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Small but Mighty: Cell Size and Bacteria

Petra Anne Levin1 and Esther R. Angert2

1Department of Biology, Washington University, St. Louis, Missouri 63130 2Department of Microbiology, Cornell University, Ithaca, New York 14853 Correspondence: [email protected]; [email protected]

Our view of bacteria is overwhelmingly shaped by their diminutive nature. The most ancient of organisms, their very presence was not appreciated until the 17th century with the inven- tion of the microscope. Initially, viewed as “bags of enzymes,” recent advances in imaging, molecular phylogeny,and, most recently,genomics have revealed incredible diversity within this previously invisible realm of life. Here, we review the impact of size on bacterial evo- lution, physiology, and morphogenesis.

umanity has always experienced the im- bolic support. Although less than 1% of bacteria Hpact of microorganisms, most obviously canbeculturedreadily inthelaboratory(Amann through their ability to cause devastating dis- et al. 1995), the biochemical versatility among ease. For the vast majority of human history, these tiny creatures exceeds that of the plants, we were unaware of their presence, much less animals, and fungi combined (Pace 1997). the fundamental microbial processes to which Anton van Leeuwenhoek’s illustrations in a we owe our existence: from the production of letter to the Royal Society of London in the late energy by our ancient bacterial endosymbionts 17th century provide one of the earliest records (the mitochondria) to the generation of oxygen of bacterial cell form (Dobell 1960). Viewed in our atmosphere. Despite their astounding through a single lens, Leeuwenhoek pioneered global abundance (1030 cells) and their sub- studies of the human microbiome, describing stantial contribution to the total biomass of motile bacilli, cocci, and spirochetes he found planet earth (Whitman et al. 1998; Kallmeyer in scrapings taken from between his teeth (and et al. 2012), our inability to see these tiny life the teeth of others). This triumph was made forms shrouded their nearly limitless diversity possible by incomparable curiosity, lens con- in mystery. It was not until the 17th century, struction, and exceptional lighting. The simple with the careful observations and reports of An- cellular structure and glassy nature of most un- ton van Leeuwenhoek, that we became aware of stained bacteria viewed with a light microscope this previously invisible world on and around generated little interest in bacterial cell biology us. Today, we know that there are more bacteria with the exception of objects of unusual con- living in our intestinal tract than stars in the trast, such as endospores described by Robert Milky Way galaxy (and that they far outnumber Koch and the wonderfully colorful and large all the people who have ever lived). We also cyanobacteria. The bacterial nature of the latter know now that we thrive because of their meta- was itself only appreciated late in the 20th cen-

Editors: Rebecca Heald, Iswar K. Hariharan, and David B. Wake Additional Perspectives on Size Control in Biology: From Organelles to Organisms available at www.cshperspectives.org Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a019216 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019216

