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Department of Microbiology and Immunology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202-9037
SUMMARY INTRODUCTION The Best Arguments The Perfect Example The Perfect Experiment The Imperfect Science To Protect and To Serve NUTRIENT ACCESS Why Are Prokaryotic Cells Small? Theoretical limits. Surface-to-volume ratio. The diffusion sphere. Intracell mixing. How Diffusion Affects Cell Shape Contrasting examples. Conclusions. Morphological Variation Variation with growth rate. Filamentation with nutritional status. Nutritionally deficient streptococci. True to form? Prosthecae as Nutrient Whiskers? Filaments and Blimps Miscellaneous Shape Effects Summary CELL DIVISION AND SEGREGATION Shape Uniformity The Cell Cycle Resists Shape Changes Summary ATTACHMENT Physicochemical Considerations Shear Forces Poles Apart: Polar Localization Cell-Cell Interactions Safety in numbers. Antisocial shapes. Biofilms: where no one stands alone. Summary DISPERSAL Float Like a Butterfly Hovercraft Subterranean Explorers: Geological Transport Rock Solid Summary MOTILITY Energetics of Motility Brownian motion. Chemotaxis: efficiencies of stalking. Size vise. Sleds and saucers. Side Effects: Motility Near Surfaces Motility versus Viscosity Thicker than water. One good turn: helical motility. The polymer maze. Motility and Polarity Summary POLAR DIFFERENTIATION Sequestration Separation Localized force. Aging. Summary PREDATION Protistan Grazing (Bacterivory) Selection for Altered Cell Dimensions Goldilocks and the bimodal effect. The long of it: selection for filaments. The short of it: selection for small cells. Wide load: selection for increased diameter. Selection for Altered Shape Prosthecate bacteria. Helices and spirals. Selection for Multicellular Complexes Size Isn't Everything Phage Effects Predatory Prokaryotes Transcellular shape attack. Summary DIFFERENTIATION Asymmetric Division Stationary Phase Rod to coccus. Rod to filament. Bifids: Two Heads Are Better than One Swarming: in Serried Ranks Assembled Out of shape. Shape regulation. Heterocysts Pathogenesis-Associated Differentiation Fungal Pathogenesis and Differentiation Shape Discrimination by the Immune System Bacterial Pathogenesis and Differentiation Uropathogenic Escherichia coli. Legionella pneumophila. Listeria monocytogenes. Helicobacter pylori. Campylobacter jejuni. Conclusions. Bacteroids: Plant-Microbe Symbiosis Unusual Symbioses Multicellular Interactions Fruiting bodies. Interlocking shapes: a puzzlement. Summary THE SHAPE OF THINGS TO COME What We Need A shape atlas. Unanswered questions. Techniques. Ecology. Evolution. Mechanisms. Going Forward ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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—Robert Morgan (221)
To be brutally honest, few people care that bacteria have different shapes. Which is a shame, because the bacteria seem to care very much. A simple way to verify this is to take a leisurely stroll through Bergey's Manual of Determinative Bacteriology (133) or The Prokaryotes (65, 313), pausing to admire the surprising and bewildering riot of shapes, sizes, and aggregates, some of which are illustrated in Fig. 1. There are cells that look like lemons, teardrops, or oblong spheroids; some are bent, curved, flat sided, triangular, bean shaped, or helical; others are rounded, squared, pointed, curved, or tapered. One is a flat square, and another is a slim, coin-like circular disk. The prosthecate bacteria radiate extensions that create star-like constellations or bulbous whiskers, all of which, though seemingly irregular, replicate faithfully. Other organisms grow as branched or unbranched filaments, live in sheathed or unsheathed chains, or aggregate in primitive or highly organized multicellular composites. The sizes of individual cells range over at least six orders of magnitude. And yet, amazingly, this short inventory barely begins to catalogue the known forms. As Zinder and Dworkin point out, our dogmatic fixation on rods, cocci, and spirals has "obscured the spectrum of enormous morphological diversity manifested by the bacteria" (380).
