Sculpting the Bacterial Cell Review

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William Margolin tuberculosis is a pleomorphic rod that often branches or swells at the poles; Staphylococcus aureus is a sphere, Prokaryotes come in a wide variety of shapes, determined and Streptococcus pneumoniae is ovoid, with pointed polar largely by natural selection, physical constraints, and caps. Then there are even more interesting ones, including patterns of cell growth and division. Because of their Streptomyces, which form fungi-like mycelial mats and aerial relative simplicity, bacterial cells are excellent models for hyphae; Caulobacter crescentus, which forms a curved rod how genes and proteins can directly determine mor- like V. cholerae but with tapered ends and a polar stalk at phology. Recent advances in cytological methods for one end. Archaea also have diverse shapes. While they bacteria have shown that distinct cytoskeletal filaments generally have similar shapes and sizes as bacteria, either composed of actin and tubulin homologs are important round or rod-like, one extreme thermophile (Haloquadratum for guiding growth patterns of the cell wall in bacteria, walsbyi) forms flat squares. Some bacteria are tiny rods, and that the glycan strands that constitute the wall are such as Bdellovibrio bacteriovorus, which hunts and invades generally perpendicular to the direction of growth. This other bacteria and grows in their cytoplasm. Others are cytoskeleton-directed cell wall patterning is strikingly remi- hundreds of microns long and over 20 microns wide, such niscent of how plant cell wall growth is regulated by micro- as Epulopiscium fishelsoni. All this diversity does not even tubules. In rod-shaped bacilli, helical cables of actin-like take into account the multicellular structures that many MreB protein stretch along the cell length and orchestrate bacteria can form, such as the long chains of Anabaena or elongation of the cell wall, whereas the tubulin-like FtsZ the flower-shaped fruiting bodies of Stigmatella aurantiaca. protein directs formation of the division septum and the re- Even bacteria without walls, such as the mycoplasmas, sulting cell poles. The overlap and interplay between these have their own distinctive shapes organized by an internal two systems and the peptidoglycan-synthesizing enzymes cytoskeletal scaffold. More detailed information about these they recruit are the major driving forces of cylindrical species and their shapes can be found in a recent compre- shapes. Round cocci, on the other hand, have lost their hensive review [5]. MreB cables and instead must grow mainly via their division Why have such morphological diversity? The answer is septum, giving them their characteristic round or ovoid partly because of selective pressures and in part from phys- shapes. Other bacteria that lack MreB homologs or even ical constraints [6]. One important selective pressure is com- cell walls use distinct cytoskeletal systems to maintain their petition for nutrients, which can be optimized by increasing distinct shapes. Here I review what is known about the the surface area relative to total cell volume. The C. cres- mechanisms that determine the shape of prokaryotic cells. centus stalk is an extreme example of increasing surface area for nutrient foraging in nutrient-poor environments [7]. Introduction Other selective pressures on shape include motility and The control of cell shape is ultimately an epigenetic process, resistance to predation [8]. with molecules exerting their effects on various physical One key physical constraint is diffusion limitation. Without constraints. Bacteria like Escherichia coli offer an excellent obvious cytoplasmic transport mechanisms, the scope of opportunity to understand shape determination at the cell sizes for most bacteria is quite narrow, sufficiently small molecular level. The cells are relatively simple, the genomes to accommodate diffusion-limited processes but large and proteomes manageable in size, and there are many enough to house the necessary macromolecules, such as mutants in a single gene that give rise to altered shape. ribosomes and chromosomal DNA, to maintain and dupli- Like plant cells, most bacteria have walls, and the shape of cate the cell. Another physical constraint is turgor pressure. their cells is largely governed by how the wall grows. For Because of relatively low water activity in the cytoplasm rela- most bacteria, then, understanding how the wall grows will tive to the outside environment, bacteria are essentially pres- be crucial for obtaining a complete picture of cell shape sure vessels, and the cell wall must be oriented to provide determination. Because the cell wall or murein (also called sufficient strength to counteract turgor forces of several the sacculus) is one large macromolecule, both the building hundred kilopascals in Gram-negative bacteria and as high and the turnover of this large structure help to determine cell as 3 megapascals in Gram-positive bacteria [9,10]. This pla- shape. A number of recent reviews cover the biochemistry ces limitations on the varieties of shapes bacterial cells can and cell biology of bacterial murein in detail [1–4]. have. However, there are examples where turgor pressure is probably low, such as in extreme halophiles where the A Great Diversity of Shapes molar levels of salt inside the cell are similar to those outside Despite usually being constrained by a cell wall, the shapes [11]. This, along with a putative