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P.A. Levin and E.R. Angert

tury (Oren 2004). For the most part, bacteria or 0.22 mL). Thiomargarita is slightly larger were viewed as primitive “bags of enzymes” un- than a Drosophila eye (Schulz et al. 1999) and til the 1990s, when the complexity of bacterial big enough to be seen by the human eye (Fig. 1). subcellular structure and regulators of cell re- A closer look at Thiomargarita reveals a central- production finally began to emerge. Tools and ly located fluid-filled vacuole, which takes up reagents developed for eukaryotic cell biol- 98% of the cell volume and is a nitrate reser- ogy (e.g., stains for DNA, membranes, and fluo- voir used to fuel sulfide oxidation. Even when rescent protein tags), once applied to bacterial accounting for the intracellular vacuole, a large cells, revealed astonishing insights including the Thiomargarita cell has a tremendous biovolume specific and even dynamic localization patterns to support (4.4 106 fL). Epulopiscium spp., of proteins, and the accuracy of chromosome intestinal symbionts of certain marine surgeon- organization. fish, are the largest known heterotrophic bacte- ria. These cigar-shaped cells are up to 600 mm 80 mm with an active cytoplasmic volume of BACTERIAL SIZE RANGE 2 106 mm3 or 0.02 mL (Angert et al. 1993). Bacillus subtilis, Staphylococcus aureus, Escheri- Unlike Thiomargarita, Epulopiscium cells con- chia coli, and Caulobacter crescentus, the prima- tain no storage vacuoles or other inert inclu- ry models for bacterial cell biology, are more sions (Fig. 1). The difference in size between or less typical in size, with individual cell vol- Candidatus Actinomarina minuta and these gi- umes between 0.4–3 mm3 (or 0.4–3.0 femto- ants is equivalent to the difference between a liters; femtoliter or fL is equal to 10215 L). Free- mouse and the Empire State Building (E. coli living marine ultramicrobacteria, appropriately could be represented by a small skunk or a rabbit named Candidatus Actinomarina minuta, have on this scale). We refer the reader to Niklas an average cell volume 1% that of E. coli (2015), wherein he discusses how cell features (0.013 mm3, range 0.6 1022 to 2.4 and geometry factor into considerations of cell 1022 fL). At the other end of the spectrum, size. the marine sediment–dwelling Thiomargarita What limits bacterial cell size? The smallest namibiensis, the “Sulfur pearl of Namibia,” is cells need enough volume to accommodate ad- a spherical organism with a volume eight or- equate genetic resources to support the cell’s ders of magnitude more than that of E. coli lifestyle (Koch 1996). The cell must also contain (750 mm in diameter, volume 2.2 108 fL the basic machinery required to express those

Figure 1. Giant bacteria. On the left is a chain of Thiomargarita namibiensis cells. In this bright-field image, sulfur granules can be seen in the cytoplasm. The panel on the right shows an exceptionally large Epulopiscium cell with two large internal offspring. Scale bars, 100 mm.

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Cell Size and Bacteria

genes as well as housekeeping proteins and port becomes numbingly unreliable. A small biochemicals to maintain its metabolism and molecule like oxygen at room temperature cellular reproduction. Genomic and metabol- would typically take about an hour to diffuse ic streamlining is seen in obligate intracellular 1 mm (Schulz and Jorgensen 2001). Similarly, symbionts, pathogens, and organelles that have the acquisition of nutrients relies on diffusion given up metabolic capabilities because those and capture of molecules at the surface of a cell. needs are supplied by the host (McCutcheon Consequently, free-living cells tend to be small, and Moran 2012; Wernegreen 2012). The loss with a large surface area relative to their cyto- of genes for sensing environmental change and plasmic volume, so that capture of nutrients, at responding to those contingencies can allow for low concentrations, can support the cell’s met- substantial genome reduction but not always a abolic needs. Cell form and function have been corresponding reduction in cell size. subjected to these constraints. For comparison, The structure and function of all large cells E. coli has a surface-area-to-volume ratio of appear bounded by the limits of diffusion 3.7 mm2 to 1 mm3, whereas the largest Thio- (Schulz and Jorgensen 2001). Encounters with margarita cell has a surface-to-volume ratio of nutrients, elimination of waste, and the timely 8.2 1023 mm2 to 1 mm3. Clearly, these large movement of biomolecules within the cell to bacteria are bending the rules. support metabolic needs all impact the ability of a large bacterium to survive in its environ- How Epulopisicum Has Overcome ment. The compartmentalization of cellular the Issue of Diffusion functions, the motor protein-facilitated traf- ficking over a complex cytoskeletal network, Most large bacteria either maintain a high sur- the expansion of genomic resources, and the face-to-volume ratio by being long and thin acquisition of endosymbionts that became en- like Spirochaeta plicatilis (a 250-mm-long, ergy-generating organelles have all been credit- 0.75-mm-diameter corkscrew-shaped bacte- ed for the advancement of the size and com- rium) or adopt a morphology in which no plexity of eukaryotic cells (Angert 2012). part of the cytoplasm is much more than a mi- cron away from the external environment. Thi- omargarita is an example of the latter, maintain- THE PROBLEM OF DIFFUSION ing a thin layer of cytoplasm surrounding a large The identification of giant bacteria required a fluid-filled vacuole. Epulopiscium spp. are the reexamination of the long-held beliefs about exception to this rule. maximum bacterial cell size. For all cells, growth Although their elongated form undoubted- and reproduction is limited by the speed of ly helps increase their surface-area-to-volume chemical communication and the availability ratio (Koch 1996), the largest Epulopiscium has of nutrients to fuel metabolism. Diffusion is a ratio of 0.6 mm2 to 1 mm3, 1/6 that of E. coli. the random, three-dimensional movement of Despite this difference, Epulopiscium maintains a molecule. For movement of molecules inside a high metabolic rate. Epulopiscium spp. use a a cell, it is the most efficient means of transport number of other structural modifications to ad- over a short distance of a few microns. Even a vance cell size. Epulopiscium cells have a highly large protein can reliably traverse this distance invaginated cell membrane that may compen- in the cytoplasm in much less than a second. sate for the small apparent surface area of the Thus, a biomolecule synthesized or entering a cell envelope (Angert 2006). Large Epulopiscium typical bacterium has a high probability that it spp. are covered with flagella and are highly will reach its site of activity almost instantly. But motile, which helps the cell maintain its posi- considering a small distance by our scale, across tion in the gut in proximity to a plentiful sup- a gap of a millimeter—approximately the length ply of nutrients. The coordinated rotation of of an Epulopiscium cell and a fraction of the cir- flagella helps stir the surrounding medium, fa- cumference of Thiomargarita—diffusive trans- cilitating movement of molecules across the cell

