Small but Mighty: Cell Size and Bacteriacshperspectives.cshlp.org/content/7/7/a019216.full.pdfSmall but Mighty: Cell Size and Bacteria Petra Anne Levin1 and Esther R. Angert2 1Department

Small but Mighty: Cell Size and Bacteria

Petra Anne Levin1 and Esther R. Angert2

1Department of Biology, Washington University, St. Louis, Missouri 631302Department 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 ancientof 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 withinthis previously invisible realm of life. Here, we review the impact of size on bacterial evo-lution, physiology, and morphogenesis.

Humanity has always experienced the im-pact of microorganisms, most obviously

through their ability to cause devastating dis-ease. For the vast majority of human history,we were unaware of their presence, much lessthe fundamental microbial processes to whichwe owe our existence: from the production ofenergy by our ancient bacterial endosymbionts(the mitochondria) to the generation of oxygenin our atmosphere. Despite their astoundingglobal abundance (�1030 cells) and their sub-stantial contribution to the total biomass ofplanet earth (Whitman et al. 1998; Kallmeyeret al. 2012), our inability to see these tiny lifeforms shrouded their nearly limitless diversityin mystery. It was not until the 17th century,with the careful observations and reports of An-ton van Leeuwenhoek, that we became aware ofthis previously invisible world on and aroundus. Today, we know that there are more bacterialiving in our intestinal tract than stars in theMilky Way galaxy (and that they far outnumberall the people who have ever lived). We alsoknow now that we thrive because of their meta-

bolic support. Although less than 1% of bacteriacan be cultured readily in the laboratory (Amannet al. 1995), the biochemical versatility amongthese tiny creatures exceeds that of the plants,animals, and fungi combined (Pace 1997).

Anton van Leeuwenhoek’s illustrations in aletter to the Royal Society of London in the late17th century provide one of the earliest recordsof bacterial cell form (Dobell 1960). Viewedthrough a single lens, Leeuwenhoek pioneeredstudies of the human microbiome, describingmotile bacilli, cocci, and spirochetes he foundin scrapings taken from between his teeth (andthe teeth of others). This triumph was madepossible by incomparable curiosity, lens con-struction, and exceptional lighting. The simplecellular structure and glassy nature of most un-stained bacteria viewed with a light microscopegenerated little interest in bacterial cell biologywith the exception of objects of unusual con-trast, such as endospores described by RobertKoch and the wonderfully colorful and largecyanobacteria. The bacterial nature of the latterwas 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

1

on January 21, 2021 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from

mailto:[email protected]




http://www.cshperspectives.org



http://cshperspectives.cshlp.org/

tury (Oren 2004). For the most part, bacteriawere viewed as primitive “bags of enzymes” un-til the 1990s, when the complexity of bacterialsubcellular structure and regulators of cell re-production finally began to emerge. Tools andreagents developed for eukaryotic cell biol-ogy (e.g., stains for DNA, membranes, and fluo-rescent protein tags), once applied to bacterialcells, revealed astonishing insights including thespecific and even dynamic localization patternsof proteins, and the accuracy of chromosomeorganization.

BACTERIAL SIZE RANGE

Bacillus subtilis, Staphylococcus aureus, Escheri-chia coli, and Caulobacter crescentus, the prima-ry models for bacterial cell biology, are moreor less typical in size, with individual cell vol-umes between �0.4–3 mm3 (or 0.4–3.0 femto-liters; femtoliter or fL is equal to 10215 L). Free-living marine ultramicrobacteria, appropriatelynamed Candidatus Actinomarina minuta, havean average cell volume �1% that of E. coli(0.013 mm3, range 0.6 � 1022 to 2.4 �1022 fL). At the other end of the spectrum,the marine sediment–dwelling Thiomargaritanamibiensis, the “Sulfur pearl of Namibia,” isa spherical organism with a volume eight or-ders of magnitude more than that of E. coli(�750 mm in diameter, volume 2.2 � 108 fL

or 0.22 mL). Thiomargarita is slightly largerthan a Drosophila eye (Schulz et al. 1999) andbig enough to be seen by the human eye (Fig. 1).A closer look at Thiomargarita reveals a central-ly located fluid-filled vacuole, which takes up�98% of the cell volume and is a nitrate reser-voir used to fuel sulfide oxidation. Even whenaccounting for the intracellular vacuole, a largeThiomargarita cell has a tremendous biovolumeto support (�4.4 � 106 fL). Epulopiscium spp.,intestinal symbionts of certain marine surgeon-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�2 � 106 mm3 or 0.02 mL (Angert et al. 1993).Unlike Thiomargarita, Epulopiscium cells con-tain no storage vacuoles or other inert inclu-sions (Fig. 1). The difference in size betweenCandidatus Actinomarina minuta and these gi-ants is equivalent to the difference between amouse and the Empire State Building (E. colicould be represented by a small skunk or a rabbiton this scale). We refer the reader to Niklas(2015), wherein he discusses how cell featuresand geometry factor into considerations of cellsize.

What limits bacterial cell size? The smallestcells need enough volume to accommodate ad-equate genetic resources to support the cell’slifestyle (Koch 1996). The cell must also containthe 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, sulfurgranules can be seen in the cytoplasm. The panel on the right shows an exceptionally large Epulopiscium cell withtwo large internal offspring. Scale bars, 100 mm.