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The second indication that form is an important physiological character is the fact that bacteria actively modify their shapes. Some changes are temporary (moving from one growth phase to another, responding to nutritional alterations, or passing through a host), some are repetitive (dimorphic or pleomorphic life cycles), and some accompany the development of specialized cells or structures (spores, heterocysts, swarmers, and elaborate multicellular assemblies). Such transitions are under explicit genetic and biochemical control, which is a compelling argument that shape is a significant element in these physiological adaptations.
The third argument entails the evolutionary progression of cell shape. Early on, Woese et al. concluded that the coccoid bacteria were spread across phylogenetic units and should be considered as degenerate forms of more complicated bacterial shapes (311, 366). More recently, Siefert and Fox (303) mapped the basic shapes onto the prokaryotic phylogenetic tree and concluded that bacterial morphology exhibits a definite historical trend, most likely beginning with a filamentous or rod-shaped cell. Certain shapes, morphological cycles, or developmental strategies are confined to particular branches of the tree, and, contrary to the widespread misconception that the first cells had to be spheroidal, coccoid cells are a near-dead-end shape that arose independently numerous times (303). Using different phylogenetic tools, two other groups arrived at similar conclusions. Gupta (111) proposed a map of prokaryotic evolution based on the distribution of DNA insertions and deletions, and Tamames et al. (324) generated an analogous tree by cataloguing gene order in a chromosomal segment devoted to septation. All these analyses indicate that morphology is significant, that it can be charted on an evolutionary scale, and that the earliest cells were probably rods or filaments, with cocci being derived and degenerate forms. Although the results of these approaches do not coincide at every point, the principal conclusions are virtually identical, lending credence to the idea that bacterial morphology is as important a selectable trait as any other biochemical adaptation.
Using nanofabrication techniques, Takeuchi et al. created micrometer-sized agarose moldings in which they trapped and grew Escherichia coli (322). By altering the contours of these traps, they forced cells to grow in a variety of shapes that persisted when the bacteria were released (322). Unexpectedly, the motility of these cells changes according to their gross morphology. Cells that are short crescents move in a straight line, as do helical cells with a long spiral pitch, whereas cells coiled like tightly wound springs move in tight circles, "going nowhere" (Fig. 2) (322). Note that the individual cells differed from one another exclusively in their overt morphology, because their shapes were imposed physically and not genetically or biochemically. Every biological facet of these cells except shape is equivalent, a feat accomplished by no other experimental system to date. The results prove that cells change their three-dimensional motions in extraordinary ways simply by adopting one shape over another, hinting that other shape-dependent behaviors await discovery.
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| NUTRIENT ACCESS |
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—T. J. Beveridge (19)
Bacteria have to eat, and diffusion is the fundamental physical factor that determines how well they do so. Cells may secrete molecules to scavenge chemicals in short supply, and those that are motile may move to where nutrients are more highly concentrated, but, however they cope, in the end virtually all prokaryotes rely entirely on diffusion to bring needed compounds to their surfaces and to mix nutrients and macromolecules in their cytoplasm. This dependence on the laws of diffusion exerts a powerful constraint on cell size and may also influence shape. Of course, bacterial size spans an enormous range, from the tiny Pelagibacter ubique (enclosing the miniscule volume of 0.01 µm3) (266) to the gargantuan Thiomargarita namibiensis and Epulopiscium fishelsoni (with internal volumes 108 to 1010 times greater) (8, 291, 292), demonstrating that diffusion alone does not dictate overall cell dimensions. Also, bacteria sharing the same niche may have vastly different shapes, indicating that the nutritional environment does not, by itself, specify shape. Nonetheless, bacterial morphology must conform to, and be circumscribed by, the general physical principles of nutrient access. It is therefore pertinent to know these limitations and the boundaries they impose.
For greater depth and incisive descriptions about how diffusion affects prokaryotic size, interested readers should consult four superb reviews (19, 170, 227, 292). In particular, the article by Schulz and Jørgensen provides a comprehensive, in-depth introduction to the subject (292), and the report of the National Research Council Space Studies Board has the most far-reaching discussions regarding physical and theoretical restraints on cell size (227).
