edgar and vollmer 2012 the physiology of bacterial cell division

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Antimicrobial Therapeutics Reviews

The physiology of bacterial cell divisionAlexander J. F. Egan and Waldemar VollmerCentre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University,Newcastle upon Tyne, United Kingdom

Address for correspondence: Waldemar Vollmer, Centre for Bacterial Cell Biology, Institute for Cell and MolecularBiosciences, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK. [email protected]

Bacterial cell division is facilitated by the divisome, a dynamic multiprotein assembly localizing at mid-cell tosynthesize the stress-bearing peptidoglycan and to constrict all cell envelope layers. Divisome assembly occurs intwo steps and involves multiple interactions between more than 20 essential and accessory cell division proteins.Well before constriction and while the cell is still elongating, the tubulin-like FtsZ and early cell division proteinsform a ring-like structure at mid-cell. Cell division starts once certain peptidoglycan enzymes and their activatorshave moved to the FtsZ-ring. Gram-negative bacteria like Escherichia coli simultaneously synthesize and cleave theseptum peptidoglycan during division leading to a constriction. The outer membrane constricts together with thepeptidoglycan layer with the help of the transenvelope spanning TolPal system.

Keywords: cell division; divisome; peptidoglycan; penicillin-binding protein; peptidoglycan hydrolyase; outer mem-brane; TolPal

IntroductionPropagating bacteria undergo cycles of growth andcell division during which all cellular compoundsare synthesized in a regulated manner and they be-come distributed to the daughter cells. A bacterialcell has only two components that are inherited asa single molecule, the chromosome and the cellwall peptidoglycan (murein) sacculus. Obviously,the single copy number, huge size (compared to thecell size), andessentiality of both componentsneces-sitate safe mechanisms for their replication/growthand distribution to the daughter cells. The saccu-lus, an integral component of the cell envelope, en-cases the cytoplasmic membrane to form a net-likeand continuous layer1,2 that is required to main-tain cell shape and osmotic stability against the tur-gor. In Gram-negative bacteria the peptidoglycanis 36 nm thick and likely a single layer, whereasin Gram-positive species it is thicker than 1020nm and contains covalently attached secondary cellwall polymers such as teichoic acids and capsularpolysaccharides.3

The chemical composition of peptidoglycan iswell known. It is composed of glycan chains madeof alternating N-acetylmuramic acid (MurNAc)and N-acetyglucosamine (GlcNAc) residues thatare connected by short peptides.4 These containboth l- and d-amino acids and, in Gram-negativebacteria such as Escherichia coli, are synthesizedas pentapeptides with the following sequence:l-Ala-d-iGlu-m-Dap-d-Ala-d-Ala (m-Dap, meso-diaminopimelic acid). Two or more peptidesprotruding from neighboring glycan chains can beconnected to form dimeric or multimeric cross-links. Moreover, in some Gram-negative species,peptides can serve as attachment sites for Lpp(Brauns lipoprotein), which anchors with its lipidmodification in the outer membrane and ensures afirm connection between the outer membrane andthe peptidoglycan.5 Many Gram-positive speciesanchor cell wall proteins to the peptides in pepti-doglycan by the enzyme sortase.6 In Gram-positivebacteria there is considerable variation in the struc-ture of the peptides, and many species struc-turally modify the glycan chains (for example,

doi: 10.1111/j.1749-6632.2012.06818.x8 Ann. N.Y. Acad. Sci. 1277 (2013) 828 c 2012 New York Academy of Sciences.

Egan & Vollmer Bacterial cell division

Figure 1. Sites of peptidoglycan synthesis during growth andcell division in different bacteria. The rod-shaped E. coli andB. subtilis elongate by insertion of new peptidoglycan (red dots)into the lateral cell wall. Coccal S. aureus cells lack an elonga-tion phase, and the ovococcus S. pneumoniae elongates from agrowth zone atmid-cell. The dark red lines indicate sites of zonalpeptidoglycan synthesis. Gram-negative bacteria synthesize andsplit the septum simultaneously, resulting in a constriction. Bycontrast, Gram-positive bacteria synthesize a complete septumbefore cell separation.

by N-deacetylation or O-acetylation)7 and/or thepeptides (by amidation).8,9

The molecular architecture of the mainly single-layered sacculus from Gram-negative bacteria hasbeen debated over the last decades.10 The currentmodel is based on the enhanced elasticity of isolated,rod-shaped E. coli sacculi in the direction of thelong axis, their thickness of 36 nm, and cryo-electrontomography imaging. Accordingly, pepti-doglycan adopts a single-layered architecture withglycan strands oriented mainly perpendicular tothe long axis of the cell. The more flexible pep-tide cross-links are oriented mainly in the directionof the long axis. Cryo-electrontomography imagessuggest some degree of disorder in the layer,11 con-sistent with the glycan strands being much shorterthan the circumference of the cell and the chemi-cal heterogeneity of the peptides.12,13 These imageshave been produced from isolated, that is, relaxed,sacculi. Presumably, in the cell, where the sacculusis stretched by the turgor, the glycan chains, andpeptides adopt a more ordered configuration thatfacilitates the activities of peptidoglycan syntheticand hydrolytic enzymes.3

The molecular mechanisms by which the pepti-doglycan layer grows during cell elongation and di-vision are largely unknown. According to a currentmodel, membrane-bound multienzyme complexesmade of peptidoglycan synthases and hydrolasesare positioned and/or regulated from inside thecell by cytoskeletal proteins and their interactionpartners, many of which are membrane bound.14,15

The actin-like MreB is required for maintainingrod shape in many species, except those which ex-hibit polar growth, for example actinobacteria andcertain -proteobacteria.14,16 MreB and associatedproteins localize on a helical path along the lengthof the cell,14 and recent high-resolution techniquesshowed them moving in dynamic patches aroundthe cell, depending on ongoing peptidoglycan syn-thesis.1719 It appears that cytoskeletal and pepti-doglycan proteins act interdependently to ensuremaintaining the diameter of the cell by a yet un-known mechanism.Cell division is orchestrated by FtsZ and more

than 12 other essential cell division proteins (Ftsproteins).20 They form the divisome complex togenerate thenewpoles of thedaughter cells. Betweenelongation and cell division, Caulobacter crescentusand to lesser extent E. coli employ a preseptal phaseof elongation in which the cells elongate by cell en-velope growth at mid-cell dependent on FtsZ.21,22