P.A. Levin and E.R. Angert

2 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019216



genes as well as housekeeping proteins andbiochemicals to maintain its metabolism andcellular reproduction. Genomic and metabol-ic streamlining is seen in obligate intracellularsymbionts, pathogens, and organelles that havegiven up metabolic capabilities because thoseneeds are supplied by the host (McCutcheonand Moran 2012; Wernegreen 2012). The lossof genes for sensing environmental change andresponding to those contingencies can allow forsubstantial genome reduction but not always acorresponding reduction in cell size.

The structure and function of all large cellsappear bounded by the limits of diffusion(Schulz and Jorgensen 2001). Encounters withnutrients, elimination of waste, and the timelymovement of biomolecules within the cell tosupport metabolic needs all impact the abilityof a large bacterium to survive in its environ-ment. The compartmentalization of cellularfunctions, the motor protein-facilitated traf-ficking over a complex cytoskeletal network,the expansion of genomic resources, and theacquisition of endosymbionts that became en-ergy-generating organelles have all been credit-ed for the advancement of the size and com-plexity of eukaryotic cells (Angert 2012).

THE PROBLEM OF DIFFUSION

The identification of giant bacteria required areexamination of the long-held beliefs aboutmaximum bacterial cell size. For all cells, growthand reproduction is limited by the speed ofchemical communication and the availabilityof nutrients to fuel metabolism. Diffusion isthe random, three-dimensional movement ofa molecule. For movement of molecules insidea cell, it is the most efficient means of transportover a short distance of a few microns. Even alarge protein can reliably traverse this distancein the cytoplasm in much less than a second.Thus, a biomolecule synthesized or entering atypical bacterium has a high probability that itwill reach its site of activity almost instantly. Butconsidering a small distance by our scale, acrossa gap of a millimeter—approximately the lengthof an Epulopiscium cell and a fraction of the cir-cumference of Thiomargarita—diffusive trans-

port becomes numbingly unreliable. A smallmolecule like oxygen at room temperaturewould typically take about an hour to diffuse1 mm (Schulz and Jorgensen 2001). Similarly,the acquisition of nutrients relies on diffusionand capture of molecules at the surface of a cell.Consequently, free-living cells tend to be small,with a large surface area relative to their cyto-plasmic volume, so that capture of nutrients, atlow concentrations, can support the cell’s met-abolic needs. Cell form and function have beensubjected to these constraints. For comparison,E. coli has a surface-area-to-volume ratio of�3.7 mm2 to 1 mm3, whereas the largest Thio-margarita cell has a surface-to-volume ratio of8.2 � 1023 mm2 to 1 mm3. Clearly, these largebacteria are bending the rules.

How Epulopisicum Has Overcomethe Issue of Diffusion

Most large bacteria either maintain a high sur-face-to-volume ratio by being long and thinlike Spirochaeta plicatilis (a 250-mm-long,0.75-mm-diameter corkscrew-shaped bacte-rium) or adopt a morphology in which nopart of the cytoplasm is much more than a mi-cron away from the external environment. Thi-omargarita is an example of the latter, maintain-ing a thin layer of cytoplasm surrounding a largefluid-filled vacuole. Epulopiscium spp. are theexception to this rule.

Although their elongated form undoubted-ly helps increase their surface-area-to-volumeratio (Koch 1996), the largest Epulopiscium hasa ratio of 0.6 mm2 to 1 mm3, �1/6 that of E. coli.Despite this difference, Epulopiscium maintainsa high metabolic rate. Epulopiscium spp. use anumber of other structural modifications to ad-vance cell size. Epulopiscium cells have a highlyinvaginated cell membrane that may compen-sate for the small apparent surface area of thecell envelope (Angert 2006). Large Epulopisciumspp. are covered with flagella and are highlymotile, which helps the cell maintain its posi-tion in the gut in proximity to a plentiful sup-ply of nutrients. The coordinated rotation offlagella helps stir the surrounding medium, fa-cilitating movement of molecules across the cell

Cell Size and Bacteria

Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019216 3



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 theinfolded cell membrane likely facilitate the cap-ture of nutrients.

Epulopiscium cells show extreme polyploidythroughout their life cycle, an adaptation thatmay also contribute to their ability to attainsuch a large mass (Ward et al. 2009). Polyploidyis a common feature of large cells. Examplesfrom eukaryotes include endoreduplication inDrosophila salivary cells and, as discussed inCzesnick and Lenhard (2015), plant cells withspecialized functions including trichomes, thefine hairlike appendages visible on the leavesand stems of certain species. Gillooly and col-leagues (2015) discuss a potential role for endo-replicationandincreases innuclearDNAcontentas a determinant of size in human cells. Largebacteria, however, have taken polyploidy to anextreme level (Angert 2012). Although the Epu-lopiscium chromosome is fairly typical in size(�4 Mb), each cell contains tens of thousandsto hundreds of thousands of copies (Mendell etal. 2008). In contrast to most bacteria, in whichthe highlyorganized chromosome or “nucleoid”occupies almost the entire cytoplasm, Epulopis-cium nucleoids are located at the peripheryof thecytoplasm (Fig. 2). This is likely an importantorganizational feature that allows Epulopisciumto respond immediately to environmental stim-uli. It may also accommodate the growth of in-ternal offspring cells (described below).