Surface-to-volume ratio. The typical argument for prokaryotes being small is that the rate for transporting nutrients into a cell is a function of the amount of exposed surface area (19, 170, 292). However, it is not surface area per se that is important but the fact that the cell can insert greater numbers of nutrient transport complexes, which in turn deliver nutrients to the cytoplasm (170).
Thus, reliance on diffusion creates the strong tendency to form smaller cells, which increases the surface-to-volume ratio and decreases the amount of cytoplasm that has to be supported by any one transporter (19, 170, 292).
The diffusion sphere. A cell's nutritional problem is complicated by the existence of a "diffusion sphere" (292) or "Reynolds envelope" (19) that adds to the cell's effective dimensions and forms a diffusion barrier around the cell. The diffusion sphere can be thought of as a thin layer of external liquid attached to, surrounding, and traveling with a bacterium and through which nutrients and waste products must pass (15, 19, 263). The existence and dimensions of this sphere are not affected by even the most turbulent conditions in natural waters (292). Because of this, the edges of the diffusion layer can be considered to be the surface area in contact with the undiluted nutrient concentrations in the external medium. The shape of this area is similar to that of the cell itself if the cell is a perfectly symmetrical sphere or smooth rod. However, the diffusion layer of a spiral cell has less "spiral" character than the cell body because parts of the diffusion sphere overlap. This means that distinctly shaped diffusion envelopes may surround cells of different shapes, potentially affecting their access to nutrients. For example, if a smooth straight rod and a thin spiral cell have equivalent diffusion spheres, the spiral cell might import more nutrients because it has more cell surface area into which it can insert transporters (Fig. 3A). The effects of alternate diffusion barriers are hypothetical, however, as I am not aware of calculations that address the consequences of spheres produced by cells of different morphologies.
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0.8 µm by 4.8 µm) will take about 0.5 s to migrate from one side wall to the cell center (a distance of 0.4 µm) or will require about 5 s to migrate from pole to pole (19). Schulz and Jørgensen calculated relatively similar "traffic times," which describe how long it takes for any two molecules to meet one another (292). Schulz and Jørgensen also calculated the "mixing time" for a 1-µm-diameter coccus and found that a small molecule takes only about 1 millisecond to appear with equal probability anywhere in cell, whereas a larger protein takes about 10 milliseconds (292). These times will change with cells of different sizes and might eventually limit particular biochemical reactions at some combination of size and shape. The trouble is that if maximizing the surface-to-area ratio were the single guiding principle governing prokaryotic morphology, then a thin, flat, disk-like cell would seem to be the best alternative (63). However, with the exception of the archaeal halobacteria (26, 35, 349, 350), there are few really flat bacteria (63). The major reason may be that the surface area provided by flat cells is not significantly greater than that of thin filamentous cells (349), and a rod-shaped cell imparts an abundance of additional benefits (discussed below). Of course, molecular considerations may also constrain the synthesis of walls with flat shapes.
Contrasting examples.
At the smallest end of the free-living bacteria, the SAR11 clade of marine Alphaproteobacteria constitute up to 25% of all ocean microbes (50% in some surface waters) and 12% of the marine prokaryotic biomass (93, 222, 266). Of these, Pelagibacter ubique has the smallest genome (93) and grows as tiny, slightly curved rods (vibrioid), with newly divided cells measuring 0.2 µm by 0.4 µm and having an estimated cell volume of 0.01 µm3 (44, 266). Because the cell is extremely thin, the surface-to-volume ratio is very high, which seems to be the rule for oligotrophic (low-nutrient milieu) organisms. Cells with such dimensions fit the model in which natural selection optimizes the surface-to-volume ratio to provide appropriate transport rates in low-nutrient conditions (93). So far, this is consistent with the idea that diffusion plays a powerful role in shaping these cells. But herein lies a conundrum. Although P. ubique is one of the most successful and numerous life forms on the planet, a cell whose size we can explain because it has a tiny volume and large surface-to-volume ratio and whose dimensions we believe to be optimized for nutrient acquisition, even so we cannot explain why P. ubique is vibrioid. There are (as yet) no obvious reasons why the cells should be curved rods. Viewed from the point of view of diffusion alone, straight rods should do just as well. Curiously, many marine microorganisms are vibrioid, with the most notable examples being members of the genus Vibrio or of freshwater genera such as Caulobacter. The reasons probably stem from forces other than diffusion considerations.