In E. coli and presumably other Gram-negative bac-teria, growth of the sacculus is also regulated fromthe outside by recently identified outer membranelipoproteins, LpoA and LpoB.23,24 However, whilemany players for sacculus growth have been iden-tified, there is little knowledge about the moleculardetails driving and regulating cell elongation anddivision.Gram-negative bacteria employ a constrictive

mode of cell division, that is, the synthesis of thenew septum, its splitting, and the invagination ofthe outer membrane occur simultaneously. By con-trast, most Gram-positive bacteria first completelysynthesize the septal cross-wall before splitting it forcell separation (Fig. 1).Antibiotic inhibition of bacterial cell division and

peptidoglycan synthesis has been covered by re-cent articles.2528 In the present review we focusmainly on the physiology of cell division in Gram-negative bacteria because most available data wereobtained from the model bacterium E. coli. Unlessotherwise noted any data and/or conclusions dis-cussed, particularly pertaining to interaction andactivity data, are derived from work on this or-ganism. We briefly summarize the peptidoglycanbiosynthetic pathway, which is the target of impor-tant antibiotics, and present the peptidoglycan en-zymes and their activities and interactions with anemphasis on peptidoglycan synthesis during cell di-vision. We also discuss cell division proteins and

Ann. N.Y. Acad. Sci. 1277 (2013) 828 c 2012 New York Academy of Sciences. 9

Bacterial cell division Egan & Vollmer

Figure 2. Peptidoglycan synthesis and hydrolysis. A nascent peptidoglycan strand is synthesized from the lipid II precursorby glycosyltransferase (GTase) reactions and is attached to peptidoglycan by a DD-transpeptidase (DD-TPase) cross-linking twopeptides. Peptidoglycan is remodeled and hydrolyzed by various hydrolases: DD- or LD-carboxypeptidases (CPases) remove aterminal amino acid from the peptides, endopeptidases (EPases) hydrolyse DD- or LD-cross-links, lytic transglycosylases (LTs)cleave within the glycan strands producing 1,6-anhydro-N -acetylmuramic acid-containingmuropeptides, and amidases hydrolyzesthe amide bond betweenMurNAc and L-Ala. Light brown,N -acetylmuramic acid; blue rod,N -acetylgucosamine; dark orange rod,1,6-anhydro-N -acetylmuramic acid.

summarize the current knowledge on the regula-tion of septum-splitting peptidoglycan hydrolasesand on the process of outer membrane invaginationduring constriction.

Peptidoglycan synthesis and hydrolysisThe biosynthesis of peptidoglycan begins inthe cytoplasm with the synthesis of thenucleotide-activated precursors UDP-GlcNAc andUDP-MurNAc. The latter is synthesized fromUDP-GlcNAc by the enzymes MurA and MurB. Theamino acid ligases MurC, MurD, MurE, and MurFcatalyze the sequential ligation of l-Ala, d-Glu,m-Dap, and d-Ala-d-Ala to UDP-MurNAc.29 D-amino acids are converted from the L-enantiomersby racemases, and the d-Ala-d-Ala dipeptide issynthesized by the DdlA/B ligases. The next stepsoccur at the inner face of the cytoplasmic mem-brane.30 The transferaseMraY forms undecaprenyl-pyrophosphoryl-MurNAc(pentapeptide) (lipid I)and then the glycosyltransferase MurG trans-fers a GlcNAc residue from UDP-GlcNAc tolipid I, forming undecaprenyl-pyrophosphoryl-MurNAc(pentapeptide)-GlcNAc (lipid II). ManyGram-positive bacteria, but not E. coli, modifylipid II or nascent peptidoglycan by the additionof amino acids to position 3 of the peptide by

Fem-transferases,31 by the amidation of carboxylicgroups in the peptide,8,9 and/or by O-acetylation/N-deacetylation of glycan residues.7 Lipid II istransported across the cytoplasmic membrane byFtsW/RodA flippases32 where it is used as substrateof the glycosyltransferase (GTase) reactions to pro-duce new glycan strands at the periplasmic face ofthe cytoplasmic membrane (Fig. 2).33 Biochemi-cal data and crystal structures indicate that GTasesact processively.34 The growing glycan strand is thedonor and lipid II the acceptor in the reaction,whichreleases the undecaprenol pyrophosphate moietyfrom the growing glycan strand.3436 Peptides pro-truding from different glycan strands are cross-linked by transpeptidase (TPase) reactions to pro-duce the net-like peptidoglycan polymer (Fig. 2).37

TPases use a pentapeptide as donor and a tri-, tetra-,or pentapeptide as acceptor. During the reaction theterminal D-alanine residue of the donor peptide isreleased.Synthesis of newpeptidoglycan and its incorpora-

tion into the sacculus is accompaniedby the removalof old material by hydrolases (MurNAc-l-alanineamidases, DD-endopeptidases and lytic transglyco-sylases), a process called peptidoglycan turnover.38

The soluble peptidoglycan turnover products aretransported into the cytoplasm where they are recy-cled for de novo peptidoglycan synthesis.39

10 Ann. N.Y. Acad. Sci. 1277 (2013) 828 c 2012 New York Academy of Sciences.


The precise molecular mechanisms by whichthe peptidoglycan layer grows during cell elon-gation and division are not known. There areseveral models. According to Burman and Park,DD-endopeptidases cleavepeptide cross-links in thesacculus to allow the insertion of two newly synthe-sized glycan strands.40 The three for one modelby Holtje assumes that three new glycan strandsare synthesized and attached underneath a singledocking strand in the existing peptidoglycan. Si-multaneous removal of the docking strand by hy-drolysis allows the insertion of the three new strandsinto the peptidoglycan layer.41 This mechanism ex-plains the observed peptidoglycan turnover as theremoval of the docking stands. Holtje also pro-posed that peptidoglycan synthases and hydrolasesformmultienzyme complexes in which all activitiesrequired for a safe peptidoglycan growth mecha-nism are coordinated.15 Although an active pep-tidoglycan enlargement complex has not yet beenisolated from cells, recent research has identifiedseveral interactionsbetweendifferent peptidoglycansynthases, peptidoglycan synthases and hydrolases,and peptidoglycan enzymes and regulatory proteins(see below).