REPRODUCTION AMONG THE GIANTS

Large bacteria show diverse reproductive strat-egies, some of which may help to maximizetheir reproductive potential as well as the pro-duction and release of large offspring. All of thelargest Epulopiscium spp. reproduce once daily,forming two or more intracellular offspring.Surprisingly, internal offspring initiation anddevelopment follows a predictable daily cycleand any given population inside a host fish iswell synchronized with respect to development.Offspring growth occurs during the day andcoincides with times when the host is feeding.

Reproduction begins with bipolar divisionof the mother cell. The polar cells are fully en-gulfed and these offspring then grow inside amembrane-bound compartment in the moth-er-cell cytoplasm, until they completely fill themother cell. At a late stage of development, themother cell appears to undergo a form of pro-grammed cell death, a process that likely con-serves the biochemical resources accumulatedduring growth (Ward et al. 2009). In the finalstages, offspring emerge through a split in themother cell envelope. Despite developmentalsynchrony, inhabitants of a single populationvary in volume by as much as a factor of five(Mendell et al. 2008).

Because of their size, unusual methods ofreproduction and the difficulty in determiningevolutionary relationships among microbesbased on phenotypic characteristics, Epulopis-cium was originally classified as a novel protist.Molecular phylogeny corrected this oversight,grouping Epulopiscium spp. with the Clostridia(Angert et al. 1993), endospore-forming Gram-positive organisms (of the Firmicutes phylum)that include the intestinal pathogen Clostridiumdifficile, Clostridium perfingens, the cause of

Figure 2. DNA in Epulopiscium sp. type B. This clus-ter of Epulopiscium cells is stained with the DNA dyeDAPI. Each of the two large mother cells at the centerof this field contains two large offspring. DNA islocated at the periphery of the cytoplasm in mothercells and offspring. At this late stage of development,the mother-cell DNA is difficult to see because muchof it has degraded. Offspring cells contain brightlystained polar structures; these are the start of “grand-daughter cells.” Scale bar, 100 mm.





gangrene, and the foodborne pathogen Clos-tridium botulinum from which we acquireBotox. Internal offspring production in Epulo-piscium spp. arose from endospore formation(Miller et al. 2012). Adapting endospore forma-tion as a mode of reproduction appears to havehappened several times in the spore-formingFirmicutes (Angert 2005).

Despite the unusual mode of reproductionshown by Epulopiscium, other giant bacteria usemore “pedestrian” strategies. Thiomargarita na-mibiensis in particular undergoes binary fissionin a single plane, arguably the most commonmode of reproduction among the bacteria. Thi-omargarita cells do not separate, however, re-maining instead as chains of cells housed in acommon mucus matrix (Fig. 1). At the sametime, closely related organisms reproduce usingother strategies, which include budding of off-spring from a sessile mother cell and the pro-duction of multiple internal offspring (Baileyet al. 2011). How reproductive form affects fit-ness of extremely large bacteria remains to bedetermined.

THE IMPACT OF ENVIRONMENTON SPECIES SIZE

Large bacteria abound in certain environmentsin which nutrients are consistently available andconcentrations are high. We believe that theseassociations give us some clues as to conditionsthat can help organisms break free from diffu-sion limitations on cell size. Giant spirochetescan be found in nutrient-rich sediments and insome intestinal systems, such as the hindgut oftermites. Large rod-shaped cells have been seenin intestinal tracts of a number of herbivores.Sulfur-oxidizing bacterial cells like Thiomarga-rita abound in marine sediments. They are near-ly ubiquitously distributed around the globe andcarry with them minerals to fuel their respira-tion. An abundant supplyof energy is acommontheme in all of these systems and may be a uni-fying feature. Polyploidy is widespread in bacte-ria but is expensive to sustain. In nutrient-richenvironments or when energy is practically lim-itless, selection for scaling down to a single copyof the genome may be relaxed and allow poly-

ploid microbes to abound. Subsequent modifi-cations to cellular architecture may then accom-modate further expansion in size.

CELL-SIZE HOMEOSTASIS

Under steady-state conditions, isogenic popu-lations of bacteria maintain cell size within sur-prisingly narrow parameters. Although a histo-gram of E. coli or B. subtilis cells sampled inmid-exponential phase is biased toward theleft, a consequence of the greater number ofsmaller newborn cells, only in rare cases arethe largest organisms more than twice the sizeof the smallest (Fig. 3).

Like eukaryotes, bacteria initiate and termi-nate one round of DNA replication for eachdivision event. In contrast to eukaryotes, how-ever, DNA replication, chromosome segrega-tion, and assembly of the division machineryare not discrete events, instead overlappingone another to a greater or lesser degree de-pending on growth rate (Fig. 4).

Importantly, at fast growth rates supportedby nutrient-rich conditions, bacteria are able tomaintain interdivision periods less than half thelength of time required for replication of theirchromosomal DNA. For example, E. coli candouble in mass and divide in as few as 20 min;however, even under ideal conditions, replica-tion of the entire 4-Mb E. coli chromosometakes �60 min. Rapidly growing cells resolvethis apparent conflict by constantly synthesizingDNA and by initiating new rounds of replica-tion before completion of the old one. Such cellsmay have 4, 8, or even 16 ongoing replicationforks (Yoshikawa et al. 1964; Cooper and Helm-stetter 1968). Although initiation is restricted toonce per division cycle, only one round of rep-lication needs to be completed before division.Within a single division cycle, initiation andtermination events, thus, do not necessarily cor-respond to the same replication fork.