internal cytoskeleton, might of bacteria are highly diverse, reflecting their large phyloge- explain why some species of halophilic archaea can assume netic range. For example, of the relatively straightforward triangular and square shapes with sharp corners that are not shapes, E. coli and Bacillus subtilis are straight rods; Vibrio seen in other species. cholerae is a curved rod; the Borrelia burgdorferi spirochete For a long time, these selective and physical forces were is a flat wave, an elongated and iterated version of the curved thought to be the main determinants of bacterial shape, in rod; Spiroplasma species are helix-shaped; Mycobacterium part because bacteria were not supposed to have cytoskeletons. The genetic and cytoskeletal determinants of bacte- Department of Microbiology and Molecular Genetics, University of rial cell size and shape have only been appreciated relatively Texas Medical School at Houston, 6431 Fannin Street, Houston, TX recently, largely because of the sequencing of multiple 77030, USA. genomes and fluorescent protein tags for visualizing protein E-mail: [email protected] localization in living cells. Special Issue R813

Figure 1. Basic growth modes of six repre- A sentative species. B. subtilis E. coli C. crescentus Shown are three species that contain MreB (A) and three species that lack MreB (B), with modes of growth summarized below for each. For each species, the arrow refers to the transition between a newborn cell and one at the ﬁnal stages of division prior to cell separation and new pole formation. For each stage, areas of the cell probably not engaged in signiﬁcant peptidoglycan synthesis are outlined in blue; areas actively synthesizing Sidewall, then septum Sidewall, then Sidewall, then peptidoglycan are outlined in other colours. septum + constriction constriction only Areas of MreB-dependent wall growth are shown in red or magenta; the magenta in B S. pneumoniae S. aureus C. glutamicum C. crescentus indicates slower growth relative to the red, because of the inhibitory effects of crescentin. Areas of FtsZ-dependent wall growth are shown in green. Solid green outlines indicate septal wall synthesis, and green dots indicate probable locations of active FtsZ-directed sidewall synthesis preceding cell division. Areas of DivIVA- dependent wall growth are shown in orange. In species with septal growth but no constriction, proper formation of the new pole Septum + sidewall Septum only Tip growth, then septum requires splitting of the septum and turgor- Current Biology dependent reshaping.

For simplicity, there are three main types of bacterial cell the wall, and a number of models have been proposed to shape to consider: cylindrical, typified by E. coli or B. subtilis, explain this type of insertion of new peptidoglycan [2]. but also including curved rods such as C. crescentus; ovococ- Recent cryo-electron tomography and atomic force micros- cal, typified by S. pneumoniae; and coccal, typified by copy data support the model in which individual glycan S. aureus (Figure 1). Cylindrical cells grow mainly by extending strands are coiled together into larger fibers, and once the length of the cylinder, and new cell poles are synthesized inserted into the existing peptidoglycan structure (the during a relatively short time window at cell division. This sacculus) under turgor pressure, stretch to assume an orien- shape has its advantages over a round shape, but requires tation roughly perpendicular to the long axis of the cell [14]. more sophisticated controls. Ovococci and the cocci both In contrast, the peptide bridges connecting glycan strands grow mainly via their division septa, but ovococci undergo are oriented parallel to the long axis. The end result, at least some length extension, whereas cocci grow exclusively via in B. subtilis, is that the glycan strands appear like a tightly division septa [12]. As a result, growing cocci are usually packed array of parallel telephone cords [15]. present as diplococci because they must divide in order to One attractive model postulates that a complex of cell wall grow, and their cell wall synthetic machinery localizes to the synthases and hydrolases inserts new peptidoglycan strands division septum. Because ovococci grow to some extent between old strands that act as templates to allow long-axis independently of their division septa, they need to switch extension without changing cell width [16]. However, such growth modes and place new division septa at the cell template-directed synthesis would seem to require an or- midpoint, similar to rod shaped cells. If cell separation is inef- dered lattice of glycan strands, whereas the evidence ficient, then these ovococci form cell chains which are typical supports a more disordered pattern [14,17]. This suggests for these species. Cocci, on the other hand, are spheroidal that existing cell wall cannot reliably provide a template for and do not form chains, because their division planes alter- new wall synthesis, and that a cytoskeletal template is nate in each generation. As a result, many cocci such as Dein- needed to maintain growth in the same pattern. Indeed, ococcus radiodurans or S. aureus form clusters or packets. In recent breakthrough work has demonstrated that cytoskel- particular, Neisseria species divide in two alternating planes, etal cables, not the chemical composition of the wall per se, whereas S. aureus divides in three alternating planes [12]. spatially direct both sidewall and septal wall synthesis.