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P.A. Levin and E.R. Angert

surface, refreshing the immediate environment. In this way, Epulopiscium is able to reduce reli- ance on diffusion of molecules down a concen- tration gradient. Transporters located on the infolded cell membrane likely facilitate the cap- ture of nutrients. Epulopiscium cells show extreme polyploidy throughout their life cycle, an adaptation that may also contribute to their ability to attain such a large mass (Ward et al. 2009). Polyploidy is a common feature of large cells. Examples from eukaryotes include endoreduplication in Drosophila salivary cells and, as discussed in Figure 2. DNA in Epulopiscium sp. type B. This clus- Czesnick and Lenhard (2015), plant cells with ter of Epulopiscium cells is stained with the DNA dye specialized functions including trichomes, the DAPI. Each of the two large mother cells at the center fine hairlike appendages visible on the leaves of this field contains two large offspring. DNA is and stems of certain species. Gillooly and col- located at the periphery of the cytoplasm in mother cells and offspring. At this late stage of development, leagues (2015) discuss a potential role for endo- the mother-cell DNA is difficult to see because much replicationandincreasesinnuclearDNAcontent of it has degraded. Offspring cells contain brightly as a determinant of size in human cells. Large stained polar structures; these are the start of “grand- bacteria, however, have taken polyploidy to an daughter cells.” Scale bar, 100 mm. extreme level (Angert 2012). Although the Epu- lopiscium chromosome is fairly typical in size (4 Mb), each cell contains tens of thousands Reproduction begins with bipolar division to hundreds of thousands of copies (Mendell et of the mother cell. The polar cells are fully en- al. 2008). In contrast to most bacteria, in which gulfed and these offspring then grow inside a thehighlyorganized chromosome or“nucleoid” membrane-bound compartment in the moth- occupies almost the entire cytoplasm, Epulopis- er-cell cytoplasm, until they completely fill the ciumnucleoids arelocated atthe peripheryofthe mother cell. At a late stage of development, the cytoplasm (Fig. 2). This is likely an important mother cell appears to undergo a form of pro- organizational feature that allows Epulopiscium grammed cell death, a process that likely con- to respond immediately to environmental stim- serves the biochemical resources accumulated uli. It may also accommodate the growth of in- during growth (Ward et al. 2009). In the final ternal offspring cells (described below). stages, offspring emerge through a split in the mother cell envelope. Despite developmental synchrony, inhabitants of a single population REPRODUCTION AMONG THE GIANTS vary in volume by as much as a factor of five Large bacteria show diverse reproductive strat- (Mendell et al. 2008). egies, some of which may help to maximize Because of their size, unusual methods of their reproductive potential as well as the pro- reproduction and the difficulty in determining duction and release of large offspring. All of the evolutionary relationships among microbes largest Epulopiscium spp. reproduce once daily, based on phenotypic characteristics, Epulopis- forming two or more intracellular offspring. cium was originally classified as a novel protist. Surprisingly, internal offspring initiation and Molecular phylogeny corrected this oversight, development follows a predictable daily cycle grouping Epulopiscium spp. with the Clostridia and any given population inside a host fish is (Angert et al. 1993), endospore-forming Gram- well synchronized with respect to development. positive organisms (of the Firmicutes phylum) Offspring growth occurs during the day and that include the intestinal pathogen Clostridium coincides with times when the host is feeding. difficile, Clostridium perfingens, the cause of