Early Work on Cell-Size Homeostasis

In 1968, William Donachie proposed that cell-size homeostasis was tied to cell-cycle progres-sion (Donachie 1968). Combining data on Sal-





monella from the Maaløe laboratory with E. colidata from Cooper and Helmstetter (Schaechteret al. 1958; Cooper and Helmstetter 1968), Do-nachie inferred that bacterial cells initiate DNAreplication on achievement of a specific mass.Donachie suggested that the growth-dependentaccumulation of a positive acting factor—or in-hibition of a negative acting factor—would besufficient to coordinate the onset of the cell cy-cle with cell size.

Subsequent work implicated the highly con-served AAAþ ATPase DnaA, a dose-dependentactivator of DNA replication, in E. coli cell-sizecontrol (Løbner-Olesen et al. 1989). Reducinglevels of DnaA delays the onset of DNA replica-tion and increases cell size, whereas increasingDnaA levels results in premature initiation andreduced cell size.

On the face of it, tying replication initiationto cell size is an ideal way to correct any defects

Cytokinesis

ReplicationInitiation

ReplicationTermination

GrowthOuter circle represents

assembly and maturationof the cytokinetic ring

DNA replication

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 newround of replication is initiated only once per cycle. At least one round of replication must be terminated beforedivision. 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.

5

10

15

20

25

30

35

40

0<3 3–4 4–5 5–6

Cell length (µm)Fr

actio

n of

cel

ls (

%)

6–7 >7

Wild type

UDP-glucosemutant

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 thehigher 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 similarlyconstrained within a twofold range. (Data courtesy of Norbert Hill.)





in cell size early in the cell cycle. Short cellswill have to grow longer before they accumulatesufficient DnaA to initiate DNA replication,whereas longer cells will initiate earlier (andthus, at smaller sizes) because critical levels ofDnaA are reached earlier in the cell cycle. Theobservation that in Saccharomyces cerevisiaeachievement of a specific size is required beforeunbudded cells can pass through START at theG1/S transition suggests that this type of controlmay be broadly applicable (Pringle and Hart-well 1981). (Intriguingly, the dnaA gene of Epu-lopiscium sp. type B encodes an exceptionallylong mononucleotide tract, a hot spot for poly-merase slippage and potential expression of atruncated and nonfunctional peptide [Mendellet al. 2008]. Although the specific contributionof this unusual gene structure to DnaA regula-tion is not known, it is possible that it plays arole in increasing Epulopiscium’s cytoplasmicvolume-to-DNA ratio by reducing DnaA levelsand delaying replication.)

Despite the appeal of this model, recentdata suggests DnaA accumulation is unlikelyto be the sole or even primary determinantof bacterial cell size. Small B. subtilis mutantsshow wild-type cell-cycle profiles with regardto initiation and DNA replication, suggestingthat initiation is independent of cell size inthis Gram-positive model organism (Hill et al.2012). Even in E. coli, in which initiation is high-ly sensitive to modest changes in the amount ofactive DnaA (Løbner-Olesen et al. 1989), regu-lating mass at initiation is only part of the story.Mutations that result in early or delayed initia-tion appear to adjust their rate of DNA synthesisto compensate for changes in initiation time(Hill et al. 2012). Together, these data suggestthe presence of a homeostatic and DnaA-inde-pendent mechanism to maintain normal inter-division periods.

Cell Division and Cell-Size Control

In most bacteria, division is initiated by as-sembly of the essential GTPase FtsZ into aring-like structure at the future division site.The “Z-ring” serves as a framework for assem-bly of the rest of the cell-division machinery.

Structural data suggests that FtsZ is an evolu-tionary precursor of tubulin, although the twoproteins share limited sequence similarity. Ap-proximately one-third of FtsZ in the cell is in theFtsZ ring at a given time with subunit turnoverrates on the order of seconds. (For reviews ofFtsZ and bacterial cell division, see Ericksonet al. 2010 and Lutkenhaus et al. 2012.)

Altering FtsZ levels has a direct impact oncell size, making the cytoskeletal protein an ide-al target for factors governing cell-size homeo-stasis. Depleting FtsZ does not effect cell growthor DNA replication, at least initially, and resultsin a rapid increase in length. Even modest re-ductions (�20%) in intracellular FtsZ concen-tration lead to a large increase (.50%) in celllength under steady-state conditions (Palacioset al. 1996).

If one imagines the cytokinetic ring as abrick building, then it is easy to see how changesin the supply of bricks would influence cell size.When FtsZ concentration is high, bricks arereadily available, reducing transport time andfacilitating assembly of a stable and functionalZ-ring. However, reductions in available FtsZslow the transport of bricks to the constructionsite, delaying assembly of the division machin-ery and with it cytokinesis. As cells continue toincrease in size at normal rates, delays in assem-bly of the cytokinetic ring translate directly intoincreases in cell size.

Because FtsZ levels are proportional to cellsize in both E. coli and B. subtilis, regardlessof growth rate (Weart and Levin 2003), stochas-tic variations in cell size should be largely mit-igated by FtsZ availability. Short cells will re-quire longer periods to accumulate sufficientFtsZ to divide—increasing their size at division.Conversely, longer cells will take less time toaccumulate enough FtsZ to support assemblyof the division machinery and consequentlyshow shorter interdivision periods reducingdaughter-cell length.