At the other end of the spectrum is the giant endosymbiont Epulopiscium fishelsoni, averaging 40 µm in width and 250 µm in length but reaching 80 µm in diameter and up to 600 µm in length (8). The salient point is that this biovolume does not surround an empty vacuole; instead, the internal volume is made up of true cytoplasm. Thus, these cells really are large; they are not just a collection of thin bacteria masquerading as a large cell. Each E. fishelsoni cell has a volume 106 times greater than that of a single E. coli cell, maintains a cytoplasm-to-genome ratio about 20 times greater than that of E. coli, and contains 37,000 to 40,000 genome equivalents (J. Mendell, personal communication). Especially important is that each unit of surface area supports a cytoplasmic volume 200 to 400 times greater than that supported by the surface of P. ubique (Table 3). Though the differences are great, the physics of diffusion must still apply. E. fishelsoni seems to moderate its size disadvantages in three ways: the organism lives in a nutrient-rich environment (the surgeonfish gut), the inner membrane contains many invaginations, and the DNA is located in a narrow band around the inside of this membrane (J. Mendell, personal communication). These features increase nutrient availability by increasing the effective surface-to-volume ratio. Nonetheless, the existence of this behemoth highlights our inability to predict, from physical principles alone, the size, let alone the shape, of individual prokaryotes.
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Variation with growth rate.
In the classic work of Schaechter et al., Salmonella enterica serovar Typhimurium produced cells that were wider when incubated in rich medium than when grown in minimal medium, and slowly growing cells were shorter than those growing more rapidly (287). Similarly, rapidly growing cells of E. coli B/r are wider than slowly growing cells, with cells having a generation time of 22 min being significantly wider (1 µm) than cells having a generation time of 72 min (0.5 µm) (226). However, not all strains respond the same way. For example, E. coli B/r becomes more elongated at higher growth rates, but E. coli B/r H266 becomes more rounded (226). A more permanent effect of growth rate on cell shape is suggested by evolutionary experiments by Lenski and Mongold, who identified a measurable shape change in E. coli during a 10,000-generation experiment (190). The change was simple, i.e., an increase in length and width leading to a doubling of cell volume, but was adaptive and heritable (190), verifying in practice that a slight shape change is correlated with the ability to outgrow competitors.
The upshot of these and other experiments is that bacterial morphology is not set in stone; i.e., the size and shape of an individual cell do not have predetermined, permanent dimensions. Instead, although the overall shape may be constrained (e.g., to be rod-like), a cell's length and width may change in response to growth conditions (228).
Filamentation with nutritional status. Perhaps the most frequent shape change due to nutritional stress is filamentation, triggered by a limitation in the availability of one or more nutrients. For example, in the absence of phosphate, cysteine, or glutathione, Actinomyces israelii grows as branched or filamentous rods, and adding back these compounds returns the cells to a regular rod-like morphology (251). When limited for biotin, Arthrobacter globiformis forms abnormally large, branched rods of variable size (365), as do other isolates when starved for manganese (56, 89). An analogous magnesium deficiency inhibits cell division and produces nonbranching filamentation in Clostridium welchii (355, 356), and in nutrient-poor conditions Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens elongate into long slim cells, unlike the short rods observed in liquid medium (302, 314). The simplest explanation for these responses is that, when the environment demands it, many bacteria can accelerate or delay cell division and septation, thereby creating shorter or longer cells, respectively.
Why do this? First, as noted above, elongating increases a cell's uptake-proficient surface without changing its surface-to-volume ratio appreciably (Fig. 3B). This may be reason enough for cells in suspension. Second, filamentation may benefit cells attached to a surface, not because elongation increases the total surface area but because it increases that specific surface area in direct contact with the solid medium (314). Steinberger et al. calculated that a perfectly spherical coccus contacts a planar solid with 17% of the cell's surface, and a rod twice as long makes contact with 20% of its surface (314). For a rod whose length is 7 times the sphere's diameter the contact surface increases to 23%, but a rod 10 times as long increases its contact area to only 24%, and further elongation has little additional effect (314). Thus, a rod seven times as long as a coccus increases its surface contact by 40%, which should be sufficient to favor rod-shaped cells if surface contact is the principal source of nutrients. Finally, filamentation may allow cells to access nutrients that would otherwise be out of reach for mechanical reasons, by increasing the possibility that part of the filament will contact a nutrient-rich zone and funnel compounds to the rest of the cell's biomass.