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Cell Size and Bacteria

gangrene, and the foodborne pathogen Clos- ploid microbes to abound. Subsequent modifi- tridium botulinum from which we acquire cations to cellular architecture may then accom- Botox. Internal offspring production in Epulo- modate further expansion in size. piscium spp. arose from endospore formation (Miller et al. 2012). Adapting endospore forma- CELL-SIZE HOMEOSTASIS tion as a mode of reproduction appears to have happened several times in the spore-forming Under steady-state conditions, isogenic popu- Firmicutes (Angert 2005). lations of bacteria maintain cell size within sur- Despite the unusual mode of reproduction prisingly narrow parameters. Although a histo- shown by Epulopiscium, other giant bacteria use gram of E. coli or B. subtilis cells sampled in more “pedestrian” strategies. Thiomargarita na- mid-exponential phase is biased toward the mibiensis in particular undergoes binary fission left, a consequence of the greater number of in a single plane, arguably the most common smaller newborn cells, only in rare cases are mode of reproduction among the bacteria. Thi- the largest organisms more than twice the size omargarita cells do not separate, however, re- of the smallest (Fig. 3). maining instead as chains of cells housed in a Like eukaryotes, bacteria initiate and termi- common mucus matrix (Fig. 1). At the same nate one round of DNA replication for each time, closely related organisms reproduce using division event. In contrast to eukaryotes, how- other strategies, which include budding of off- ever, DNA replication, chromosome segrega- spring from a sessile mother cell and the pro- tion, and assembly of the division machinery duction of multiple internal offspring (Bailey are not discrete events, instead overlapping et al. 2011). How reproductive form affects fit- one another to a greater or lesser degree de- ness of extremely large bacteria remains to be pending on growth rate (Fig. 4). determined. Importantly, at fast growth rates supported by nutrient-rich conditions, bacteria are able to maintain interdivision periods less than half the THE IMPACT OF ENVIRONMENT length of time required for replication of their ON SPECIES SIZE chromosomal DNA. For example, E. coli can Large bacteria abound in certain environments double in mass and divide in as few as 20 min; in which nutrients are consistently available and however, even under ideal conditions, replica- concentrations are high. We believe that these tion of the entire 4-Mb E. coli chromosome associations give us some clues as to conditions takes 60 min. Rapidly growing cells resolve that can help organisms break free from diffu- this apparent conflict by constantly synthesizing sion limitations on cell size. Giant spirochetes DNA and by initiating new rounds of replica- can be found in nutrient-rich sediments and in tion before completion of the old one. Such cells some intestinal systems, such as the hindgut of may have 4, 8, or even 16 ongoing replication termites. Large rod-shaped cells have been seen forks (Yoshikawa et al. 1964; Cooper and Helm- in intestinal tracts of a number of herbivores. stetter 1968). Although initiation is restricted to Sulfur-oxidizing bacterial cells like Thiomarga- once per division cycle, only one round of rep- rita abound in marine sediments. They are near- lication needs to be completed before division. ly ubiquitouslydistributed around the globeand Within a single division cycle, initiation and carry with them minerals to fuel their respira- termination events, thus, do not necessarily cor- tion. An abundant supplyof energy is acommon respond to the same replication fork. theme in all of these systems and may be a uni- fying feature. Polyploidy is widespread in bacte- Early Work on Cell-Size Homeostasis ria but is expensive to sustain. In nutrient-rich environments or when energy is practically lim- In 1968, William Donachie proposed that cell- itless, selection for scaling down to a single copy size homeostasis was tied to cell-cycle progres- of the genome may be relaxed and allow poly- sion (Donachie 1968). Combining data on Sal-