How to Build a Rod: Two Distinct Modes of Growth Bacterial Actin Cables Drive Cell Elongation Growth and division of rod shaped bacteria such as E. coli Rod-shaped E. coli cells can be converted to roughly sphe- can be readily explained by sequential switching between roidal shape by treatment with beta-lactam antibiotics two modes of growth. In newborn cells that have just divided, such as mecillinam, which inhibit PBP2, or by inactivating peptidoglycan is synthesized along the sidewall, resulting in the function of several genes, including the genes for PBP2 the elongation of the cell to ultimately twice the length of the or RodA [18,19]. These round cells expand and lyse under newborn cell. At the time of cell division, the synthesis appa- many growth conditions, but with slow growth or increased ratus switches from sidewall peptidoglycan synthesis to divi- activity of the cell division protein FtsZ (see below), they sion septum synthesis. These two modes of growth probably can grow and divide asymmetrically via a cleavage-furrow compete with one another [13]. like mechanism, forming heart-shaped dividing cells Work over many years has shown that peptidoglycan (Figure 2). They also can revert back to rod shape once the insertion is evenly distributed over many sites throughout inactivation is relieved. Strikingly, round E. coli cells divide Current Biology Vol 19 No 17 R814

in alternating perpendicular planes like other cocci [20], paralogs lead to shape abnormalities, indicating that all three perhaps because their division plane is specified by the contribute to B. subtilis rod shape. asymmetric geometry of the parent cell [21]. Gram-positive The prediction, then, is that cables formed by MreB homo- rods such as B. subtilis also have a RodA protein that is logs (including Mbl cables of B. subtilis) directly recruit cell required for proper cell shape [22]. Even an ovococcus wall synthetic enzymes, thus ensuring that cell wall synthesis such as Streptococcus thermophilus requires RodA to main- occurs in a helical pattern. This appears to be the case in tain ovococcal shape and prevent cells from becoming B. subtilis, where MreB cables interact directly with PBP1 spherical [23]. and several other peptidoglycan synthetic enzymes [39]. Another gene required to maintain rod shape is mreB However, other interactions are probably indirect. The [24,25]. MreB and RodA work together to help synthesize mreB gene usually is in an operon with two other conserved the glycan strands of peptidoglycan [26]. A number of obser- genes, mreC and mreD.InC. crescentus, MreC, an integral vations on MreB together led to a breakthrough in our under- membrane protein, interacts with MreB and peptidoglycan standing of bacterial cell shape: first, that MreB is homolo- synthetic enzymes and forms helical patterns in the mem- gous to actin [27]; second, that MreB forms cables in brane [40–43]. Interestingly, at any given time, the MreB and a helical pattern that extended much of the length of the MreC helices do not colocalize, but instead one helix alter- cell [28]; third, that these helical patterns are similar to the nates with the other, with PBP2 colocalizing mostly with the pattern of new peptidoglycan insertion, defined by labeling MreC helix [35]. This suggests that whereas MreB cables with fluorescent vancomycin [29]; and fourth, that MreB is continuously move by treadmilling [44–46] to specify where conserved among many bacteria, but only those that are the next wall needs to be synthesized, MreC helices may rod shaped. remain relatively fixed at the current site of wall synthesis, These results suggested that MreB cables either direct bound to peptidoglycan synthetic complexes, and lag behind the pattern of new wall growth or provide physical support MreB helices. Less is known about MreD, but it has been to continuously maintain a cylindrical wall. Support for the shown to interact with MreC [47,48]. Importantly, inhibition first model came from studies with an MreB-specific drug, of MreB cable formation does not prevent cell wall synthesis, A22 [30]. When A22 is added to rod-shaped cells, it rapidly as rod-shaped cells expand their walls uniformly to become disrupts the MreB cables, yet the cell shape is unaltered for round under these conditions. This indicates that the cell the short term [31]. Eventually, after additional time, the wall wall synthetic complexes are always active to some degree, grows abnormally, creating round cells. The lack of cell and need to be constantly constrained in space by the cyto- shape change immediately after complete disruption of skeleton in order to inhibit the default state of uniform wall MreB cables strongly indicates that MreB cables do not expansion. It is not yet known how peptidoglycan synthetic provide an essential physical support but instead direct complexes move, but the models for peptidoglycan insertion the pattern of new wall growth. This is remarkably analo- and replacement suggest that the complexes are probably gous to microtubule-directed cellulose synthesis in plant continuously assembled and disassembled, and not proces- cells [32]. sive like plant cellulose synthases are thought to be. Fluores- How do the MreB cables specify the growth pattern? To cence photobleaching analysis of PBP3 in C. crescentus answer this, we must understand how the MreB cable shows a high turnover rate, consistent with this idea [49]. pattern itself is generated and what the proteins recruited If the MreB cable localization pattern directs the pattern of by them do. In E. coli, MreB cables form a double helix wall growth, then what directs the MreB cable pattern? In with a wide pitch that extend the length of the cell during somewhat of a surprise, it turns out that inactivating the growth [33]. At the time of cell division, the cables then transmembrane proteins MreC, MreD or RodA can perturb collapse to form two MreB rings that