Nutritionally deficient streptococci. In 1961, Frenkel and Hirsch isolated a streptococcus that grew with a range of unusual morphologies (80). When grown in nutrient-limiting conditions, these isolates had thickened cell walls and often grew as true filaments instead of as cocci (283). These were first described as "nutritionally variant streptococci" (283) but are now known as "nutritionally deficient streptococci" (NDS) (28, 42). When visualized by electron microscopy, 14 NDS strains were observed to be shape variable, having thickened cell walls and improper septation (29). At first thought to be variants of normal viridans streptococci, the organisms were later assigned to two new Streptococcus species, Streptococcus defectivus and S. adjacens (283), and still later were identified by 16S RNA analysis to be in a new genus altogether, Abiotrophia (159), along with a third new species, Abiotrophia elegans (272). Since their discovery, NDS strains have been isolated from diverse clinical sources (28, 42), even though they are difficult to identify because of their bizarre morphologies, which include rods and filaments with irregularly spaced bulbous swellings (28).
The shape changes of the NDS represent yet another response to nutritional status. The morphological aberrations of NDS can be manipulated by altering the vitamin B6 concentration: lower concentrations induce more rod-like, filamentous, bulging, and aberrant morphologies (42). In fact, most NDS revert to the classical coccoid form when supplied with appropriate nutrients (cysteine, thiols, or vitamin B6) (28, 42). The filamentous cells have incomplete septa (42), perhaps because vitamin B6 is required to convert L-alanine to D-alanine for peptidoglycan synthesis (283). In any case, the NDS represent yet another example of bacteria responding to nutrient deprivation by controlled filamentation.
True to form? The behavior of NDS organisms raises an intriguing possibility. Some bacteria we know as pleomorphic exhibit these morphologies because they are deprived of essential nutrients during culture in vitro, yet they have uniform shapes in the presence of a required nutrient. It may be that some of the shapes with which we are most familiar are artifacts of our culturing methods, in somewhat the same sense that other organisms are said to be "nonculturable." On the other hand, perhaps the ability to adopt aberrant shapes is useful for bacteria in their natural habitats. In any case, nutritionally dependent cell shape variations should provoke us to report the natural, in vivo shapes of the organisms we study and to ask if bacterial shape accommodates itself to a cell's nutritional status or other aspects of its surroundings.
100 to 150 nm in diameter, and the width of the central pore is only 10 to 20 nm (253). Because of this tight squeeze, it is not surprising that the internal channel is mostly free of cytoplasmic proteins (137), which means that the cell surface can be extended substantially with only a miniscule increase in cell volume.
Prosthecae increase the surface area available for nutrient absorption in a nutrient-poor environment because the stalk can collect nutrients and direct them, by diffusion, into the cell body (220, 228, 253). This idea arose from the observation that decreasing phosphate concentrations provoke the growth of longer prosthecae in Caulobacter, Asticcacaulis, Hyphomicrobium, and Rhodomicrobium (254, 255). When grown in limiting phosphate, the stalks of Caulobacter and Rhodomicrobium elongate from their usual length of 1 to 3 µm to as much as 20 µm (33, 61, 97, 289). The response is under direct genetic control, because Caulobacter mutants produce elongated stalks even in the presence of sufficient phosphate (33, 97). These mutations map to the pst genes responsible for high-affinity phosphate transport, which strengthens the link between phosphate uptake and regulation of stalk growth (97). Thus, the longer the stalk, the more easily the cell can access exogenous phosphate, which suggests that the stalk plays a prominent and perhaps specialized role in phosphate uptake (254). Further strengthening this supposition is the behavior of Ancalomicrobium, which adopts several morphological types depending on the prevailing nutritional conditions. When nutrient concentrations are high, the cells are spherical or rod shaped; at intermediate concentrations, the cells are knobby rods; and at low nutrient concentrations, the rod-like cells have multiple protruding filamentous branches (61). Since, unlike Caulobacter, Ancalomicrobium does not use its prosthecae for attachment, these length changes are probably related directly to the need for increased surface area for nutrient transport.