Activities and interactions ofpeptidoglycan synthasesPeptidoglycan synthases fall into three cate-gories; bifunctional GTase/TPase (class A penicillin-binding proteins (PBPs)), monofunctional TPase(class B PBPs), and monofunctional GTase.37 E. colihas three bifunctional PBPs (PBP1A, PBP1B, andPBP1C). PBP1A andPBP1Bhavemajor and semire-dundant roles in peptidoglycan synthesis. PBP2 andPBP3 are monofunctional TPases essential for cellelongation and cell division, respectively. The func-tions of the nonessential PBP1C and themonofunc-tional GTase MtgA are not known, although thelatter localizes to the cell division site.42 All pepti-doglycan synthases are anchored to the cytoplasmicmembrane, with their catalytic sites residing outsidethe cytoplasm.The activities of class A PBPs from E. coli have

been demonstrated in vitro with their natural sub-strate. PBP1A is capable of polymerizing lipid II toforma cross-linkedpeptidoglycanproduct that con-tains glycan strands with an average length of 20disaccharide units and with 1826% of the pep-tides present in cross-links.43 The catalytic amino

acids Glu-94 and Ser-473 are essential for GTaseand TPase activity, respectively. Remarkably, in thepresence of purified peptidoglycan sacculi, PBP1Ais capable of attaching a fraction of the newly madepeptidoglycan to the sacculi via transpeptidationreactions.43 Transpeptidation-mediated attachmentof nascent peptidoglycan chains occurs in the cellduring peptidoglycan growth.40,44

PBP1B exists as two main isoforms, PBP1B andPBP1B , that differ in the length of their shortcytoplasmic parts. PBP1B is a truncated versionoriginating from a translational start at Met-46 ofPBP1B.45 A third version, PBP1B, is generatedby an artificial cleavage of PBP1B by the outermembrane protease OmpT during cell fractiona-tion.46 PBP1B dimerizes both in the cell and in vitrowith a KD of 0.13 M.47,48 It exhibits GTase andTPase activities with the native lipid II substrate invitro. The enzyme is most efficient at conditions fa-voring its dimerization, suggesting that it might beactive as dimer in the cell. In vitro, PBP1B produceda peptidoglycan product with an average glycanstrand length of >25 disaccharide units, with up to50% of the peptides being part of cross-links.47 Thecatalytic amino acid residues Glu-233 and Ser-510are essential for the GTase and TPase activities, re-spectively.47,49 Other conserved residues within theGTase domain of PBP1B (Asp-234, Phe-237, His-240, Thr-267, and Gln-271) are essential for bothGTase activity and in vivo functioning.50 PBP1B isamong the few class A PBPs with a known crystalstructure, which includes the transmembrane helixand, next to the GTase and TPase domains, a smallUvrB-like domain situated between both catalyticdomains called UB2H.36

Bifunctional PBPs coordinate their GTase andTPase activities. Both, PBP1A and PBP1B requireongoing GTase reactions for efficient cross-linkingTPase activity.43,47 Hence, it is possible that thegrowing glycan strands produced by the GTase sitedelivers a pentapeptide donor peptide into the activesite of the TPase for the formation of a cross-link;this hypothesis is supported by the crystal structureof PBP1B showing that the GTase donor site forbinding of the growing glycan chain and the TPasesite are located on the same face of the molecule.36

The GTase activity can be affected by other factors:the GTase activity of PBP1B is stimulated by LpoBand FtsN23,47 and that of PBP1A by PBP2,51 whereasfull-length S. pneumoniae PBP2a produced longer



glycan strands than the truncated version lackingthe transmembrane anchor.52

Class B PBPs have an N-terminal membraneanchor followed by a noncatalytic domain and aTPase domain. The noncatalytic domain might berequired for the correct folding of the protein, asisolated TP domains of class B PBPs are intrinsi-cally unstable.53 In addition, the noncatalytic do-mainmight act as a pedestal to optimally positionthe TPase domain to interacting proteins and thepeptidoglycan layer.54 The TPase activity of a classB PBP had not been demonstrated until recently forE. coliPBP2,which is active in thepresenceofPBP1Aandpeptidoglycan sacculi.51 Under these conditionsPBP2 contributes to the attachment of newly madepeptidoglycan chains to sacculi by TPase reactions.Cells with a temperature-sensitive PBP2 versiongrown at elevated temperature or cells treated withthe PBP2-specific -lactam mecillinam grow intospheres and eventually lyse, indicating the essentialrole of PBP2 in cell elongation. PBP2 localizes tothe lateral wall and to mid-cell at an early stage ofseptation, but it leaves mid-cell before completionof cell division, indicating a role in cell elongationand early septation.55 InC. cresentus but not E. coli amild osmotic upshift causes the relocation of PBP2tomid-cell, indicating that changing environmentalconditions may affect growth modes in bacteria.56

H. pylori PBP2 interacts with the cell elongationprotein MreC,57 which, in E. coli, forms a com-plex withMreB andMreD.58 PBP3 (also called FtsI)localizes to the septum and its activity is essentialfor cell division.59 Although purified PBP3 binds arange of-lactams and is capable of transferring thedonor moiety of an artificial thioester substrate to aD-amino acid acceptor,60 its activity with a naturalpentapeptide donor and a peptidoglycan acceptorhas yet to be established. The interactions of PBP3with cell division proteins are discussed below.Several interactions between different peptido-

glycan synthases and between peptidoglycan syn-thases and other proteins have been detected.61

Pseudomonas aeruginosa PBP2 interacts with aCa2+-binding EF-hand motif of the peptidoglcyanhydrolase SltB1.62 E. coli PBP1B interacts with PBP3in vitro with a KD of 0.4 M, and the two proteinscan be cross-linked in the cell.63 The interaction wasconfirmed by bacterial two-hybrid system, whichfurther showed that the first 56 amino acids of PBP3are sufficient for the interaction with PBP1B. An in-

teraction between PBP1B andPBP3 in the cell is alsoconsistent with the following two observations: (i) afraction of the cellular PBP1B pool of wild-type cellslocalizes to mid-cell, depending on the presence,but not activity, of PBP3,63 and (ii) overexpressionof PBP1B suppresses the thermosensitive growth ofpbpB2158 (Ts) mutant cells, presumably by stabiliz-ing the labile PBP3 versionbydirect proteinproteininteraction.64 Hence, PBP1B and PBP3 likely pro-vide the main peptidoglycan synthesis activity forcell division, while PBP1A and PBP2 are likely mostactive during cell elongation.