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P.A. Levin and E.R. Angert

40 35 Wild type 30 25 UDP-glucose 20 mutant 15 10 Fraction of cells (%) Fraction 5 0 <3 3–4 4–5 5–6 6–7 >7 Cell length (µm)

Figure 3. Histogram of Escherichia coli cell size during growth in nutrient-rich medium. The size of wild-type (black) cells is restricted within a narrow twofold range. The graph is biased slightly toward the left owing to the higher number of newborn cells, the consequence of binary fission. UDP-glucose biosynthesis mutants ( pgm::kan) (hashed lines) are smaller than wild-type on average; however, the size of these cells is similarly constrained within a twofold range. (Data courtesy of Norbert Hill.)

monella from the Maaløe laboratory with E. coli Subsequent work implicated the highly con- data from Cooper and Helmstetter (Schaechter served AAAþ ATPase DnaA, a dose-dependent et al. 1958; Cooper and Helmstetter 1968), Do- activator of DNA replication, in E. coli cell-size nachie inferred that bacterial cells initiate DNA control (Løbner-Olesen et al. 1989). Reducing replication on achievement of a specific mass. levels of DnaA delays the onset of DNA replica- Donachie suggested that the growth-dependent tion and increases cell size, whereas increasing accumulation of a positive acting factor—or in- DnaA levels results in premature initiation and hibition of a negative acting factor—would be reduced cell size. sufficient to coordinate the onset of the cell cy- On the face of it, tying replication initiation cle with cell size. to cell size is an ideal way to correct any defects

Cytokinesis Replication Initiation

Replication Termination

DNA replication Outer circle represents assembly and maturation Growth of the cytokinetic ring

Figure 4. Circular depiction of the cell cycle in rapidly growing (mass doubling time ,60 min) bacterial cells. Note that DNA replication and growth are more or less constant—a consequence of multifork replication. A new round of replication is initiated only once per cycle. At least one round of replication must be terminated before division. Although the division machinery is assembled well before cytokinesis (the FtsZ ring is extant for 90% of mass doubling time in cells cultured in very rich medium), constriction itself takes only a few minutes.

