We discuss here the best-studied bacterial actin-like cytoskeletal proteins, i.e., MreB, ParM, and MamK.
(ii) MreB polymerization and depolymerization.
The MreB protein of
Thermotoga maritima self-assembles into long polymeric filaments in vitro (
51,
214). Although the cellular abundance of MreB in
T. maritima is not known, polymerization of
T. maritima MreB is rapid at the normal cellular concentrations of
E. coli MreB (∼30,000 molecules per cell [
108]) and
B. subtilis MreB (∼8,000 molecules per cell [
95]). Each filament is composed of two side-by-side linear polymers that differ in appearance from the helical double-stranded filaments of F-actin (
214). The double-stranded MreB filaments are likely to comprise the helical MreB structures of intact cells.
Polymerization occurs equally well in the presence of ATP and GTP (
50,
214), thereby differing from actin polymerization, which occurs only in the presence of ATP. MreB polymerization stimulates ATPase activity, but during the course of MreB polymerization there is a lag between polymerization and phosphate release (
50). This implies that ATP hydrolysis occurs after MreB monomers are incorporated into filaments and that ATP binding, rather than hydrolysis, is required for addition of subunits to the growing polymer, thereby resembling actin polymerization (
164).
The filaments interact to form bundles that undergo a gelation process, leading to formation of a solid-like structure (
51). The bundled MreB structure is more rigid than the equivalent F-actin structure, with properties that are more often associated with eukaryotic intermediate filaments than with actin (
51). These include high elasticity, low critical concentrations for polymerization, and a high propensity for bundling. These properties would be useful if the MreB cytoskeleton played a true skeletal role in supporting cell shape. However, the significant changes in cellular distribution of the MreB cytoskeleton that take place within the rod-shaped cells (see above) indicate that the rigidity of organized MreB polymers does not play an essential role in maintaining the rod shape of the cell. This conclusion is supported by studies with the drug A22 [
S-(3,4-dichlorobenzyl)isothiourea] (
88), which leads to loss of the MreB coiled structures and conversion of the cell from a rod to a sphere. When A22 was added, the rod-like shape of
C. crescentus was not altered until long after disappearance of the MreB helical structures (
61), arguing against the idea that rigidity of the bundled protofilaments is necessary for maintenance of cell shape.
All current information on the structure and polymerization properties of MreB filaments is based on studies of MreB from the thermophilic organism T. maritima, where the cellular organization of the protein has not yet been studied. Therefore, it will be important to confirm that MreB from organisms such as B. subtilis, E. coli, Caulobacter crescentus, and Rhodobacter sphaeroides, where almost all biological studies have been performed, behaves similarly to the T. maritima protein.
(iii) Cellular functions of MreB and MreB homologs.
(a) Cell shape determination. Since the original discovery of the
mreB (
mu
rein cluster
B) gene in a search for mutants that are sensitive to amdinocillin (
220), genes coding for MreB and MreB-related actin homologs have been shown to be present in almost all rod-shaped species and absent from species that grow as cocci (
27). MreB-depleted cells usually grow as spheres, suggesting that MreB may play a role in cell shape control in rod-shaped bacteria.
It has long been known that the primary determinant of bacterial cell shape is the murein (peptidoglycan) exoskeleton, which is located outside of the plasma membrane. The murein sacculus retains the shape of bacterial cells even when purified away from other cellular components, and in the absence of cell wall murein, rod-shaped cells become spherical. This makes it clear that the sacculus is the shape-determining structure of the cell (
184). The
mreB operon is part of a large cluster of genes involved in murein synthesis, implying a possible relation between MreB and biosynthesis of the rigid murein exoskeleton.
The shape of rod-shaped cells is dependent on enzymes responsible for longitudinal murein growth. The rod shape is also dependent on the presence of MreB or MreB homologs, since depletion of these proteins leads to loss of the normal rod shape, with formation of spherical cells, or, in the case of loss of Mbl, to markedly deformed cells with large bulges and irregular increases in cell width (
95). It has been suggested that Mbl controls cylindrical cell wall synthesis (
27). Several other cellular proteins are also implicated in establishment of the rod shape, since loss of these proteins also leads a rod-to-sphere transition. Among others, these include the murein biosynthetic enzyme penicillin-binding protein 2 (PBP2) and the RodA protein of
E. coli, which are discussed below.
It is likely that MreB and its homologs regulate the shape of rod-shaped cells by organizing murein biosynthetic enzymes into a helical pattern that is oriented along the long axis of the cell, leading to the pattern of murein synthesis that is responsible for the rod shape. This was initially suggested by studies with fluorescein-labeled vancomycin. Vancomycin blocks the cross-linking of newly synthesized glycan-pentapeptide chains into the murein sacculus by covalently attaching to the terminal
d-alanine of the pentapeptide murein biosynthetic precursors (
12,
17,
27). Fluorescein-labeled vancomycin therefore has been used as a marker for the cellular pattern of murein biosynthesis. The vancomycin studies showed that the immediate precursors of mature murein in
B. subtilis (
27) are organized in a coiled pattern that extends along the long axis of the cell and resembles the distribution patterns of MreB and Mbl (Fig.