Outer membrane lipoprotein activatorsof PBPsIn E. coli and presumably other Gram-negative bac-teria peptidoglycan synthesis is controlled not onlyfrom inside the cell by cytoskeletal structures ulti-mately linked toMreB and FtsZ, but also by recentlydiscovered outer membrane lipoproteins LpoA andLpoB. These proteins are essential for activating,from outside the sacculus, their cognate peptidogly-can synthase PBP1A and PBP1B, respectively.23,24

The cell requires either PBP1A-LpoA or PBP1B-LpoB for growth. Hence, the depletion of one of thelpo genes in the absence of the other, or in the ab-sence of a noncognate PBP, results in the lysis of thecell.TheLpoproteins interactwith small, noncatalytic

regions in their cognate PBP; for LpoB the non-catalytic region is the UB2H domain between thePBP1B GTase and TPase domains.24 Interestingly,both Lpo proteins stimulate in vitro the TPase ac-tivity of their cognate, but not noncognate, PBP.24

LpoB also stimulates the GTase activity of PBP1B.23

It appears that PBP1A and PBP1B activity requirestimulation by LpoA and LpoB, respectively, forproper functioning in the cell, which explains thelytic phenotype of lpoA/lpoB-depleted cells. Lpoproteins localize independently of, but with thesame preference for the side wall or mid-cell posi-tion as, their cognate PBP. LpoA has been shown tolocalize predominantly to the lateral wall and LpoBto the lateral wall and mid-cell.24 The septal local-ization of LpoB depends on the activity of PBP3,with LpoB mid-cell localization being diminishedafter inhibition of PBP3 with aztreonam. Presum-ably, LpoB requires ongoing septal peptidoglycansynthesis for mid-cell localization.24



Why are peptidoglycan synthases activated byouter membrane proteins? The following hypoth-esis is based on the crystal structure of PBP1B thatshows the LpoB-interactingUB2Hdomain to locatenot further than 60 A away from the cytoplasmicmembrane.24,65 Hence, with a distance from the cy-toplasmic membrane to the peptidoglycan layer of90 A, the PBP1B-LpoB interaction site would belocated in the space between the cytoplasmic mem-brane and the peptidoglycan, making it necessaryforLpoB to reach fromtheoutermembrane throughthe pores in the peptidoglycan to activate PBP1B.According to this hypothesis, the activation of PBPsby Lpo proteins becomes responsive to the state ofthe pores in the peptidoglycan network. The Lpo-mediated activation of PBPswould bemore efficientif the peptidoglycan were stetched and the poreswerewider.Conversely, the activationofPBPswouldbe less efficient if the peptidoglycan were more re-laxed and theporeswere smaller.Hence, peptidogly-can growth is activated when required, that is, whenit is stretched. This homeostatic regulation wouldallow the cell to maintain constant peptidoglycansurface density and thickness, andwould contributeto the adjustment of peptidoglycan synthesis rate tothe overall growth rate of the cell. However, mostlikely there are further mechanisms contributing tothe regulation of peptidoglycan growth.65

Components of the divisome and theirinteractionsThe cell division complex (divisome) is assembled atmid-cell to synthesize and cleave the septum and toseparate the cell into two daughters. Herein we con-sider aprotein as adivisomecomponent if it localizesto mid-cell in an FtsZ-dependent manner and if itparticipates in the process of cell division, whetheror not it is essential. The assembly of the divisomeis initiated by the GTP-dependent polymerizationof the tubulin-like FtsZ in a head-to-tail associa-tion.6668 Individual subunits form filaments andarches that combine to a ring-structuretermedZ-ringproximal to the inner face of the cytoplas-mic membrane at the prospective division site.69,70

TheZ-ring ishighlydynamicandFtsZ subunits con-stantly exchange with free, cytosolic FtsZ moleculesin a time scale of a few seconds.71 In vitro, rings ofFtsZ can form and spontaneously open and depoly-merize, as observed by atomic force microscopy.72

FtsZ is stabilized at mid-cell position by several pos-

itive regulators (see below). In addition, there are atleast twomechanisms that, together, prevent Z-ringformation and cell division away frommid-cell. TheMinC/MinD/MinE proteins prevent Z-ring assem-bly near the poles and their absence causes polardivisions, leading to DNA-free minicells. In E. colithe MinC/MinD inhibitor complex of Z-ring for-mation oscillates from pole-to-pole, driven by themembrane-bound ATPase MinE, resulting in theaverage concentration of MinC/MinD being small-est at mid-cell, thus allowing Z-ring assembly.73,74

The crystal structure of MinDMinE complexes areconsistent with a model of MinE moving betweenmembrane-bound MinD molecules driven by in-duced conformational changes.75

Another negative regulator of FtsZ assembly, theB. subtilis nucleoid occlusion factor Noc and itsE. coli functional analog SlmA prevent cell divisionnear the chromosomal DNA to avoid the closing ofthe septum through the nucleoid, which would belethal.76,77 Recent structural and mechanistic datasuggest that SlmA dimers bind to specific sites onthe chromosome and interact with FtsZ to disruptits polymerization.78,79 The B. subtilis Noc proteinhas 70 binding sites on the chromosome and co-ordinates chromosome segregation with division.80

Noc might not interact directly with FtsZ, indicat-ing its mechanism of action is different from that ofSlmA, possibly involving downstream componentsof the divisome.81 The nucleoid occlusion mecha-nism and Min proteins also ensure the remarkablyhigh precision by which E. coli cells define theirmid-cell position, leading to daughter cells with anaverage length deviation of only a few percent.82,83