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Cell Size and Bacteria

in cell size early in the cell cycle. Short cells Structural data suggests that FtsZ is an evolu- will have to grow longer before they accumulate tionary precursor of tubulin, although the two sufficient DnaA to initiate DNA replication, proteins share limited sequence similarity. Ap- whereas longer cells will initiate earlier (and proximately one-third of FtsZ in the cell is in the thus, at smaller sizes) because critical levels of FtsZ ring at a given time with subunit turnover DnaA are reached earlier in the cell cycle. The rates on the order of seconds. (For reviews of observation that in Saccharomyces cerevisiae FtsZ and bacterial cell division, see Erickson achievement of a specific size is required before et al. 2010 and Lutkenhaus et al. 2012.) unbudded cells can pass through START at the Altering FtsZ levels has a direct impact on G1/S transition suggests that this type of control cell size, making the cytoskeletal protein an ide- may be broadly applicable (Pringle and Hart- al target for factors governing cell-size homeo- well 1981). (Intriguingly, the dnaA gene of Epu- stasis. Depleting FtsZ does not effect cell growth lopiscium sp. type B encodes an exceptionally or DNA replication, at least initially, and results long mononucleotide tract, a hot spot for poly- in a rapid increase in length. Even modest re- merase slippage and potential expression of a ductions (20%) in intracellular FtsZ concen- truncated and nonfunctional peptide [Mendell tration lead to a large increase (.50%) in cell et al. 2008]. Although the specific contribution length under steady-state conditions (Palacios of this unusual gene structure to DnaA regula- et al. 1996). tion is not known, it is possible that it plays a If one imagines the cytokinetic ring as a role in increasing Epulopiscium’s cytoplasmic brick building, then it is easy to see how changes volume-to-DNA ratio by reducing DnaA levels in the supply of bricks would influence cell size. and delaying replication.) When FtsZ concentration is high, bricks are Despite the appeal of this model, recent readily available, reducing transport time and data suggests DnaA accumulation is unlikely facilitating assembly of a stable and functional to be the sole or even primary determinant Z-ring. However, reductions in available FtsZ of bacterial cell size. Small B. subtilis mutants slow the transport of bricks to the construction show wild-type cell-cycle profiles with regard site, delaying assembly of the division machin- to initiation and DNA replication, suggesting ery and with it cytokinesis. As cells continue to that initiation is independent of cell size in increase in size at normal rates, delays in assem- this Gram-positive model organism (Hill et al. bly of the cytokinetic ring translate directly into 2012). Even in E. coli, in which initiation is high- increases in cell size. ly sensitive to modest changes in the amount of Because FtsZ levels are proportional to cell active DnaA (Løbner-Olesen et al. 1989), regu- size in both E. coli and B. subtilis, regardless lating mass at initiation is only part of the story. of growth rate (Weart and Levin 2003), stochas- Mutations that result in early or delayed initia- tic variations in cell size should be largely mit- tion appear to adjust their rate of DNA synthesis igated by FtsZ availability. Short cells will re- to compensate for changes in initiation time quire longer periods to accumulate sufficient (Hill et al. 2012). Together, these data suggest FtsZ to divide—increasing their size at division. the presence of a homeostatic and DnaA-inde- Conversely, longer cells will take less time to pendent mechanism to maintain normal inter- accumulate enough FtsZ to support assembly division periods. of the division machinery and consequently show shorter interdivision periods reducing daughter-cell length. Cell Division and Cell-Size Control Just as DnaA accumulation alone is in- In most bacteria, division is initiated by as- sufficient to account for cell-size homeostasis, sembly of the essential GTPase FtsZ into a FtsZ accumulation alone is insufficient to trig- ring-like structure at the future division site. ger division. Although twofold overexpression The “Z-ring” serves as a framework for assem- of FtsZ reduces cell size by 10% in both in E. bly of the rest of the cell-division machinery. coli and B. subtilis, it does not significantly im-