surround the cytoki- the cytoplasmic MreB helix. This suggests that the localiza- netic Z ring (Figure 2; see also below). After cell division, tion of at least some of these proteins is interdependent and these MreB rings lead to the assembly of new MreB cables that the integrity of the transmembrane proteins stabilise in the daughter cells to continue the MreB pattern [34]. MreB cables [47,50]. As MreB lacks an obvious membrane- MreB undergoes similar redistribution in C. crescentus [35]. anchoring domain, the other proteins may at the very least Because formation of the division septum switches cell help to tether MreB cables to the membrane. Many wall growth from an axial to centripetal mode, the redistribu- membrane-associated proteins localize in a helical pattern tion pattern is consistent with MreB being needed to trigger [51–54], and even lipids localize in such patterns [55]. This new peptidoglycan synthesis at the required time and place. indicates that the tendency of protein polymers such as In support of this idea, MurG, which provides lipid II precur- FtsZ or MreB to form helices inside cells [56] cannot be the sors in the last cytoplasmic step in peptidoglycan synthesis, sole explanation for the patterns. localizes to both the sidewall and division septum in E. coli The helical pattern of labeling by vancomycin or ramopla- and thus is part of both sidewall and septum synthesis nin, which both label nascent glycan strands [57], indicates machines [36]. that synthesis of new cell wall is helical, supporting the orig- Unlike most bacteria, B. subtilis has three MreB paralogs. inal prescient model of Mendelson [58]. As a result, insertion One of these, called Mbl for ‘MreB-like’, is essential for cylin- of new cell wall may result in a multiple-start helix, such that drical growth and forms a long double helix similar to that everything that localizes to the membrane, even the mem- formed by E. coli MreB that colocalizes with MreB helices brane itself, is influenced by the underlying helical architec- [37]. MreB interacts with Mbl but forms shorter helices. ture. In support of this idea, even outer membrane proteins MreC interacts with Mbl but not MreB, consistent with Mbl and lipopolysaccharide of E. coli localize in a helix [59,60]. function being analogous to E. coli MreB [37]. Finally, MreBH If the helical ‘grooves’ are relatively imprecise, which is con- also forms helical cables and recruits a cell wall hydrolase, sistent with the direct imaging of the peptidoglycan strands LytE, to those cables, suggesting that MreBH specializes in described above, then the patterns will localize with different regulating cell wall turnover [38]. Mutations in any of the pitches. Special Issue R815

Figure 2. Roles for the actin (MreB) and tubulin (FtsZ) cytoskeletons in shaping A E. coli cells. Depletion (vertical triangles) of only MreB (A) MreB FtsZ or only FtsZ (B) during growth disrupts each cytoskeleton and causes cells to either slowly become spheroidal (A) or form cylindrical ﬁla- ments (B) over time (red downward arrows). Predivisional cells with normal levels of MreB (red) and FtsZ (green) have a Z ring sur- rounded by MreB rings (top rows in A and B). After cell growth and division over time (rightward black arrows), FtsZ and MreB relocalize into helical patterns, and prepare to divide at the next division site (yellow dashes). During MreB depletion (A), cells will form Z arcs (green) and divide, often asymmetrically, at those arcs to form a diplococcus, which then over time (rightward black arrows) sepa- rates to form a single round cell capable of dividing again in a perpendicular plane (yellow dashed line). These MreB-lacking cells will continue to grow and divide as spheroids. In contrast, FtsZ-depleted cells (B) continue to grow their sidewall and elongate their MreB B cables but fail to divide, thus forming long ﬁla- ments. The MreB double helix is shown as MreB FtsZ a single helix for simplicity. Micrographs rep- resenting some of the steps are shown next to the appropriate diagram.

Protein factors also regulate MreB assembly. For example, the tubulin homolog FtsZ (see below) is required for the collapse of MreB cables from extended helices to rings near the future division site [34,41], although the mechanism for this FtsZ-dependent relocalization is not known. Re- cently, another conserved bacterial shape protein called RodZ was discovered which interacts with MreB and is crucial for proper assembly of MreB cables [61–63]. Like MreB, RodZ Current Biology usually localizes as a helix, and its localization pattern requires MreB, indicating that RodZ and MreB localization are dependent therefore must divide using another as yet unknown mecha- on each other. As RodZ crosses the membrane once, its nism [67]. Although FtsZ primarily localizes to the division cytoplasmic domain probably contacts MreB, whereas its septum, when a cylindrical cell such as E. coli or B. subtilis extracytoplasmic domain may contact peptidoglycan is not dividing or preparing to divide, FtsZ localizes as synthesis enzymes. Inactivation of RodZ in C. crescentus a dynamic coil in the cytoplasm [68,69] (Figure 2). In C. cres- or E. coli results in huge, misshapen cells. Overproduction centus, FtsZ relocalizes from a focus at one cell pole to mid- of RodZ causes similar shape abnormalities, indicating that cell upon chromosome segregation [70]. Just as MreB reloc- RodZ levels need to be in an optimal range. Overproduction alizes from sidewall to septum depending on the temporal of MreB also leads to a dominant-negative effect on cell and spatial requirements for peptidoglycan-synthesis, FtsZ shape, but overproducing both MreB and RodZ mostly relocalization may serve the same function. In support of restores normal rod shape, suggesting that the MreB:RodZ this, FtsZ participates in both septal synthesis and a subset ratio is crucial. Future studies will be needed to understand