The division site selection is robust and works evenin E. coli cells grown in nanofabricated channels toirregular cell shape.84

Cell division in Gram-negative bacteriaThe divisome proteins of E. coli (Table 1) assembleat mid-cell in two steps (Fig. 3):85 FtsZ, FtsA, ZipA,ZapA, ZapB, ZapC, ZapD, and FtsEX assemble earlyat the future division site and well before a con-striction is visible. Their localization coincides withthe phase of preseptal elongation, and it has beensuggested that once assembled the early cell divi-sion proteins control cell wall elongation complexescontaining PBP1A and PBP2.61 Immediately beforeconstriction the divisomematures by incorporatingFtsK, FtsQ, FtsL, FtsB, FtsW, PBP3 (FtsI)-PBP1B,



Table 1.Divisome proteins of E. coli

Function/category Protein (gene)a Role/remarksb

Cytoskeletal protein FtsZ (ftsZ) tubulin-like, polymerizes with GTP,forms the Z-ring at mid-cell

Membrane attachment of

FtsZ and regulation of

Z-ring dynamics

FtsA (ftsA), ZipA (zipA), ZapA(zapA), ZapB (zapB), ZapC

(zapC), ZapD (yacF/zapD)

Membrane attachment of FtsZpolymers (FtsA, ZipA)

Stabilization of Z-ring andregulation of its dynamics (ZapA,

ZapB, ZapC, ZapD)

Divisome maturation and

stability, PG-binding

FtsK (ftsK ), FtsQ (ftsQ), FtsL(ftsL), FtsB (ftsB)

Recruitment of downstreamdivisome proteins (FtsK, FtsQLB)

and DNA transport (FtsK) FtsW (ftsW ) Lipid II flippase (FtsW) FtsN (ftsN), DamX (damX),DedD (dedD), RlpA (rlpA)

PG binding (FtsN, DamX, DedD,RlpA) and divisome stability

(FtsN)

PG synthesis (and its

regulation)

PBP1B (mrcB), PBP3 (ftsI),MtgA (mtgA)

Synthesis of PG (PBP1B, PBP3,MtgA)

LpoB (lpoB) Activation of PBP1B (LpoB)PG hydrolysis (and its

regulation)

AmiA (amiA), AmiB (amiB),AmiC (amiC)

Septal PG cleavage for daughter cellseparation (AmiA, AmiB, AmiC)

FtsE (ftsEc), FtsX (ftsX c), EnvC(envC), NlpD (nlpD)

Control of septal PG cleavage(FtsEX, EnvC, NlpD)

OM invagination Pal (pal), TolA (tolA), TolB(tolB), TolQ (tolQ), TolR (tolR)

OM invagination and stabilityduring division (TolA,TolB, TolQ,

TolR, Pal) PG binding (Pal)

aGenes essential for cell division are written in bold.bSee the text for detailed discussion and references.cNot essential at high osmolarity of the growth medium.

andFtsN,whilePBP2 leaves the cell division site.55,85

How the steps in divisome assembly are temporallycontrolled is largely unknown but likely involvesmultiple proteinprotein interactions between itscomponents. The interactions of relevant divisomeproteins are summarized in Figure 4. The timing ofmid-cell arrival of cell division proteins is similar inC. crescentus,with some differences, for example therelatively late arrival of FtsW and FtsB.86

ZipA and the actin-like FtsA are essential forcell division. They bind to the same C-terminalregion in FtsZ, stabilize the Z-ring, and anchor itto the cytoplasmic membrane.87,88 FtsA polymer-izes bi-directionally and forms membrane-attachedprotofilaments.89,90 The membrane potential stim-ulates the attachment of FtsA and other cell mor-phogenesis proteins like MreB and MinD to the

membranes, which explains why compounds af-fecting the membrane potential delocalize theseproteins and cause growth and cell division de-fects.91 Other cytoplasmic Z-ring associated pro-teins, ZapA, ZapB, ZapC, and ZapD, are dispens-able for cell division. ZapA and ZapD interact withFtsZ, stimulating protofilament association and sta-bilizing the Z-ring.9294 Cells with reduced FtsZlevel cannot divide when they lack ZapA, and adouble mutant lacking zapA and zapD has an in-creased cell length, consistent with a slight divisiondefect.92,93 ZapB interacts with ZapA and formsspontaneous filaments in vitro.95,96 ZapB-mCherrylocalizes inside the Z-ring and presumably stabi-lizes the Z-ring via ZapA.97 ZapA and ZapB arerequired for mid-cell anchoring of MatP, a proteinthat structures the chromosomal terminus region



Figure 3. Hierarchical recruitment of cell division proteins.The divisome is build from inside the cell, with FtsZ and theearly cell divisionproteins localizingwell before septation starts.The black arrows indicate dependency on mid-cell localizationmediated in most cases by direct proteinprotein interaction.The gray arrows show further direct interactions involving pep-tidoglycan enzymes. The late cell division proteins include thelipid II flippase FtsW (green), peptidoglycan synthases (blue),and peptidoglycan hydrolases (red). PBP1B-LpoB are not es-sential for cell division, as their function can be taken over byPBP1A-LpoA. Peptidoglycan hydrolases including the amidasesare not required for cell division but for separation of daughtercells.

into a macrodomain.98 The relocation of the ter-minus macrodomain from the cell pole to the divi-sion site via MatPZapA/ZapB interactions occursprior to the replication of the terminus region andis required for proper nucleoid segregation.98 ZapCinteracts with FtsZ and localizes to mid-cell whereit stabilizes the Z-ring by promoting FtsZ polymerbundling and by suppressing the GTPase activityof FtsZ.99,100 In bacteria other than E. coli thereare different combinations of FtsZ-ring stabilizingproteins.101