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P.A. Levin and E.R. Angert

pact the timing of division (Ward and Lutken- as twofold depending on growth rate haus 1985; Weart and Levin 2003; Hill et al. (Schaechter et al. 1958). Cells cultured in nutri- 2012). The inability of excess FtsZ to trigger ent-rich medium with doubling times of division suggests the presence of as-yet uniden- 20 min were more than twice the size of their tified factors required to license FtsZ assembly counterpartsculturedin nutrient-poor medium at the nascent division site. How these factors with mass doubling times well over an hour. Sig- are integrated with cell-cycle progression re- nificantly, the relationship between growth rate mains an open question. It is also worth noting and size held true regardless of whether growth that FtsZ is one of many components of the was slowed by limiting carbon, phosphate, or cytokinetic machinery, some of which are also nitrogen suggested that doubling time—rather rate limiting for division and, thus, potential than the specific nutrient content of the medi- candidates for the homeostatic regulators of um—was the primary determinant of cell size. cell size (Lutkenhaus et al. 2012). However, more recent work suggests that bacte- Although a model in which cell-size homeo- rial size is a complex phenomenon, dependent stasis is dependent on accumulation of a rate- on a precisely orchestrated set of regulatory cir- limiting cell-cycle component is consistent cuits, each responsive to a different and poten- with what we know about division in E. coli, tially overlapping nutrient-dependent signals. B. subtilis, and Caulobacter, it is harder to see In E. coli and B. subtilis, UDP-glucose, a how it would apply to organisms that divide by nucleotide sugar synthesized in two steps from budding or tip growth as well as those that show glucose-6, serves as an intracellular signal for nonlinear growth patterns. Individual E. coli carbon availability and growth rate. Defects cells elongate at a more or less constant rate un- in either the phosphoglucomutase required der steady-state conditions (Wang et al. 2012). for interconversion of Glc-6 and Glc-1 and the In contrast, Mycobacterium smegmatis tip elon- pyrophosphorylase responsible for synthesizing gation occurs at different rates depending on UDP-glc from Glc1 reduce E. coli and B. subtilis whether the cell inherited an old pole that is cell size by as much as 30% under nutrient- primed for growth, or a new pole that is not rich conditions (Weart et al. 2007; Hill et al. (Aldridge et al. 2012). In cases involving growth 2013). Consistent with a growth-rate-depen- asymmetry in daughter cells, other mechanisms dent mechanism, loss of UDP-glc synthesis has must be at play. The mechanisms governing little impact on size in cells cultured in nutrient- cell-size homeostasis are equally mysterious poor medium. Its proximity to central carbon for those organisms that reproduce by budding, metabolism makes UDP-glc an ideal signal for such as Hyphomicrobium, Planctomyces spp., carbon availability. etc.; colonial organisms that adjust ploidy levels UDP-glucose levels are sensed and transmit- and cell length during developmental transi- ted to the division machinery by two, unrelated tions, such as Streptomycetes; bacteria that un- glucosyltransferases, OpgH and UgtP, in E. coli couple periods of DNA replication and growth and B. subtilis, respectively. Although UDP-glu- from rounds of cytokinesis as in some cyano- cose stimulates interactions among OpgH, bacteria and Bdellovibrio; and, finally, those that UgtP, and FtsZ, the molecular mechanisms reproduce by internal daughter-cell production, by which they function are highly divergent. such as Epulopiscium (Angert 2005). OpgH is an integral membrane protein. Genetic and biochemical data suggest that UDP-glc binding drives a conformational change that re- NUTRIENT-DEPENDENT REGULATION veals a binding site for FtsZ. By sequestering OF CELL SIZE FtsZ monomers, OpgH effectively reduces the Nutrient availability is the primary determinant amount of FtsZ available for assembly into the of cell size in many bacteria. In a classic paper, cytokinetic ring. UgtP, on the other hand, is Schaechter, Maaløe, and Kjeldgaard deter- only transiently associated with the membrane. mined that Salmonella cell size varies as much UgtP interacts with itself or FtsZ in what ap-

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Cell Size and Bacteria

pears to be a mutually exclusive manner. UgtP At the same time, carbon availability is un- homo-oligomerization is favored in the absence likely to be the sole determinant of cell size. As of UDP-glucose, whereas increases in intracel- Schaechter et al. showed in their seminal 1958 lular UDP-glucose levels during growth in nu- paper, limiting not only carbon, but also other trient-rich medium promote interaction with nutrients including nitrogen and phosphate, FtsZ (Chien et al. 2013). UgtP levels are high leads to reductions in both growth rate and cell during growth in nutrient-rich medium, but mass (Schaechter et al. 1958). Moreover, rapidly low in nutrient-poor medium, further reliev- growing cells defective in UDP-glucose bio- ing division inhibition during slow growth synthesis are still significantly larger than their (Weart et al. 2007). UgtP inhibits FtsZ assembly slow-growing counterparts (Weart et al. 2007; but has no impact on GTP hydrolysis, suggest- Hill et al. 2013). These observations strongly ing that it may have an activity similar to mi- suggest the presence of additional intracellular crotubule severing proteins like Katanin (Chien signaling molecules and cognate sensors, some et al. 2013). of which are likely coupled to nitrogen and It is striking that two such evolutionarily phosphate utilization, that also contribute to divergent organisms (E. coli and B. subtilis are growth rate-dependent increases in cell size. more distantly related than Homo sapiens and Precisely why cells increase size in response S. cerevisiae) use UDP-glucose and unrelated to nutrient-dependent increases in growth rate glucosyltransferases to help coordinate cell size is not known. However, analysis of FtsZ ring with growth rate and nutrient availability. formation and chromosome segregation in OpgH and UgtP are both “moonlighting” pro- wild-type and short mutant strains suggests teins with roles in cell-envelope biogenesis. that increases in cell size during rapid growth OpgH takes part in the synthesis of the osmo- help ensure that there is sufficient room to ac- regulated periplasmic glucans (OPGs), large commodate the additional DNA generated by glucans found in between the inner and outer multifork replication. Both E. coli and B. subtilis membranes of Gram-negative bacteria, whereas maintain a constant cell-mass-to-DNA ratio re- UgtP synthesizes the Di-Glc-diacylglycerol an- gardless of growth rate (Sargent 1975; Donachie chor for lipoteichoic acid, a major anionic com- and Begg 1989; Sharpe et al. 1998). This appears ponent of the Gram-positive cell wall (Lazarevic true for giant bacteria as well (Mendell et al. et al. 2005; Lequette et al. 2008). Both the OPGs 2008). Because chromosome segregation and and LTAsare thought to play a role in the cellular division are not coupled as they are in eukary- response to changes in osmolarity, a stress re- otes, defects in bacterial cell size lead to an in- sponse that leads to a temporary arrest of cell crease in the frequency of FtsZ rings and divi- division. Synthesis of OPG or LTAis the primary sion septa over unsegregated nucleoids under source of diacylglycerol (DAG) in E. coli and conditions supporting rapid growth (Weart B. subtilis (Zhang and Rock 2008). Although et al. 2007; Hill et al. 2013). (Viability is pre- diacylglycerol is a well-studied secondary mes- served in these cases by DNA transferases that senger in eukaryotes, it has not yet been impli- pump chromosomal DNA away from the invag- cated in signaling in bacteria. One possibility inating septum.) is that DAG accumulation serves as a proxy for cell-envelope biogenesis, transmitting infor- CONCLUDING THOUGHTS mation about actual growth rate (rather than nutrient availability) to the division machinery. As with our appreciation of the vital and diverse Intriguingly, FabH, an enzyme responsible for roles that bacteria play in the earth’s biogeo- catalyzing an early step in fatty acid biosynthe- chemical cycles, we are only beginning to un- sis, has also been implicated in the nutrient- derstand the molecular and physical forces that dependent control of cell size; although, wheth- govern bacterial size. Cataloging the diverse er, FabH plays a direct or indirect role has yet to nature of bacterial life—both in physical and be established (Yao et al. 2012). metabolic terms—and characterizing the more