of sidewall synthesis by recruiting peptidoglycan-synthesis the mechanism by which RodZ regulates MreB cable machinery via PBP 2 [71–73] (Figure 1). However, FtsZ assembly and cell shape. cannot direct cylindrical growth without MreB, probably The bacterial tubulin cytoskeleton coordinates growth, because only MreB can properly organize the growth cell division, and cell size. FtsZ, the bacterial tubulin pattern. Interestingly, both FtsZ and MreB help to distribute homolog [64], assembles into a cytokinetic ring called the the peptidoglycan synthetic machinery evenly, because Z ring at the site of cell division [65,66]. The Z ring is used E. coli or S. aureus mutants that lack both proteins synthe- for cytokinesis in most bacteria, although some families size their cell wall in large patches [74,75]. Therefore, both such as the Planctomycetes and Chlamydia lack FtsZ and actin and tubulin cables are important for tethering the Current Biology Vol 19 No 17 R816

peptidoglycan synthetic machinery to ensure a properly dis- into filaments without nucleotide. A single crescentin fila- persed, nonrandom distribution of new wall material. In ment normally localizes to the membrane at the inner curve cocci that lack MreB and grow by their division septum, of C. crescentus cells. Because a crescentin filament can FtsZ is likely the sole means of recruitment and tethering initiate assembly of a new filament on any part of the cell of the peptidoglycan-synthetic machinery. membrane, its inner curve location is the result of its action, FtsZ is important for bacterial shape in two other respects. not the cause. The available evidence suggests that a cres- The first is that its orchestration of septal wall synthesis centin filament constrains wall growth only on the side determines the shape of the cell poles (Figure 1). Cell poles of the cell where it is present. Crescentin filaments are of C. crescentus are tapered because, as mentioned above, stretched, because release from the membrane results in FtsZ contributes significantly to cell wall extension near contraction of the filament into a helix. the division site, resulting in mostly constriction instead of These results suggest that the stretched crescentin fila- septum formation in this species. In contrast, B. subtilis ment may locally inhibit new wall synthesis by locally divides mainly by septation without constriction, resulting decreasing strain in the peptide crosslinks of the peptido- in its typically squared off poles. As B. subtilis is Gram-posi- glycan, resulting in increased growth of the wall on the oppo- tive and thus lacks an outer membrane, there is no need for site side and ultimately curving the cell [85]. In support of this coordinated constriction. E. coli has round poles because it model, exogenously produced crescentin in E. coli induces divides with a combination of septum formation and con- severe coiling of the cells. The model also predicts that striction of inner and outer membranes, which are all coordi- other proteins that localize to the membrane, form polymers, nated [76]. Once poles are formed during cell division, they and interact with MreB cables might also be able to distort generally do not change their shape, and their peptidoglycan cell shape. Interestingly, altered expression or mutants of is inert (see below). Polar morphology in E. coli can be MinE, MreB, FtsZ and FtsA can all cause similar dramatic altered, but usually only by mutations in FtsZ or FtsZ-depen- coiling of E. coli cells under certain conditions [86–89], al- dent peptidoglycan-modifying enzymes that perturb the though there is no evidence so far of direct interaction geometry of the septum [77–80]. between MreB and these other proteins. Moreover, when FtsZ can also control cell size, an important component of C. crescentus cells are forced to assume unnatural shapes shape. For example, E. coli increases cell size — mostly via by growing them in a microfabricated chamber, they main- increased length — as nutrients become more plentiful and tain the shapes when removed [85]. This is consistent with growth rate increases [81]. Cell division also requires cells earlier findings suggesting that the shape of the bacterial to attain a threshold length. However, even in rich medium, sacculus is determined by its growth pattern and remains mutants of E. coli or B. subtilis have been discovered that rigid [90,91]. make cells w20% shorter than normal at the same growth To test if crescentin might interact with MreB, crescentin rate. The B. subtilis mutants are in UgtP, a component of was exogenously produced in Agrobacterium tumefaciens, a glucolipid pathway for cell wall biosynthesis [82]. In addi- which lacks MreB but is rod-shaped. The presence of cres- tion to its enzyme activity, UgtP negatively regulates FtsZ centin had no effect on shape [92]. This and other evidence assembly, resulting in delayed Z ring formation and larger demonstrates that interaction of crescentin with the cell cells, but only in rich medium. In poor growth medium, cells membrane requires that it bind to MreB cables; if MreB are small whether or not they have UgtP. But in rich medium, cables are perturbed, crescentin filaments become dis- lack of UgtP results in the assembly of more Z rings per cell lodged from the membrane and lose their shape-changing mass, and shorter than normal cells. Therefore, this pathway properties. The potentiation of crescentin function by MreB uncovers the first known link between bacterial metabolic cables is similar to the organization of intermediate filaments activity and cell size. In E. coli, a gain-of-function mutation in eukaryotic cells by actin and microtubules [93]. in the actin-like cell division protein FtsA also results in Although difficult to find by amino-acid similarity alone, w20% shorter cells in rich medium, by increasing FtsA– bacterial intermediate-filament-like proteins are widespread