2G). The coiled vancomycin pattern was dependent on the presence of the MreB homolog Mbl (
27). In species that lack an
mbl gene, it is likely that MreB or another MreB homolog carries out this function. Consistent with these results, studies of murein deposition in
E. coli cells (
32) also suggest a coiled pattern of new murein incorporation into the sacculus (Fig.
2H).
The following experiments indicate that the Mbl-dependent helical pattern of murein synthesis reflects a helical organization of the murein biosynthetic enzymes needed for longitudinal cell growth. First, the biosynthetic murein transpeptidase PBP2, which is required for rod shape, is distributed in a coiled pattern along the cell cylinder, similar to the distribution patterns of MreB and Mbl. This has been shown in
C. crescentus (
35,
38,
53) and is likely also true in
E. coli (
30). Second, the coiled distribution pattern of PBP2 is dependent on the presence of MreB and MreC (
35,
53), implying that the helical MreB and MreC cytoskeletal structures (discussed further below) play an essential role in determining the cellular organization of PBP2.
MreC appears to act as a bridge between the MreB cytoskeleton and the murein biosynthetic machinery, as shown in studies of
E. coli and
B. subtilis (
107,
113). The MreD protein is also likely to be a component of the bridging complex in organisms that contain an
mreD gene, such as
E. coli and
B. subtilis. Thus,
E. coli MreC interacts with MreB and MreD in bacterial two-hybrid assays, whereas MreB does not interact with MreD (
107). This suggests that MreC may be intercalated between MreB and MreD in a putative multiprotein complex. In addition, several lines of evidence suggest that MreB, MreC, MreD, PBP2, and perhaps other murein biosynthetic enzymes are components of a structure that mediates the effects of the MreB cytoskeleton on the topology of murein synthesis (Fig.
2F). First, formation of normal MreB helical cytoskeletal structures requires the presence of
mreC and
mreD in
E. coli and
B. subtilis (
29,
107). Second, several PBPs which code for murein biosynthetic enzymes were recovered by affinity chromatography of a
C. crescentus cell extract on an MreC column, suggesting a link between MreC and multiple elements of the murein biosynthetic machinery in this organism (
35). Third,
C. crescentus MreC and PBP2 (
35,
38) and
B. subtilis MreC and MreD (
113) are present in helical patterns that resemble those of the MreB and Mbl coiled elements. It has been suggested that RodA, which is required for maintenance of the rod shape and for the enzymatic activity of PBP2 (
87,
196), may be another component of the MreBC complex (
107). MreC has been thought to be the transmembrane link in this complex (
107), because sequence analysis predicts a transmembrane organization. However, it has recently been reported that cell fractionation studies indicate a periplasmic location for the
C. crescentus MreC protein (
35). Further studies will be needed to clarify the cellular location of MreC in the various organisms under study.
The interaction between MreB and the MreC-based structure in vivo appears to be transient, since, in contrast to MreB, the distribution of the MreC helical pattern does not significantly change during the cell cycle (
35). The observation that disruption of the MreB helical structures by A22 did not have immediate effects on the localization of MreC and PBP2 also supports the idea that MreB is not an essential part of the basic MreC-PBP complex (
35,
38).
MreC affinity chromatography identified approximately 19 candidates to be MreC-associated proteins (
35). These included eight presumed outer membrane proteins, nine cytoplasmic proteins, and small amounts of several PBPs. Studies of GFP derivatives of five of the outer membrane proteins showed clusters of labeled protein that were interpreted to indicate a spiral, punctate, or banding distribution pattern similar to that seen with MreC and PBP2 (
35). Based on these results, it was suggested that the PBPs and outer membrane proteins might be part of an MreC-based complex anchored in the inner membrane that could provide a link between the internal MreB cytoskeleton and the outer layers of the cell envelope. If this is correct, the failure to recover MreB from the MreC affinity column might be attributed to problems in solubilization of the MreB cytoskeletal structures or to the presence of intermediate linking proteins between MreB and MreC. Further work will be needed to fully interpret these observations.