Just as DnaA accumulation alone is in-sufficient to account for cell-size homeostasis,FtsZ accumulation alone is insufficient to trig-ger division. Although twofold overexpressionof FtsZ reduces cell size by �10% in both in E.coli and B. subtilis, it does not significantly im-





pact the timing of division (Ward and Lutken-haus 1985; Weart and Levin 2003; Hill et al.2012). The inability of excess FtsZ to triggerdivision suggests the presence of as-yet uniden-tified factors required to license FtsZ assemblyat the nascent division site. How these factorsare integrated with cell-cycle progression re-mains an open question. It is also worth notingthat FtsZ is one of many components of thecytokinetic machinery, some of which are alsorate limiting for division and, thus, potentialcandidates for the homeostatic regulators ofcell size (Lutkenhaus et al. 2012).

Although a model in which cell-size homeo-stasis is dependent on accumulation of a rate-limiting cell-cycle component is consistentwith what we know about division in E. coli,B. subtilis, and Caulobacter, it is harder to seehow it would apply to organisms that divide bybudding or tip growth as well as those that shownonlinear growth patterns. Individual E. colicells elongate at a more or less constant rate un-der steady-state conditions (Wang et al. 2012).In contrast, Mycobacterium smegmatis tip elon-gation occurs at different rates depending onwhether the cell inherited an old pole that isprimed for growth, or a new pole that is not(Aldridge et al. 2012). In cases involving growthasymmetry in daughter cells, other mechanismsmust be at play. The mechanisms governingcell-size homeostasis are equally mysteriousfor those organisms that reproduce by budding,such as Hyphomicrobium, Planctomyces spp.,etc.; colonial organisms that adjust ploidy levelsand cell length during developmental transi-tions, such as Streptomycetes; bacteria that un-couple periods of DNA replication and growthfrom rounds of cytokinesis as in some cyano-bacteria and Bdellovibrio; and, finally, those thatreproduce by internal daughter-cell production,such as Epulopiscium (Angert 2005).

NUTRIENT-DEPENDENT REGULATIONOF CELL SIZE

Nutrient availability is the primary determinantof cell size in many bacteria. In a classic paper,Schaechter, Maaløe, and Kjeldgaard deter-mined that Salmonella cell size varies as much

as twofold depending on growth rate(Schaechter et al. 1958). Cells cultured in nutri-ent-rich medium with doubling times of�20 min were more than twice the size of theircounterparts cultured in nutrient-poor mediumwith mass doubling times well over an hour. Sig-nificantly, the relationship between growth rateand size held true regardless of whether growthwas slowed by limiting carbon, phosphate, ornitrogen suggested that doubling time—ratherthan the specific nutrient content of the medi-um—was the primary determinant of cell size.However, more recent work suggests that bacte-rial size is a complex phenomenon, dependenton a precisely orchestrated set of regulatory cir-cuits, each responsive to a different and poten-tially overlapping nutrient-dependent signals.

In E. coli and B. subtilis, UDP-glucose, anucleotide sugar synthesized in two steps fromglucose-6, serves as an intracellular signal forcarbon availability and growth rate. Defectsin either the phosphoglucomutase requiredfor interconversion of Glc-6 and Glc-1 and thepyrophosphorylase responsible for synthesizingUDP-glc from Glc1 reduce E. coli and B. subtiliscell size by as much as 30% under nutrient-rich conditions (Weart et al. 2007; Hill et al.2013). Consistent with a growth-rate-depen-dent mechanism, loss of UDP-glc synthesis haslittle impact on size in cells cultured in nutrient-poor medium. Its proximity to central carbonmetabolism makes UDP-glc an ideal signal forcarbon availability.

UDP-glucose levels are sensed and transmit-ted to the division machinery by two, unrelatedglucosyltransferases, OpgH and UgtP, in E. coliand B. subtilis, respectively. Although UDP-glu-cose stimulates interactions among OpgH,UgtP, and FtsZ, the molecular mechanismsby which they function are highly divergent.OpgH is an integral membrane protein. Geneticand biochemical data suggest that UDP-glcbinding drives a conformational change that re-veals a binding site for FtsZ. By sequesteringFtsZ monomers, OpgH effectively reduces theamount of FtsZ available for assembly into thecytokinetic ring. UgtP, on the other hand, isonly transiently associated with the membrane.UgtP interacts with itself or FtsZ in what ap-





pears to be a mutually exclusive manner. UgtPhomo-oligomerization is favored in the absenceof UDP-glucose, whereas increases in intracel-lular UDP-glucose levels during growth in nu-trient-rich medium promote interaction withFtsZ (Chien et al. 2013). UgtP levels are highduring growth in nutrient-rich medium, butlow in nutrient-poor medium, further reliev-ing division inhibition during slow growth(Weart et al. 2007). UgtP inhibits FtsZ assemblybut has no impact on GTP hydrolysis, suggest-ing that it may have an activity similar to mi-crotubule severing proteins like Katanin (Chienet al. 2013).