FtsZ interactions and generally enhancing the integrity of as judged by the presence of central segmented coiled coil the Z ring [83], although no metabolic link has yet been es- domains and the ability to assemble spontaneously into fila- tablished in this case. In conclusion, regulation of Z ring ments [94]. One such homolog is FilP of Streptomyces coeli- activity by nutritional and other inputs can determine the color, which also forms intracellular cytoskeletal filaments. cell length at division, which has an obvious impact on cell In the absence of FilP, cell growth and morphology were shape. abnormal, and the hyphae became susceptible to mechanical breakage [94]. Therefore, like eukaryotic intermediate Caulobacter crescentus as a Model for Bacterial filament proteins, at least one bacterial homolog seems to Shape Control impart mechanical strength to the cell, although the mecha- C. crescentus is genetically tractable and has an obvious nism is unclear. Another, the cytoplasmic filament protein of asymmetric shape: it is a curved rod with tapered poles, Treponema spirochetes, spans the entire cell length and and predivisional cells have a flagellum at one pole and seems to be important for cell division [95]. a thin extension of the cell body, called a stalk, at the oppo- The C. crescentus stalk is also of interest because it acts site pole. These properties make C. crescentus an excellent as a nutrient antenna and can grow considerably longer model system to study bacterial cell shape and polarity. than the cell body under certain conditions. Inactivation of A knockout of a gene called creS, encoding crescentin, PBP2, or depletion of MreB or RodA, results in a stalk elon- converts the normally curved rods to straight rods that still gation defect [96]. These results are consistent with the have polar stalks and flagella [84]. Although not obvious idea that the stalk is a very narrow continuation of the cell from its primary sequence, crescentin has a similar domain body, and that MreB cables most likely act indirectly, structure to that of eukaryotic intermediate filaments, and possibly via PBP2, at the cell pole to initiate and maintain indeed purified crescentin can spontaneously assemble this small diameter (about 5 times smaller than the cell itself). Special Issue R817

Figure 3. Phylogeny of some shape-determining proteins across representative Family Species Shape MreB MreC MreD RodZ DivIVA bacteria. The species are grouped by family. For each γ-proteo Escherichia coli -++++ species, typical cell shape is shown (not drawn to scale), along with the presence or γ-proteo Vibrio cholerae -++++ absence of the protein as encoded in the genomic sequence. The presence or absence γ-proteo Francisella tularensis of MreB, MreC, MreD or RodZ in different ----- species as identiﬁed by STRING COG was β-proteo Neisseria gonorrhoeae described in [61]. STRING COG was also ----- used to identify DivIVA homologs. DivIVA is absent in all the Gram-negative species β-proteo Bordetella pertussis -++++ listed, but some members of the Gram- negative d-proteobacteria contain DivIVA α-proteo Sinorhizobium meliloti ----- orthologs. α-proteo Caulobacter crescentus -++++

α-proteo Rickettsia typhi -+-++

FtsZ is also required for formation of ε-proteo Helicobacter pylori ---++ cross-band structures in the stalk [43]. Firmicutes Bacillus subtilis +++++ Phylogeny of Shape It is likely that rod-shaped bacteria Firmicutes Staphylococcus aureus ++++- arose first in evolution, and cocci have evolved multiple times from them by Firmicutes Streptococcus pyogenes ++--- losing crucial genes, including those for the cytoskeleton [97,98]. Phyloge- Mollicutes Mycoplasma pneumoniae ----- netic comparisons are completely consistent with the idea that cocci are Actino- Corynebacterium +---- bacteria glutamicum the default shape of bacterial cells that have lost the MreB cytoskeleton; MreB Spirochetes Borrelia burgdorferi -+-++ is almost never found in cocci. As MreB probably acts to shape the cell Cyano- Anabaena variabilis -++++ bacteria wall via MreC, it comes as no surprise that essentially all species that have Current Biology MreB also have MreC (exceptions include Wolbachia endosymbionts of insects and Thermotoga maritima, which have MreB but no requires that new cell poles be initiated along the sidewall MreC) [61]. MreD is often present in bacteria with MreB and of an existing filament, which is not observed in most rod- MreC, but is absent in others such as the g-proteobacteria. shaped bacteria. The apparent trigger of new pole formation RodZ is usually only present in species that have MreB, which is DivIVA, a protein found in many Gram-positive bacteria is consistent with its role as an MreB assembly factor; [100].InStreptomyces, DivIVA forms foci along hyphal side- however, RodZ is somewhat less conserved than that of walls at incipient branches, and overproduction of DivIVA MreB, with some species containing MreB but lacking can force new zones of cell wall to be assembled [101] RodZ [61]. The distribution of some cell shape proteins in (Figure 4A). The importance of DivIVA for growth in Strepto- several divergent species is shown in Figure 3. myces is underscored by the dispensability of MreB for cell There are a couple of surprises from the phylogeny of wall extension in these species, although MreB is essential shape proteins. One is that MreC and MreD are sometimes for spore formation [102]. present in species with no MreB, such as Gram-positive Another related species, Corynebacterium glutamicum,is cocci. This suggests that MreB was specifically lost in these roughly rod-shaped and yet lacks MreB, MreC and MreD. species, but that MreC and MreD still retain a role in cell wall How can it maintain a rod shape without MreB cables? Like synthesis. The other surprise is that a fairly large number of Streptomyces, C. glutamicum uses polar targeting of DivIVA rod-shaped species