Interestingly, loss of either MreB or MreC causes PBP2 to lose its helical organization and instead to localize near midcell in
C. crescentus (
38). This requires the essential division protein FtsZ, suggesting that the cellular localization of PBP2, and presumably also its site of action, may be regulated by an interplay between the cell division machinery and the MreB/MreC cytoskeleton. This is consistent with the observation that PBP2 localizes to midcell at the time of septation in
E. coli (
30). The unrelated PBP2 of
Staphylococcus aureus also localizes to midcell (
161).
A full understanding of the role of the cytoskeleton in organizing the murein biosynthetic machinery is complicated by observations such as the following. (i) Localization studies of all 11 vegetative PBPs of
B. subtilis failed to show a helical distribution (
182). Unless these results reflect technical limitations, this implies that the association of murein biosynthetic enzymes with the cytoskeleton may vary from species to species or may be limited to a small subset of the enzymes. (ii) Growth of
B. subtilis in the presence of 25 mM Mg
2+ restored a normal rod morphology and normal helical distribution of nascent murein to
ΔmreB cells (
54). Therefore, although there is inferential evidence that the MreB cytoskeleton may participate in determination of cell shape by providing a scaffold for the helical distribution of murein biosynthetic enzymes along the length of the cell, this effect either is indirect or operates through another scaffolding protein, perhaps MreC. High Mg
2+ levels could stabilize the scaffolding partner to permit it to function in the absence of MreB. These observations show that the MreB cytoskeleton is not essential for cell shape determination in
B. subtilis despite the fact that depletion of MreB results in a change of cell shape.
(b) Cell polarity. The poles of rod-shaped cells differ in several respects from the remainder of the cell body. These differences include the specific polar localization of a number of membrane-associated proteins (
90), the presence of polar flagella or pili in certain species (
185), the lack of turnover of murein and of externally labeled surface proteins at the cell poles (
31,
33), the absence of zones of adhesion between the inner membrane and the murein-outer membrane layer at the poles (
23,
131), and anatomic changes (the bacterial birth scar) at the newly formed cell pole (
130). MreB has been implicated in one of these aspects of cell polarity, the localization of specific proteins to one or both cell poles.
Proteins that play a role in regulating the
C. crescentus differentiation cycle are differentially targeted to one or both cell poles (reviewed in reference
59). These include the membrane histidine kinases, PleC, DivJ, and CckA. MreB is required for the polar targeting of these proteins (
60). After depletion of MreB, polar localization of the proteins is lost and they become diffusely distributed within the cell.
MreB is also required for the polar localization of proteins in other organisms. In
E. coli and related gram-negative bacteria, membrane-associated proteins involved in chemotaxis, motility, secretion, and virulence are normally targeted to one or both cell poles (
185). When several of these proteins were expressed in
E. coli, depletion of MreB led to a change of polar targeting. The proteins include the
E. coli aspartate chemoreceptor (
188), the
Shigella flexneri virulence protein IcsA (
151,
188), and the
Vibrio cholerae type II secretion protein EpsM (
151). It is likely that MreB also will be shown to play a role in the polar targeting of other proteins.
However, not all polar proteins require MreB for their localization. Thus, the assembly of the
E. coli Min proteins into membrane-associated polar zones (
188,
205) and the polar localization of the
C. crescentus TipN protein (
111) appear to be independent of MreB. These may be exceptions to the general rule that MreB is involved in localization of polar proteins, reflecting the special role that the Min proteins play in establishing the position of the cellular division site (
175) and the special role of TipN in marking the cell pole and in determination of cell polarity (
83,
111). It is not known how MreB accomplishes the polar targeting of proteins. The MreB helical filaments could participate directly in moving the proteins to the poles by providing tracks for the active translocation of specific cargo proteins, probably in collaboration with substrate-specific carrier and/or motor proteins. Segments of the MreB filaments might themselves move toward the poles (
194) as part of the translocation process. Alternatively, MreB might act indirectly by positioning polar targets for protein localization. For example, the polar end of the MreB cytoskeleton might initiate the localization or organization of other polar components that would then act as polar binding sites for a family of substrate proteins. The targets need not be proteins, since both the specialized membrane lipid composition of the poles (
141) and the presence of a segregated polar murein compartment (
33) could contribute to the target sites.
(c) Chromosome segregation. During the normal cell cycle, daughter nucleoids move rapidly to opposite ends of the cell, leading to their equipartition into the two daughter cells (
74). This process is perturbed when MreB is depleted in
E. coli and
C. crescentus (
60,
108). This is manifested by the production of cells in which multiple nucleoids are irregularly distributed within the cytoplasm (Fig.