Surprisingly, in light of the surfeit of indirect evidence, specific experimental support for the proposition that prosthecae function in phosphate transport has been hard to come by. The basic problem is to show that the required transporters exist in the stalk and are active. The prosthecae of Asticcacaulis biprosthecum can actively transport all 20 amino acids (323) and contain a glucose uptake system (185, 257), but the accumulated glucose is not metabolized, leaving the usefulness of the transport system in question (257). C. crescentus stalks contain mostly outer membrane and periplasmic nutrient binding proteins but have a deficit of cytoplasmic proteins (137, 346), which is "consistent with the hypothesis that the stalk plays a role in nutrient uptake" (137). These stalks do, in fact, import phosphate-ester into the periplasm and hydrolyze it (346). Calculations indicate that long stalks can import material at a higher rate per unit volume than can filamentous cells of the same length, meaning that stalk formation can supply more nutrients per unit of cell mass (346). So far, these data represent the best experimental support for the idea that stalk elongation enhances nutrient accumulation and does so more efficiently than classical cell filamentation.
Improving nutrient uptake is only one potential function for the stalks of prosthecate bacteria, some of which attach themselves to solid substrates by means of adhesins at the tips of their appendages (33, 253, 255). Immobilized prosthecae may orient cells in a flowing liquid and expose them to bulk nutrients, they may reduce overall buoyancy and orient cells near air-water interfaces, or they may elevate the cell body so that daughter cells are dispersed more readily (see "DISPERSAL" below) (253, 255, 346). An interesting question is whether phosphate limitation is created by the competition for nutrients among neighboring cells in a biofilm. A pack of competing cells might effectively lower the effective concentration of phosphate or other nutrients available to any single cell, triggering prosthecate bacteria to elongate their stalks so that the cells rise above the mass of competing biofilm into a less competitive environment (253, 255, 346). In addition, prosthecae may decrease the settling time of cells in the water column (see "DISPERSAL" below). Thus, prosthecae may enhance a cell's access to nutrients in several ways and can be considered one of the morphological strategies for nutrient acquisition.
The giant sulfur-oxidizing bacteria Thioplaca, Beggiatoa, and Thiomargarita spp. store sulfur as inclusion bodies in a thin layer of cytoplasm surrounding an enormous central vacuole in which they store nitrate (290). To get to these compounds, Beggiatoa and Thioplaca cells form filaments (290). Thioplaca cells adhere to one another in mucus sheaths that are inserted several centimeters into the sediment, and the cells access both nutrients by shuttling up and down (290). Beggiatoa filaments grow only in thin horizontal zones in the sediment where sufficient concentrations of the two compounds overlap (290). A second strategy is exemplified by Thiomargarita, which forms chains of spherical cells, each of which averages 100 to 300 µm in diameter, with some reaching 750 µm (0.75 mm!) (291). The cells are trapped and buried in sediments and are therefore cut off from nitrate but are in contact with sulfide (290). Every so often, after weeks or months, the sediments are resuspended by eruptions of methane or by other means. While resuspended, the cells come into contact with nitrate, which they accumulate in the voluminous central vacuole to tide them over when they inevitably settle back to the sea floor and are reburied (290). In effect, Thiomargarita is a blimp, rising and falling through different strata, collecting and storing electron donors and acceptors against periods of starvation (291). The huge size and balloon-like vacuole of Thiomargarita are morphological adaptations that permit this unique lifestyle.
Other cell shapes may give their owners flexibility in coping with dramatic changes in osmotic pressure. Javor et al. described box-shaped halophilic archaea shaped like irregular rectangles, squares, trapezoids, or triangles and others that are flat, round, or ovoid (144). They argued that "in their natural environment these cells are more likely than most other prokaryotes to experience abrupt large increases in internal osmotic pressure when rain or high tides dilute the salt ponds" and hypothesized that these "flat shapes and relatively soft cell walls allow a large increase in their internal volume with a relatively small change in their cell envelope shape" (144). The idea has not been tested (as far as I know), but the tendency of certain shapes to deform without lysing may represent another morphological adaptation to environments dominated by diffusion and osmotic pressure.