lack MreB, along with MreC and MreD. to drive tip growth, which is mediated by PBP1a, PBP1b, and Clues are now emerging about how these bacteria maintain RodA [103]. Staining of C. glutamicum cells with vancomycin their rod shape without MreB cables. showed that young cells stained at the poles while older cells stained at the septum [29]. This is consistent with the idea Cell Poles and Cytoskeletal Control of Tip Growth that DivIVA initiates tip growth, then switches growth to the Some bacteria form branches, grow at their cell poles division septum when cells reach a critical length (Figure 1). without the benefit of MreB cables, or both. One example This is analogous to the switch from sidewall to septal of an MreB-containing species that branches extensively is growth in MreB-containing bacteria. In support of this model, the filamentous fungus-like Streptomyces, which grow vege- depletion of DivIVA in C. glutamicum results in nearly round tatively as a mycelial filamentous mat and branch extensively shape, whereas overproduction of DivIVA results in swollen [99]. During starvation, Streptomyces then send up aerial poles, indicative of locally increased peptidoglycan hyphal branches to make spores. The branching process synthesis at polar sites of DivIVA localization [104]. The Current Biology Vol 19 No 17 R818

Figure 4. Cytoskeletons trigger new shapes A B in bacteria. Shown are two examples of new shapes resulting from bacterial cytoskeletal proteins. (A) In Streptomyces, DivIVA (orange balls) forms large assemblies at the poles of hyphae, possibly recognizing a region of sharp membrane curvature, but also forms seem- ingly random foci along the sidewall. As Div- IVA tends to self-assemble, larger foci are easily generated (middle panel). These larger assemblages probably trigger branch formation, because a DivIVA focus usually forms under a new pole prior to visible branch initiation. A DivIVA lattice is subsequently main- tained at the new branch tip (green) and is required for continued tip extension. (B) Cells of Mycoplasma pneumoniae lack peptidoglycan, and would be spheroidal without a cytoskeleton (top). Cytoskeletal proteins form the terminal organelle, which is impor- Current Biology tant for gliding motility (red rightward arrow); the resulting cell extension (red) upon movement and subsequent duplication of the organelle at the opposite pole (not shown) gives these wall-less cells a roughly cylindrical shape with narrow tips. closely related Mycobacterum smegmatis also needs DivIVA forming a lattice structure. Positive membrane curvature to grow as pleomorphic rods [105]. In the unrelated S. pneu- inside bacterial cells is rare, but is a distinct feature of the moniae, which also lacks MreB but is ovoid, DivIVA localizes outer surface of the developing endospore, where SpoVM at the major site of peptidoglycan synthesis — the division localizes and functions in building the spore coat. As septum — and probably is involved in septal peptidoglycan mentioned earlier, the future division plane of E. coli may synthesis [106]. depend on cell shape cues [21]. We will probably soon be The role of DivIVA as a local organizer of tip growth is remi- learning about more proteins that use similar shape cues niscent of the role and localization of the apical body (Spit- for their localization and/or activity, which might help to rein- zenko¨ rper) of filamentous fungi. This function is not as far- force and maintain certain types of shapes. fetched as it would seem, because DivIVA has properties Many species of the a-proteobacteria are rod-shaped, and of a cytoskeletal element. The carboxy-terminal coiled-coil yet lack MreB, MreC, MreD, RodZ and DivIVA. An unrelated domain of DivIVA has some homology with tropomyosins, g-proteobacterium and the cause of tularemia, Francisella which, intriguingly, localize preferentially to fungal Spitzen- tularensis, also lacks these proteins and yet is rod-shaped ko¨ rper [107]. Moreover, purified DivIVA protein forms ‘doggy [61]. Because vancomycin does not penetrate the outer bone’ structures in vitro that probably translate into a membrane of these Gram-negative cells to be able to bind membrane-associated lattice in vivo [108]. How this putative to its periplasmic target, we do not yet know if they grow by DivIVA lattice recruits cell-wall synthesizing enzymes is not tip extension like corynebacteria. However, some a-proteo- yet known, given that no MreC and MreD are present in bacteria lacking the known shape proteins above share a C. glutamicum and related species. However, these species strong tendency with corynebacteria and mycobacteria to may use protein kinases to regulate the activity of peptido- form branches instead of filaments when their cell division glycan synthesizing enzymes [109]. machinery is perturbed [113–116]. This suggests that cells How does DivIVA localize to the cell poles and division of these species also expand by tip growth instead of exten- septum? Recent evidence suggests that DivIVA recognizes sion along the cell cylinder, and inhibiting cell division forces regions of high negative membrane curvature [110]. This is new poles to accommodate the mass increase. It is likely supported by its ability to localize along arcs at cell poles that these a-proteobacterial species have a DivIVA-like cyto- of other species that normally lack DivIVA, such as E. coli skeletal protein that localizes to cell poles and organizes a pu- and even fission yeast [111]. The DivIVA foci in Streptomyces tative peptidoglycan-synthesis complex there. Curiously, filaments that trigger formation of a new pole may initially C. crescentus, which is also an a-proteobacterium but has form randomly, and then form a lattice at the higher curvature MreB, synthesizes a protein, TipN, that is conserved in some once the branch has initiated. Alternatively, the foci may of the above species. TipN localizes to the division