It is striking that two such evolutionarilydivergent organisms (E. coli and B. subtilis aremore distantly related than Homo sapiens andS. cerevisiae) use UDP-glucose and unrelatedglucosyltransferases to help coordinate cell sizewith growth rate and nutrient availability.OpgH and UgtP are both “moonlighting” pro-teins with roles in cell-envelope biogenesis.OpgH takes part in the synthesis of the osmo-regulated periplasmic glucans (OPGs), largeglucans found in between the inner and outermembranes of Gram-negative bacteria, whereasUgtP synthesizes the Di-Glc-diacylglycerol an-chor for lipoteichoic acid, a major anionic com-ponent of the Gram-positive cell wall (Lazarevicet al. 2005; Lequette et al. 2008). Both the OPGsand LTAs are thought to play a role in the cellularresponse to changes in osmolarity, a stress re-sponse that leads to a temporary arrest of celldivision. Synthesis of OPG or LTA is the primarysource of diacylglycerol (DAG) in E. coli andB. subtilis (Zhang and Rock 2008). Althoughdiacylglycerol is a well-studied secondary mes-senger in eukaryotes, it has not yet been impli-cated in signaling in bacteria. One possibilityis that DAG accumulation serves as a proxy forcell-envelope biogenesis, transmitting infor-mation about actual growth rate (rather thannutrient availability) to the division machinery.Intriguingly, FabH, an enzyme responsible forcatalyzing an early step in fatty acid biosynthe-sis, has also been implicated in the nutrient-dependent control of cell size; although, wheth-er, FabH plays a direct or indirect role has yet tobe established (Yao et al. 2012).

At the same time, carbon availability is un-likely to be the sole determinant of cell size. AsSchaechter et al. showed in their seminal 1958paper, limiting not only carbon, but also othernutrients including nitrogen and phosphate,leads to reductions in both growth rate and cellmass (Schaechter et al. 1958). Moreover, rapidlygrowing cells defective in UDP-glucose bio-synthesis are still significantly larger than theirslow-growing counterparts (Weart et al. 2007;Hill et al. 2013). These observations stronglysuggest the presence of additional intracellularsignaling molecules and cognate sensors, someof which are likely coupled to nitrogen andphosphate utilization, that also contribute togrowth rate-dependent increases in cell size.

Precisely why cells increase size in responseto nutrient-dependent increases in growth rateis not known. However, analysis of FtsZ ringformation and chromosome segregation inwild-type and short mutant strains suggeststhat increases in cell size during rapid growthhelp ensure that there is sufficient room to ac-commodate the additional DNA generated bymultifork replication. Both E. coli and B. subtilismaintain a constant cell-mass-to-DNA ratio re-gardless of growth rate (Sargent 1975; Donachieand Begg 1989; Sharpe et al. 1998). This appearstrue for giant bacteria as well (Mendell et al.2008). Because chromosome segregation anddivision are not coupled as they are in eukary-otes, defects in bacterial cell size lead to an in-crease in the frequency of FtsZ rings and divi-sion septa over unsegregated nucleoids underconditions supporting rapid growth (Weartet al. 2007; Hill et al. 2013). (Viability is pre-served in these cases by DNA transferases thatpump chromosomal DNA away from the invag-inating septum.)

CONCLUDING THOUGHTS

As with our appreciation of the vital and diverseroles that bacteria play in the earth’s biogeo-chemical cycles, we are only beginning to un-derstand the molecular and physical forces thatgovern bacterial size. Cataloging the diversenature of bacterial life—both in physical andmetabolic terms—and characterizing the more





tractable aspects of their physiology has led toadvances in our understanding not only of bac-teria, but also fundamental aspects of biologycommon to all forms of life.

Significantly, mechanisms that have beenimplicated in the control of cell size in eukary-otes including endoreduplication (reviewed inLee et al. 2009), nutrient signaling (reviewedin Davie and Petersen 2012), and, of course,cell-cycle control (see reviews by Jorgensenand Tyers 2004; Goranov and Amon 2010) arerepeating themes in the control of bacterialcell size. We anticipate that the application oftechniques—both the tried and true (e.g., theawesome power of microbial genetics), and thenew (e.g., microfluidics [Wang et al. 2012],high-throughput image analysis [Cabeen et al.2009; Russell et al. 2013], and single-cell metab-olomics [Zenobi 2013]) will reveal insights intocell-size control in bacteria that apply to all.

REFERENCES�Reference is also in this collection.

Aldridge BB, Fernandez-Suarez M, Heller D, Ambravanes-waran V, Irimia D, Toner M, Fortune SM. 2012. Asym-metry and aging of mycobacterial cells lead to variablegrowth and antibiotic susceptibility. Science 335: 100–104.

Amann RI, Ludwig W, Schleifer KH. 1995. Phylogeneticidentification and in situ detection of individual micro-bial cells without cultivation. Microbiol Rev 59: 143–169.

Angert ER. 2005. Alternatives to binary fission in bacteria.Nat Rev Microbiol 3: 214–224.

Angert ER. 2006. The enigmatic cytoarchitecture of Epulo-piscium spp. In Microbiology monographs (ed. Shively JM),pp. 285–301. Springer, Berlin.

Angert ER. 2012. DNA replication and genomic architec-ture of very large bacteria. Annu Rev Microbiol 66: 197–212.

Angert ER, Clements KD, Pace NR. 1993. The largest bacte-rium. Nature 362: 239–241.

Bailey JV, Salman V, Rouse GW, Schulz-Vogt HN, Levin LA,Orphan VJ. 2011. Dimorphism in methane seep-dwell-ing ecotypes of the largest known bacteria. ISME J 5:1926–1935.