2E, cell 1) (
108) and of anucleate cells (Fig.
2E, cell 2) or cells in which an incompletely partitioned nucleoid is guillotined by the septum (2E, cell 3). The possibility that the nucleoid segregation defects are caused by the spherical shape of the MreB-depleted cells has been excluded by the observation that expression of certain mutant alleles of
mreB that do not interfere with the rod shape of the cell is still associated with nucleoid segregation defects (
106,
108).
Further evidence that MreB is needed for normal chromosome partition came from studies of the segregation of the origin and terminus regions of newly replicated chromosomes of
E. coli and
C. crescentus (
61,
108). In wild-type cells, the newly replicated
oriC regions rapidly move to opposite ends of the cell. Separation of the terminus regions takes place later. In contrast, in MreB-depleted cells, the normal movement of the newly replicated
oriC regions to opposite ends of the cell does not occur, and the terminus regions appear to adhere together (
106,
108).
Biochemical evidence that the
oriC region is the chromosomal target of MreB came from studies of
C. crescentus cell extracts by Gitai and coworkers showing that MreB and DNA from the origin-proximal region could be chemically cross-linked and were coimmunoprecipitated with anti-MreB antiserum (
61). This strongly implies that MreB is associated with the origin-proximal region of the chromosome, either directly or via other proteins that correspond to the centromere-binding kinetochore proteins of eukaryotic cells. These results imply that the association of the MreB cytoskeleton with the
oriC region plays an important role in moving the daughter chromosomes towards opposite cell poles.
The mechanism responsible for the chromosomal movement is not known. Based on the present evidence, it appears likely that the
oriC region directly or indirectly interacts with the MreB cytoskeleton and is then translocated along the helical MreB structures from midcell to the cell poles, perhaps accompanied by movement of segments of the MreB filaments (
194). The energy requirement for translocation could be met by a separate motor protein, by coupling the ATPase activity of MreB to movement of the DNA, or by changes in chromosome folding. It is not known whether active movement of other chromosomal regions by a similar translocation mechanism is also involved in the segregation process. This may not be needed, since the active compaction of chromosomal DNA after
oriC is moved to the poles could complete the process of nucleoid separation (
147,
181). It also is not known whether the redistribution of MreB that occurs during the cell cycle is related to the
oriC translocation events (
53,
191).
The previous suggestion that RNA polymerase (RNAP) contributes to the motive force for chromosome separation (
37) is supported by the recent finding that inactivation of RNAP in
E. coli leads to a defect in nucleoid separation in DAPI (4′,6′-diamidino-2-phenylindole)-stained cells, similar to the effect of MreB depletion or inactivation (
106). This appears to be primarily associated with a failure of the terminus regions to segregate, although there may also be some effect on origin segregation. Of special interest, immunoprecipitation experiments using anti-MreB antibody indicated that MreB and RNAP are physically associated in cell extracts and in in vitro reconstitution experiments (
106). These results suggest that RNAP is associated with the MreB cytoskeleton and works together with MreB in the chromosomal segregation process. It has been pointed out that a significant force can be generated by a stationary RNAP molecule during transcription, and this could contribute to moving DNA during the segregation process (
37,
56).
A mechanism must also exist to explain the vectorial nature of the translocation process, in which the two newly replicated
oriC regions are moved to opposite ends of the cell. The directionality could be provided by a double-helical MreB cytoskeletal structure (Fig.
2B) if the helical strands were of opposite polarity. This would provide two tracks that point in opposite directions, each providing a one-way highway for the
oriC cargo. In another model the directionality would be imparted by the orientation of the newly replicated
oriC regions as they exit from the replication apparatus (
37). The two models are not mutually exclusive, and other mechanisms are also possible. A fuller understanding of the role of the MreB cytoskeleton in chromosome segregation will require a more detailed understanding of the macromolecular organization of the MreB helical arrays, the polarity of filament assembly and disassembly, and the basis of the MreB-
oriC interaction. In contrast to the results for
E. coli and
C. crescentus, it is not clear whether MreB or one of the other actin homologs, Mbl or MreBH, is required for chromosome segregation in
B. subtilis (
54,
193). Thus, depletion of MreB in
B. subtilis in the absence of polar effects on downstream genes did not lead to significant defects in chromosome segregation (
54), and depletion of MreB or Mbl led to only mild segregation defects (
194). Whether the third
B. subtilis actin homolog, MreBH, influences chromosome segregation remains to be determined. It also will be of interest to see what mechanism is used for chromosome segregation in coccal species, which lack MreB homologs.