site and trigger local membrane curvature that then stabilizes DivIVA persists in the daughter cells at the newly formed cell pole assembly at the curve. This is a potential example of shape [117,118]. When overproduced in C. crescentus, TipN triggers inducing and reinforcing a distinct protein localization ectopic poles to form, similar to the phenotype during recovery pattern, basically the converse of the concept of cytoskeletal from MreB depletion [96],suggestingthatTipNmaybe localization driving growth. involved in tip extension in the related MreB-lacking species. Another example of shape inducing localization in bacteria What might be the molecular mechanism for initiation of is the recent discovery that a 26-amino acid B. subtilis branches in species that lack a DivIVA-like organizer? membrane-associated protein, SpoVM, localizes specifically Recent work on E. coli offers some hints. The sidewall of to regions of high positive curvature [112], possibly by E. coli is constantly growing, indicating that peptidoglycan Special Issue R819

turnover is high. This peptidoglycan is the flexible skin of the Finally, archaea lack typical peptidoglycan walls and most cell that can change its shape in response to turgor pressure instead have surface layers, or S-layers, consisting of a thanks to autolytic enzymes and cytoskeletal proteins protein lattice on the outside of the cell [134]. Some bacteria, described above. In contrast, the peptidoglycan at the divi- such as C. crescentus, also have S-layers. One possibility is sion septum and resulting cell poles exhibits little if any turn- that S-layers comprise an exoskeleton that determines over [119], making it relatively inert. This property may be shape of archaeal cells. However, species with different a result of different orientation of the peptidoglycan strands shapes, such as the spheroidal Haloferax volcanii and the after splitting of the division septum, which discourages rod-shaped Halobacterium salinarum, have similar S-layer autolytic cleavage [10]. The cell wall is therefore a mosaic components, suggesting that S-layers do not themselves of all new and all old domains [71,120]. direct cell shape [135]. This is probably analogous to bacte- The current model proposes that branches (new cell poles) rial peptidoglycan, which passively maintains the shape occur in the sidewall because of a patch of inert peptido- determined actively by the cytoskeleton [134]. Many puta- glycan, and that MreB, RodA, PBP2 and other components tive cytoskeletal proteins are present in archaea, including of the wall elongation machinery normally prevent unwanted actin homologs present in some wall-less archaea [136].It patches of inert peptidoglycan from forming [121].InE. coli is likely that these and other cytoskeletal proteins are crucial lacking the PBP5 carboxypeptidase, which trims side chains for cell shape determination in this kingdom, although from the peptidoglycan and keeps peptidoglycan from the difficulty in working with archaea will make progress becoming inert, cells can assume a variety of bizarre shapes, slower. including multiple branches, which are consistent with growth around the inert peptidoglycan, much like a tree grows Conclusions and Outlook around a blocking object [122]. The lack of wall turnover at the It is clear that the cytoskeleton of bacteria actively organizes cell pole may create a more static environment for many cell shape. In walled bacteria, the patterns of synthesis and membrane proteins to localize specifically to cell poles degradation of the cell wall ultimately determine cell archi- [123]. Examples of proteins that may localize to cell poles tecture when under turgor pressure. In bacteria lacking by this mechanism include bacterial chemoreceptors [124] walls, other cytoskeletal structures can dynamically change and ActA/IcsA, which are required to nucleate actin tails at the shape of membranes, much like in protozoan or ani- one cell pole of Listeria or Shigella species to trigger mobility mal cells. In turn, changes in shape can provide feedback inside the eukaryotic host and evade host defenses [125,126]. to the bacterial cytoskeleton, and localized mechanical stresses may as well, as they do in plant cells [137]. Actin Thinking outside the (Peptidoglycan) Box and tubulin homologs are prominent in the bacterial cyto- What happens in prokaryotes without typical peptidoglycan skeleton, but other proteins such as RodZ, DivIVA, and inter- walls? Membranes by themselves usually assume sphe- mediate filament homologs are emerging as important sup- roidal shapes unless they are organized by a cytoskeleton. porting players. Although we know many of the players and Nevertheless, there are several examples of cells without how some of them interact, the mechanisms by which cyto- walls of any kind that have some semblance of shape. The skeletal components dictate cell shape still need to be most notable are the mycoplasmas, which completely lack worked out. While it is generally true that cell architecture cell walls, and yet often assume non-spheroidal shapes is inherited [138], it is also true that rod shaped cells can because of a unique internal cytoskeleton visible by ultra- emerge from round cells or from round dormant spores, structural analysis [127,128]. Mycoplasma pneumoniae, for which must depend on de novo assembly of cytoskeletal instance, uses cytoskeletal proteins to assemble new structures. Membrane lipid biosynthesis also must be terminal organelles to extend its tips when it is gliding along regulated to match growth of the cytoplasm and wall, and surfaces, and assumes a rod-like cell shape [129,130] (Fig- may respond to feedback mechanisms [139,140]. It will ure 4B). Therefore, an internal cytoskeleton is required for require much more work to understand the molecular motility, resulting in a distinctive cell shape. 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