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Perhaps the most important reason to maintain a uniform morphology is so that chromosomes and cytoplasmic material can be partitioned equally between daughter cells at division (Fig. 4A) (69). The chromosome is most important, but the allocation of near-equal amounts of cytoplasm is also vital. Although the intrinsic variability of distribution ensures that individual daughter cells will never be perfectly equivalent to one another, extrinsic factors also play a role, and the cell can minimize some of these (279, 310). Towards this end, a regular shape would seem to be the best way to ensure that an equal amount of "stuff" is allocated to each daughter, because a symmetrical cell can be halved accurately by mechanisms that measure length or volume (69, 124). In an irregular cell, misplaced septation might leave one cell with both chromosomes or with more than its fair share of other components. In this regard, the actual shape itself would not be important; instead, segregation-driven selection would favor a cell with bilateral twofold symmetry. This requirement for equitable segregation may be the strongest selective pressure for shape uniformity.
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In complementary work, the anucleate cell screen was used to isolate the antibacterial compound A22, which forces rod-shaped cells to grow as spheres (139). When so treated, E. coli produces a higher percentage of anucleate cells (2.4%) than is present in wild-type rods (0.03%), typical of a segregation defect (139). The mechanism of action of A22 is not via PBP 2 inhibition (139) but by inhibition of the MreB protein (94). In both A22-treated cells and PBP 2 mutants, anucleate cells are smaller than normal and are probably created by asymmetric cell division (139). Consistent with this interpretation, chromosome segregation is impaired in mreB mutants of E. coli (175). Whereas wild-type cells faithfully segregate equal numbers of chromosomes to each rod-shaped daughter, MreB mutants are spheroidal and partition their chromosomes randomly so that some newborn cells contain no chromosomes at all (175). Thus, cell shape does seem to affect symmetrical cell division and chromosomal segregation (139).
There is a caveat to interpreting the above results. Although a uniform shape appears to be important for chromosomal segregation, MreB may play a more direct role in segregation beyond its role in maintaining a cell's rod shape. Expressing certain missense mutants of MreB in E. coli disturbs chromosomal segregation even though the cells retain their rod shapes, leading Kruse et al. to conclude that "it is not the shape of the spherical cells per se that causes the chromosome segregation defect" (175). Likewise, PBP 2 mutants may perturb segregation by affecting MreB activity. It may be impossible to disentangle these two considerations (shape change versus impaired partitioning), because the two may be intimately intertwined. Even so, cell shape is clearly an important contributor, either directly or indirectly, in determining proper segregation.
A good example of this principle is E. coli, in which concentrations of the requisite division proteins are carefully balanced for its normal rod shape. In almost all eubacteria cell division is regulated by the FtsZ protein, which polymerizes to form a physical ring around the girth of a cell at the site where septation will occur (Fig. 4B) (69, 201). In E. coli, successful cell division depends on a constant and critical concentration of FtsZ combined with the proper proportions of Z-ring-stabilizing and -destabilizing proteins (275, 282). Significantly, small changes in the concentrations of FtsZ or other essential division proteins disrupt cell growth (see references cited in reference 57). Thus, division is inhibited if FtsZ is underproduced, extra divisions occur if the protein is overproduced (193, 353), and no division occurs if FtsZ levels are adequate but the FtsZ/FtsA ratio is incorrect (57). These facts prompted Dewar and Dorazi to conclude that "even small fluctuations in the levels of essential cell division proteins can severely disrupt cell growth" (57).