FtsEX is an ATP-binding cassette (ABC) trans-porter homolog that binds to FtsZ via the ATP-binding protein FtsE.102104 Mid-cell localization ofFtsE depends on the presence of FtsZ andZipA. FtsEhas been shown to be essential for cell divisionwhencells grow in low-osmolarity growth medium;105,106

its function in cell division is to recruit and regulatepeptidoglycan hydrolases (see below).In order to start septation, a number of essen-

tial late cell division proteins, FtsK, FtsQ, FtsL,FtsB, FtsW, PBP3, and FtsN, assemble to the Z-ring almost simultaneously and in an interdepen-dent fashion (Fig. 3).85,107 Several other proteins notessential for septation also localize to mid-cell de-pending on Fts proteins, including the peptidogly-can synthase PBP1B63 and the hydrolases AmiB andAmiC108,109 and their regulators LpoB,24 EnvC, andNlpD.110 Presumably, these peptidoglycan enzymesare not essential for cell division/cell separation be-cause their function can be taken over by redun-dant enzymes. For example, in the absence of thebi-functional peptidolgycan synthase PBP1B, thehomologous PBP1A becomes essential and showsenhanced localization at mid-cell, where it presum-ably takes over PBP1Bs role in septal peptidoglycansynthesis.51

FtsK is amultifunctional protein involved in bothchromosome segregation and cell division. In bacte-rial two hybrid assays FtsK interacts with FtsZ, FtsQ,FtsL, and PBP3.111113 The cytoplasmic domainof FtsK forms hexamers to directionally transportDNA, required for decatenation of sister chromo-somes, together with the Xer recombinases.114,115

This function of FtsK is only required in the fractionof cells with catenated chromosomes. The mem-brane/periplasmic part of FtsK is essential for celldivision, presumably by stabilizing late-cell divisionproteins at the septum, which explains the existenceofmutations in ftsA and ftsQ that allow for septationin the absence of FtsK.112,116,117

FtsK is required for septal recruitment of apreformed FtsQFtsLFtsB complex.107,118 Each ofthese proteins is a bitopic membrane protein witha small cytoplasmic part and periplasmic domains.The crystal structure of the periplasmic domainsof FtsQ identified two distinct regions: (i) a PO-TRA (polypeptide transport associated) domain, ofwhich the second -strand is essential for FtsQmid-cell localization, and (ii) the C-terminal -domain of FtsQ that is essential for the recruitmentof FtsL, FtsB, and FtsW.119,120 The C-terminal do-main of the periplasmic part of FtsL is required foritsmid-cell localization via interactionwith FtsQ.121

FtsQ-FtsL-FtsB 1:1:1 or 2:2:2 complexes have beenmodeled,122 the latter being consistent with thecrystal structure of an FtsQ dimer.120 Small angle



Figure 4. Proteinprotein interactions of divisome proteins. Interactions are shown individually. Solid black lines represent directinteractions identified in vitro and in the cell; dashed gray lines represent interactions shown solely by bacterial two-hybrid assays.Rectangular arrows enclosing proteins indicate homodimerization or multimerization. Numbers refer to reference numbers in thereference list. The referencing is not exhaustive for well-studied interactions (like FtsZ-FtsZ or FtsZ-FtsA).

X-ray scattering of a homologous DivIB(FtsQ)-FtsL-DivIC(FtsB) complex from the Gram-positiveStreptococcus pneumoniae is consistent with a 1:1:1complex.123 In E. coli FtsQ has been shown to inter-act with FtsW and PBP3 by bacterial two-hybridanalysis.111,113 The requirement for FtsQ can bebypassed by expressing fusion proteins of ZapA-FtsL and ZapA-FtsB, either of which is sufficient forthe recruitment of FtsW and PBP3.124

FtsW is member of the SEDS (shape, elongation,division, and sporulation) family of integral mem-brane proteins that also includes the essential cellelongation protein RodA and the sporulation pro-tein SpoVEof theGram-positiveB. subtilis.125 FtsW,an integral transmembrane protein with 10 trans-membrane regions, has recently been identified asthe long-searched transporter (flippase) for the lipidII precursor.32 Consistent with this function, FtsWinteracts with both PG synthases, PBP3 and PBP1B,and is required for recruiting PBP3 to the divi-some, presumably together with PBP1B.63,126128

The TPase PBP3 also interacts with FtsL,113 FtsQ,113

PBP1B,63 and FtsN.129 PBP3 can be recruited to thedivisome independently of the FtsQLB complex viaan FtsW-ZapA fusion, and targeting a FtsW-PBP3fusion to mid-cell restored the recruitment of Ft-sQLB in cells depleted of FtsA.124 These data suggestthat FtsW-PBP3 and FtsQLB complexes form inde-pendently of one another before recruitment to thedivisome, which is consistent with interaction stud-ies using Forster Resonance Energy Transfer (FRET)andco-immunoprecipitation.126,127 InC. crescentus,PBP3s early localization to the Z-ring relies largelyon a functional TPase domain, indicating that TPasesubstrates, or an active state of the enzyme, mightbe required.130

FtsN is an essential cell division protein thathas been thought to be restricted to enterobacte-ria. However, recent work in C. crescentus showedthat FtsN-like proteins are more widely distributedamong the proteobacteria.131 In E. coli, FtsN in-teracts with both early and late cell division



proteinsFtsA, ZapA, PBP3, FtsW, and PBP1Bas shown by various in vitro (surface plas-mon resonance, affinity chromatography) andin vivo (co-immunoprecipitation, FRET) tech-niques.126,127,129,132 Bacterial two-hybrid analysisshows additional interactions with FtsQ.111,113 FtsNis a bitopic membrane protein with a short cy-toplasmic part and one transmembrane region.The periplasmic part contains three -helices fol-lowed by a proline/glutamate-rich unstructured re-gion and a globular C-terminal SPOR peptidogly-canbindingdomain.133 While the SPORdomainhasbeen shown to interact with peptidoglycan in vitroit is not essential for cell division.134 Presumably,the peptidoglycan-binding function of FtsNs SPORdomain can be taken over by the SPOR domain-containing proteins DamX, DedD, and RlpA, whichlocalize to cell division site, as mutants lacking mul-tiple SPOR domain proteins show cell division de-fects.135,136 The precise role of FtsN during celldivision is not known. Its periplasmic region, com-prising the three partially formed -helices (aminoacids 6267, 8093, and117123), is essential for celldivision for unknown reasons. FtsN interacts withbothPBP1BandPBP363,129 and stimulates the activ-ity of PBP1B, presumably by stabilizing the dimericform of PBP1B129 consistent with the hypothesisthat a main functional role for FtsN in cell divisionis to coordinate the peptidoglycan synthases activeduring septation. Additional functions of FtsN havealso been suggested. For example, FtsN could pro-vide a signal for completion of divisome assemblyto the cytoskeletal components, a role supported byits cytoplasmic interaction with the 1c subdomainof FtsA132 and the existence of mutants with alteredFtsA that can divide without FtsN.137 FtsN has alsobeen implicated in a divisome stabilizing function,which is supported by the observation that FtsN-depletion leads to the disassembly of the already as-sembled divisome components, including the earlyproteins.138