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P.A. Levin and E.R. Angert

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Cell Size and Bacteria

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Small but Mighty: Cell Size and Bacteria

Petra Anne Levin and Esther R. Angert

Cold Spring Harb Perspect Biol 2015; doi: 10.1101/cshperspect.a019216 originally published online June 8, 2015

Subject Collection Size Control in Biology: From Organelles to Organisms

Cell-Size Control Mechanical Forces and Growth in Animal Tissues Amanda A. Amodeo and Jan M. Skotheim Loïc LeGoff and Thomas Lecuit Indeterminate Growth: Could It Represent the Biological Scaling Problems and Solutions in Ancestral Condition? Amphibians Iswar K. Hariharan, David B. Wake and Marvalee Daniel L. Levy and Rebecca Heald H. Wake The Systemic Control of Growth Intracellular Scaling Mechanisms Laura Boulan, Marco Milán and Pierre Léopold Simone Reber and Nathan W. Goehring Genome Biology and the Evolution of Cell-Size Growing an Embryo from a Single Cell: A Hurdle Diversity in Animal Life Rachel Lockridge Mueller Patrick H. O'Farrell Size Scaling of Microtubule Assemblies in Early Organ-Size Regulation in Mammals Xenopus Embryos Alfredo I. Penzo-Méndez and Ben Z. Stanger Timothy J. Mitchison, Keisuke Ishihara, Phuong Nguyen, et al. The Influence of Genome and Cell Size on Brain Size Control in Plants−−Lessons from Leaves and Morphology in Amphibians Flowers Gerhard Roth and Wolfgang Walkowiak Hjördis Czesnick and Michael Lenhard The Opposing Actions of Target of Rapamycin Nuclear DNA Content Varies with Cell Size across and AMP-Activated Protein Kinase in Cell Growth Human Cell Types Control James F. Gillooly, Andrew Hein and Rachel Sravanth K. Hindupur, Asier González and Michael Damiani N. Hall Small but Mighty: Cell Size and Bacteria Subcellular Size Petra Anne Levin and Esther R. Angert Wallace F. Marshall

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