Several E. coli mutants provide examples of how shape may affect this aspect of cell division. Mutants lacking some of the penicillin binding proteins deviate only slightly from wild-type shape during growth, but they eventually stop dividing and continue to grow in length and girth until they lyse (230, 338; unpublished results). This is consistent with an inability to produce enough septation proteins to accommodate their increased cell diameter. Additional verification is provided by E. coli strains lacking PBP 2, which grow as ever-enlarging spheres (342). In these balloon-like cells, septal Z rings either never form or, if they begin to form, do not proceed completely around the cell circumference (342). Such mutants may be rescued by overproducing the proteins FtsA, FtsZ, and FtsQ (342). The easiest explanation is that the problems created by a larger cell circumference are overcome by expressing the septal ring proteins in sufficient numbers so they can polymerize to create a complete, septation-proficient Z ring (342).
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5-nm range over which the secondary Gibbs free energy minimum allows reversible binding (269, 335, 336) (Fig. 5). Without other aid, bacteria cannot cross this barrier to reach the primary minimum that would give the strongest binding (269).
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Although individual planktonic cells cannot influence the characteristics of the surface to which they attach, they can optimize their shape to combat shear forces in two ways: by decreasing the magnitude of shear and by increasing the number of physical contacts with the external surface. The strength of the parallel component of surface shear determines if a cell remains attached or is removed, and the magnitude of this parallel force is proportional to the square of the radius of the particle (261, 336). Larger particles are affected more strongly than are smaller particles because more surface area is exposed to direct flow as diameter increases (Fig. 6B) (261, 336). A coccus, because of its spherical symmetry, exposes the same surface area to oncoming fluid flow no matter how the cell is attached. However, a rod-shaped cell can orient itself so that it is broadside to the flow or so that only the face of one pole is facing the onrushing current. Therefore, for cells of equal mass, rod-shaped cells should be able to withstand greater shear forces if they align themselves lengthwise to the direction of current. This exposes a smaller circular surface area to liquid flow while allowing adherence along the length of the cell. Few experiments address this subject directly in bacteria, but the results are consistent with these considerations. When grown under high shear force, E. coli elongates without a significant change in its diameter and is more likely to grow in chains (66). Both responses increase the surface area available for attachment while keeping constant the cross-sectional area that is susceptible to shear forces. A different response is exhibited by B. subtilis. In a high-shear environment cells of this organism are smaller by about half in each dimension, which reduces total shear because the organism's cross-sectional area decreases to one-fourth its original value but its length decreases by only half, so that its attachment-to-shear ratio doubles (284). Finally, cells may enhance the number of contacts by growing as filaments or in chains, intertwining with surface elements to resist detachment. Overall, therefore, rods and filamentous cells should have an advantage in environments with sizeable shear. Of course, as with every other physiological trade-off, these arguments presuppose that all other considerations are equal, which may not be the case. There are other options for improving attachment, and shape may not always be the dominant factor. For example, in Acinetobacter the coccal phase attaches to surfaces more firmly than does the rod phase (142). The point here is that shape represents an additional tool in the cell's arsenal of attachment strategies.
Another possible stabilizing strategy would be to interact with many other cells while attaching. Additional points of horizontal attachment to cells on all sides would increase the number of attachment points and at the same time decrease the effect of shear stress on individual bacteria. An extreme version of this strategy is practiced by leaf cells in Arabidopsis (82). These polymorphic cells interdigitate with one another exactly like puzzle pieces to withstand being dislodged by wind- and water-derived shear forces (82). The crowding effect should be similar for bacterial cells and may help explain the prevalence of environmental biofilms (see below).
The genus Simonsiella provides a fascinating example of a morphological adaptation for surface attachment to a familiar niche. Simonsiella spp. are filamentous, aerobic bacteria that are part of the natural oral flora of many mammals (123). Eight or more daughter cells are attached to one another to form short filaments, but the dimensions of each cell are unusual. When measured with respect to length of the filament, the cells are short and flat (0.5 to 1.3 µm) but quite wide (1.9 to 6.4 µm) (123). Each Simonsiella cell is slightly curved over its width, and the concatenation of these curved cells creates a ribbon-shaped filament that is bent so that one side is concave and the other side convex (Fig. 6C) (123). This morphology seems to be functional and important, because Simonsiella attaches to oral epithelial cells only with the ventral, concave side of the filament (123). Here we see what appears to be a notable surface-maximizing strategy: a large bacterial surface molde