FtsP (SufI) is a recently characterized solubleperiplasmic protein involved in cell division, al-though its precise role is unknown. It localizes tothe division sites dependent on the presence of FtsZ,FtsQ, FtsL, and FtsN.139 FtsP is dispensable un-der normal growth conditions but is required fordivisome stability when cells grow at various stressconditions, including oxidative stress and DNAdamage.140 The crystal structure of FtsP shows

structural similarity to themulticopper oxidase pro-tein family, but does not bind the metal ion.139

Cell division proteins specific to Gram-positivebacteriaIn this section we highlight several recent find-ings specific to Gram-positive bacteria. These havea thick septum peptidoglycan with distinct zonesof high and low electron densities, as visualizedby cryo-electrontomography,141,142 and they com-plete septal cross-wall synthesis before daughter cellseparation (Fig. 1). Gram-positive species containmost of the essential division proteins discussedabove, with a few exceptions such as FtsK, FtsP, andFtsN.Conserved eukaryotic-type Ser/Thr protein ki-

nases regulate various cellular processes includingcell division in Gram-positive species. The Strepto-coccus pneumoniae protein kinase StkP is requiredfor proper septal cell wall synthesis by yet unknownmechanisms.143,144 It contains several PASTA do-mains, which are also present in some PBPs andhave been suggested to bind to peptidoglycan frag-ments and -lactams.145 StkP phosphorylates thecell divisionproteinDivIVA,which is found inmanyGram-positive species that undergo elongation anddivision modes of PG synthesis, and both proteinslocalize to mid-cell and the poles. In Bacillus subtilisDivIVA regulates the septation site by positioningand stabilizing the FtsZ inhibitorMinC/MinDat thecell poles. Mutants lacking DivIVA lose the topo-logical control over MinC/MinD and form mini-cells derived from aberrant septation events at thepoles.146 DivIVA localizes to the new poles imme-diately after daughter cell separation and indepen-dentofdivisomeproteins toprevent aberrantZ-ringassembly after division.147,148 Interestingly, DivIVAhas an amphiphatic alpha-helix for membrane at-tachment, and finds the new cell pole localization byvirtue of its affinity to curved membranes, a featurethat explains its polar localization in a number ofheterologous organisms like E. coli and yeast.149

The cytoplasmic cell division protein EzrA ispresent in B. subtilis and S. aureus, but EzrA de-pletion causes a severe cell division phenotypeand an increase in cell diameter only in the lat-ter spherical-shaped species.150152 EzrA has a N-terminal membrane anchor and interacts with FtsZ.It acts as a negative regulator of Z-ring assemblyby decreasing GTP binding affinity and increasing



the GTPase activity of FtsZ, thus increasing FtsZ-depolymerization.151,153,154 Its localization to thedivisome during division suggested that it regulatesZ-ring dynamics during constriction.153 EzrA hasalso been shown to play a role in the switch fromlateral to septal cell wall synthesis in B. subtilis, to-gether with GpsB.155 This is achieved by positioningthe class A PBP1 (encoded by the ponA gene), themajor PG synthase of B. subtilis, through directproteinprotein interactions. EzrAandGpsB recruitPBP1 to the divisome, and GpsB is responsible forthe removal of PBP1 from the mature cell pole afterdivision making it available for cell elongation.155

Another recently identifiedmember of theB. sub-tilis divisome, not found in Gram-negative bacteria,is SepF. Cells lacking SepF form grossly distorted di-vision septa.156 SepF is recruited to the divisome bydirect interaction with FtsZ. It negatively regulatesthe GTPase activity of FtsZ and thus stabilizes FtsZfilaments.156,157 Interestingly, in vitro SepF formsrelatively large rings with a diameter of 50 nm andit is able to bundle FtsZ filaments into long, regularstructures which resemblemicrotubules. Therefore,it has been proposed that SepF is required for theregular arrangement and bundling of FtsZ filamentsfor proper septum placement.158

Peptidoglycan hydrolases for growthregulation and cell separationPeptidoglycan hydrolases are ubiquitous en-zymes.159 Some of them lyse target bacteria as partof the host defense or in bacterial interactions.160,161

Others are produced by bacteria to remodel or turnover their own peptidoglycan and to facilitate cellseparation during or after cell division.DD-Carboxypeptidases trim pentapeptides in

newly made peptidoglycan to tetrapeptides, whichcan be further shortened to tripeptides byLD-carboxypeptidases and to dipeptides toDL-carboxypeptidases (Fig. 2). The activities ofthese enzymes might spatiotemporally regulate theincorporation of new peptidoglycan into the saccu-lus by controlling the amount of donor and acceptorpeptides for transpeptidation, perhaps explainingthe observed shape defects in carboxypeptidasemu-tants of E. coli,Helicobacter pylori, andCampylobac-ter jejuni.162164 In E. coli DD-carboxypeptidaseshelp to properly orient the Z-ring in the cell di-vision plane, and their absence causes cells toform branched cell shapes.165 PBP5, the major

DD-carboxypeptidase in E. coli, contributes to themaintenance of the cell shape and diameter.166 Itlocalizes to foci in the lateral wall and to the celldivision site presumably by recognizing active areasof PG synthesis.167

Lytic transglycosylases, DD-endopeptidases andN-acetylmuramyl-l-alanine amidases (amidases)are responsible for peptidoglycan turnover and sep-aration of daughter cells. Their cleavage sites inpeptidoglycan are shown in Figure 2. The E. coliDD-endopeptidases PBP4 and PBP7 hydrolyze DDcross-links,168 whereas MepA is capable of cleav-ing both DD and LD cross-links.169 The E. coliDD-endopeptidases have auxiliary roles in cell mor-phogenesis,170,171 in contrast to the three H. pyloriDD-endopeptidases Csd1, Csd2, and Csd3, whichcontribute to the generation of helical cell shapepresumably by localized relaxation of peptide cross-links.172