Cabeen MT, Charbon G, Vollmer W, Born P, Ausmees N,Weibel DB, Jacobs-Wagner C. 2009. Bacterial cell curva-ture through mechanical control of cell growth. EMBO J28: 1208–1219.

Chien AC, Zareh SK, Wang YM, Levin PA. 2013. Changes inthe oligomerization potential of the division inhibitorUgtP co-ordinate Bacillus subtilis cell size with nutrientavailability. Mol Microbiol 86: 594–610.

Cooper S, Helmstetter CE. 1968. Chromosome replicationand the division cycle of Escherichia coli B/r. J Mol Biol31: 519–540.

� Czesnick H, Lenhard M. 2015. Size control in plant—Les-sons from leaves and flowers. Cold Spring Harb PerspectBiol doi: 10.1101/cshperspect.a019190.

Davie E, Petersen J. 2012. Environmental control of cell sizeat division. Curr Opin Cell Biol 24: 838–844.

Dobell C. 1960. Antony van Leeuwenhoek and his “little an-imals.” Dover, New York.

Donachie WD. 1968. Relationship between cell size and timeof initiation of DNA replication. Nature 219: 1077–1079.

Donachie WD, Begg KJ. 1989. Cell length, nucleoid separa-tion, and cell division of rod-shaped and spherical cellsof Escherichia coli. J Bacteriol 171: 4633–4639.

Erickson HP, Anderson DE, Osawa M. 2010. FtsZ in bacte-rial cytokinesis: Cytoskeleton and force generator all inone. Microbiol Mol Biol Rev 74: 504–528.

� Gillooly JF, Hein A, Damiani R. 2015. Nuclear DNA contentvaries with cell size across human cell types. Cold SpringHarb Perspect Biol doi: 10.1101/cshperspect.a019091.

Goranov AI, Amon A. 2010. Growth and division—Not aone-way road. Curr Opin Cell Biol 22: 795–800.

Hill NS, Kadoya R, Chattoraj DK, Levin PA. 2012. Cell sizeand the initiation of DNA replication in bacteria. PLoSGenet 8: e1002549.

Hill NS, Buske PJ, Shi Y, Levin PA. 2013. A moonlightingenzyme links Escherichia coli cell size with central metab-olism. PLoS Genet 9: e1003663.

Jorgensen P, Tyers M. 2004. How cells coordinate growth anddivision. Curr Biol 14: R1014–R1027.

Kallmeyer J, Pockalny R, Adhikari RR, Smith DC, D’HondtS. 2012. Global distribution of microbial abundance andbiomass in subseafloor sediment. Proc Natl Acad Sci 109:16213–16216.

Koch AL. 1996. What size should a bacterium be? A questionof scale. Annu Rev Microbiol 50: 317–348.

Lazarevic V, Soldo B, Medico N, Pooley H, Bron S, KaramataD. 2005. Bacillus subtilis a-phosphoglucomutase is re-quired for normal cell morphology and biofilm forma-tion. Appl Environ Microbiol 71: 39–45.

Lee HO, Davidson JM, Duronio RJ. 2009. Endoreplication:Polyploidy with purpose. Genes Dev 23: 2461–2477.

Lequette Y, Lanfroy E, Cogez V, Bohin JP, Lacroix JM. 2008.Biosynthesis of osmoregulated periplasmic glucans inEscherichia coli: The membrane-bound and the solubleperiplasmic phosphoglycerol transferases are encoded bythe same gene. Microbiology 154: 476–483.

Løbner-Olesen A, Skarstad K, Hansen FG, von MeyenburgK, Boye E. 1989. The DnaA protein determines the initi-ation mass of Escherichia coli K-12. Cell 57: 881–889.

Lutkenhaus J, Pichoff S, Du S. 2012. Bacterial cytokinesis:From Z ring to divisome. Cytoskeleton (Hoboken) 69:778–790.

McCutcheon JP, Moran NA. 2012. Extreme genome reduc-tion in symbiotic bacteria. Nat Rev Micro 10: 13–26.

Mendell JE, Clements KD, Choat JH, Angert ER. 2008. Ex-treme polyploidy in a large bacterium. Proc Natl Acad Sci105: 6730–6740.





Miller DA, Suen G, Clements KD, Angert ER. 2012. Thegenomic basis for the evolution of a novel form of cellularreproduction in the bacterium Epulopiscium. BMC Ge-nomics 13: 265.

� Niklas KJ. 2015. A phyletic perspective on cell growth.Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a019158.

Oren A. 2004. A proposal for further integration of thecyanobacteria under the bacteriological code. Int J SystEvol Microbiol 54: 1895–1902.

Pace NR. 1997. A molecular view of microbial diversity andthe biosphere. Science 276: 734–740.

Palacios P, Vicente M, Sanchez M. 1996. Dependency ofEscherichia coli cell-division size, and independency ofnucleoid segregation on the mode and level of ftsZ ex-pression. Mol Microbiol 20: 1093–1098.

Pringle JR, Hartwell LH. 1981. The Saccharomyces cerevisiaecell cycle. In The molecular biology of the yeast Saccharo-myces: Life cycle and inheritance (ed. Strathern JN, JonesEW, Broach JR), pp. 97–142. Cold Spring Harbor Labo-ratory Press, Cold Spring Harbor, NY.

Russell AB, LeRoux M, Hathazi K, Agnello DM, Ishikawa T,Wiggins PA, Wai SN, Mougous JD. 2013. Diverse type VIsecretion phospholipases are functionally plastic antibac-terial effectors. Nature 496: 508–512.

