell polarity has been restored in a random fashion. 55. Soufo HJ, Graumann PL: Actin-like proteins MreB and Mbl from  Bacillus subtilis are required for bipolar positioning of replication origins. Curr Biol 2003, 13:1916-1920. Fluorescence-microscopic evidence is presented for the involvement of MreB in chromosome segregation in B. subtilis. Depletion of MreB results in mislocalisation of chromosomal origins even before the cell shape is affected. 56. Espeli O, Nurse P, Levine C, Lee C, Marians KJ: SetB: an integral  membrane protein that affects chromosome segregation in Escherichia coli. Mol Microbiol 2003, 50:495-509. A genetic approach leads to the surprising discovery of a membraneintegral sugar transporter, SetB, with a function in chromosome segregation. SetB localises in a helical pattern reminiscent of MreB and is found by two-hybrid analysis to interact with MreB. The role of SetB in chromosome segregation remains obscure but seems to be unrelated to its role in sugar transport. 57. Liu JY, Miller PF, Willard J, Olson ER: Functional and biochemical characterization of Escherichia coli sugar efflux transporters. J Biol Chem 1999, 274:22977-22984. 58. Moller-Jensen J, Jensen RB, Lowe J, Gerdes K: Prokaryotic DNA segregation by an actin-like filament. EMBO J 2002, 21:3119-3127. 59. Moller-Jensen J, Borch J, Dam M, Jensen RB, Roepstorff P,  Gerdes K: Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism. Mol Cell 2003, 12:1477-1487. This paper shows how the components of the R1 plasmid partitioning system interact to bring about active plasmid segregation. Plasmid molecules are located at opposite tips of filaments formed by the actin-like ParM protein, suggesting that force for plasmid transport is generated by an insertional ParM polymerisation mechanism similar to that displayed by actin in eukaryotes. 60. Ausmees N, Kuhn JR, Jacobs-Wagner C: The bacterial  cytoskeleton: an intermediate filament-like function in cell shape. Cell 2003, 115:705-713. Reports the first discovery of a bacterial intermediate filament protein, crescentin, which has a function in shape determination in vibroid bacteria. Crescentin assembles into a curved cytoplasmic filament that lines the inner membrane of Caulobacter crescentus. Purified crescentin forms fibres that resemble eukaryotic intermediate filaments in the electron microscope. 61. Grimm R, Singh H, Rachel R, Typke D, Zillig W, Baumeister W: Electron tomography of ice-embedded prokaryotic cells. Biophys J 1998, 74:1031-1042. 62. Theriot JA: The polymerization motor. Traffic 2000, 1:19-28. 63. Huisman O, D’Ari R, Gottesman S: Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation. Proc Natl Acad Sci USA 1984, 81:4490-4494. 64. Cordell SC, Lowe J: Crystal structure of the bacterial cell division regulator MinD. FEBS Lett 2001, 492:160-165. 65. Hayashi I, Oyama T, Morikawa K: Structural and functional studies of MinD ATPase: implications for the molecular recognition of the bacterial cell division apparatus. EMBO J 2001, 20:1819-1828.

Current Opinion in Cell Biology 2005, 17:75–81