Sargent MG. 1975. Control of cell length in Bacillus subtilis.J Bacteriol 123: 7–19.

Schaechter M, Maaloe O, Kjeldgaard NO. 1958. Depen-dency on medium and temperature of cell size and chem-ical composition during balanced grown of Salmonellatyphimurium. J Gen Microbiol 19: 592–606.

Schulz HN, Jorgensen BB. 2001. Big bacteria. Annu RevMicrobiol 55: 105–137.

Schulz HN, Brinkhoff T, Ferdelman TG, Marine MH, TeskeA, Jorgensen BB. 1999. Dense populations of a giant sul-fur bacterium in Namibian shelf sediments. Science 284:493–495.

Sharpe ME, Hauser PM, Sharpe RG, Errington J. 1998. Ba-cillus subtilis cell cycle as studied by fluorescence micros-copy: Constancy of cell length at initiation of DNA rep-lication and evidence for active nucleoid partitioning. JBacteriol 180: 547–555.

Wang P, Robert L, Pelletier J, Dang WL, Taddei F, Wright A,Jun S. 2012. Robust growth of Escherichia coli. Curr Biol20: 1099–1103.

Ward JE Jr, Lutkenhaus J. 1985. Overproduction of FtsZinduces minicell formation in E. coli. Cell 42: 941–949.

Ward RJ, Clements KD, Choat JH, Angert ER. 2009. Cytol-ogy of terminally differentiated Epulopiscium mothercells. DNA Cell Biol 28: 57–64.

Weart RB, Levin PA. 2003. Growth rate-dependent regula-tion of medial FtsZ ring formation. J Bacteriol 185: 2826–2834.

Weart RB, Lee AH, Chien AC, Haeusser DP, Hill NS, LevinPA. 2007. A metabolic sensor governing cell size in bac-teria. Cell 130: 335–347.

Wernegreen JJ. 2012. Strategies of genomic integration with-in insect-bacterial mutualisms. Biol Bull 223: 112–122.

Whitman WB, Coleman DC, Wiebe WJ. 1998. Prokary-otes: The unseen majority. Proc Natl Acad Sci 95: 6578–6583.

Yao Z, Davis RM, Kishony R, Kahne D, Ruiz N. 2012. Reg-ulation of cell size in response to nutrient availability byfatty acid biosynthesis in Escherichia coli. Proc Natl AcadSci 109: E2561–E2568.

Yoshikawa H, O’Sullivan A, Sueoka N. 1964. Sequential rep-lication of the Bacillus subtilis chromosome: III. Regula-tion of initiation. Proc Natl Acad Sci 52: 973–980.

Zenobi R. 2013. Single-cell metabolomics: Analytical andbiological perspectives. Science 342: 1243259.

Zhang YM, Rock CO. 2008. Membrane lipid homeostasis inbacteria. Nat Rev Microbiol 6: 222–233.





8, 20152015; doi: 10.1101/cshperspect.a019216 originally published online JuneCold Spring Harb Perspect Biol

Petra Anne Levin and Esther R. Angert Small but Mighty: Cell Size and Bacteria

Subject Collection Size Control in Biology: From Organelles to Organisms

Cell-Size ControlAmanda A. Amodeo and Jan M. Skotheim

Mechanical Forces and Growth in Animal TissuesLoïc LeGoff and Thomas Lecuit

Ancestral Condition?Indeterminate Growth: Could It Represent the

H. WakeIswar K. Hariharan, David B. Wake and Marvalee

AmphibiansBiological Scaling Problems and Solutions in

Daniel L. Levy and Rebecca Heald

The Systemic Control of GrowthLaura Boulan, Marco Milán and Pierre Léopold

Intracellular Scaling MechanismsSimone Reber and Nathan W. Goehring

DiversityGenome Biology and the Evolution of Cell-Size

Rachel Lockridge Muellerin Animal LifeGrowing an Embryo from a Single Cell: A Hurdle

Patrick H. O'Farrell

EmbryosXenopusSize Scaling of Microtubule Assemblies in Early

Nguyen, et al.Timothy J. Mitchison, Keisuke Ishihara, Phuong

Organ-Size Regulation in MammalsAlfredo I. Penzo-Méndez and Ben Z. Stanger

Morphology in AmphibiansThe Influence of Genome and Cell Size on Brain

Gerhard Roth and Wolfgang WalkowiakFlowers

Lessons from Leaves and−−Size Control in Plants

Hjördis Czesnick and Michael Lenhard

Controland AMP-Activated Protein Kinase in Cell Growth The Opposing Actions of Target of Rapamycin

N. HallSravanth K. Hindupur, Asier González and Michael

Human Cell TypesNuclear DNA Content Varies with Cell Size across

DamianiJames F. Gillooly, Andrew Hein and Rachel

Small but Mighty: Cell Size and BacteriaPetra Anne Levin and Esther R. Angert

Subcellular SizeWallace F. Marshall

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved


http://cshperspectives.cshlp.org/cgi/collection/

http://cshperspectives.cshlp.org/cgi/collection/


Documents

Small but Mighty: Cell Size and Bacteriacshperspectives.cshlp.org/content/7/7/a019216.full.pdfSmall but Mighty: Cell Size and Bacteria Petra Anne Levin1 and Esther R. Angert2 1Department