E. coli has seven lytic transglycosylases, includ-ing the periplasmic Slt70 and the outer membranelipoproteins MltA, MltB, MltC, MltD, MltE, andMltF. These muramidases cleave the -1,4 glyco-sidic bond between MurNAc and GlcNAc, form-ing a 1,6-anhydro ring on the MurNAc residue.173

There are some differences in the specificity of lytictransglycosylases. Slt70 only cleaves glycan strandswith peptides but not glycan strands lacking these,whereas MltA is able cleave either type.174 A num-ber of crystal structures of Slt70 and soluble ver-sions of MltA, MltB, and MltE have allowed de-duction of the mechanism of the glycan strandcleavage. They possess a catalytic glutamate residueand lack the conserved catalytic aspartate presentin lysozyme, which, unlike lytic transglycosylases,hydrolyzes the glycosidic bond between GlcNAc andMurNAc.175178 Lytic transglycosylases interact withPBPs suggesting that they are components of pepti-doglycan synthesis complexes.179,180

E. coli has four amidases, the soluble periplasmicAmiA,AmiB, andAmiC181 and the outermembranelipoprotein AmiD.182,183 They hydrolyze the amidebond betweenMurNAc and l-Ala and therefore re-lease the peptides from the glycan strand. AmiB andAmiC localize to the division site, whereas AmiAlocalizes more diffuse within the periplasm.108,109

The latter is a zinc metalloenzyme active on poly-meric peptidoglycan and requiring at least tetrasac-charide sized fragments.184 AmiA, AmiB, and AmiChave major roles in the cleavage of the septum



Figure 5. Different presumed interaction states of TolPal proteins. The cartoon shows the proteins of the TolPal complex andtheir domains as linked ovals or spheres. The three panels show the proposed interaction states of TolPal proteins between thecytoplasmic membrane (CM) and outer membrane (OM). Panel I shows Pal bound to the peptidoglycan (PG) sacculus and TolBbinding with its N-terminal loop region (shown as a green line) to the IM-anchored TolA, which forms a complex with TolQ andTolR membrane proteins. In panel II Pal binds to the C-terminal domain of TolB after their respective dissociation from PG andTolA. Panel III shows Pal bound to domain III of TolA after dissociation from TolB. It is not yet clear if TolB interacts with TolAand/or Pal to form a trimeric PalTolATolB complex, and the existence of a TolAPal interaction is disputed. In vitro data supportthe states depicted in panels I and II, while in vivo cross-linking data suggest that all three states are possible. Thus, either a transienttrans-envelope complex links the OM, PG, and IM (state III) or the cycling between states I and II maintains sufficient contactbetween the envelope layers for proper invagination. The role of YbgF is not yet known. Alone, YbgF forms a homotrimer, thoughits interaction with domain II of TolA via its C-terminal domain has a 1:1 stoichiometry.

during cell division to allow the separationofdaugh-ter cells.181 Mutants lacking two or more amidasesform chains of nonseparated cells and have in-creased outermembrane permeability. Both pheno-types become more severe in amidase mutants ad-ditionally lacking DD-endopeptidases and/or lytictransglycosylases.185,186 Remarkably, about 1/3 ofthe newly made septal peptidoglycan is immedi-ately removed during septum synthesis presumablyby the septum-splitting hydrolases.187 This is consis-tent with the finding that AmiC is capable of remov-ing fluorescent peptides incorporated into the pep-tidoglycan during growth from the division site.188

Regulation of septum cleaving amidasesHow the potentially dangerous peptidoglycanhydrolases are regulated in the cell is not well un-derstood. Recent work from the Bernhardt labora-tory has provided significant understanding of theregulation of septum cleaving amidases. These datasuggest that the amidases require specific activa-tor proteins, EnvC or NlpD, that are themselvesrecruited to the divisome and are activated by FtsNor FtsEX.109 The periplasmic EnvC activates AmiA

and AmiB, and the outer membrane lipoproteinNlpD activates AmiC.109 Importantly, the expres-sion of a mis-localized EnvC causes cell lysis, pre-sumably by activating AmiA and AmiB at inappro-priate sites away from mid-cell.109 FtsN is requiredfor mid-cell localization of AmiB but not of EnvC,which is recruited earlier by FtsEX. FtsN is also re-quired for mid-cell localization of AmiC and itsactivatorNlpD.189 Importantly, the recruitment andactivation of the amidases to the divisome requiresactive PBP3. EnvC and NlpD, but not the amidases,localize in cephalexin-treated cells with blockedPBP3, indicating that PBP3-catalyzed septal PG syn-thesis precedes the recruitment and activation of theamidases.189

Both EnvC and NlpD possess a catalytically inac-tive C-terminal LytM domain. EnvC has a coiledcoil region that is essential for its recruitment to thedivisome,110 which also requires the large periplas-mic region in FtsX. Interestingly, FtsEs ATPasefunction is required for EnvC-mediated activationof AmiA and AmiB, suggesting that hydrolysis ofbonds in PG for septum cleavage is coupled to hy-drolysis of ATP.190 The activation of AmiB requires



a conformational change to remove an -helix toopen the active site cleft for substrate binding, amechanism that appears to be conserved betweenseptum-cleaving amidases.191

Outer membrane constriction during celldivisionThe outer membrane protects Gram-negative bac-teria against many antibiotics and antibacterial en-zymes by preventing access to their periplasmic orintracellular target.192 In E. coli the outer mem-brane is firmly attached to the peptidoglycan bythe highly abundant outer membrane lipoproteinLpp (Brauns lipoprotein), a fraction of which is co-valently linked to peptidoglycan.5,193 Other abun-dant outer membrane proteins, such as OmpA andthe lipoprotein Pal, interact noncovalently with thepeptidoglycan layer. The deletion of pal or lpp genesresults in reduced outermembrane integrity and in-creased release of outer membrane vesicles into thegrowth medium.194,195 Overexpression of pal res-cues lpp null mutants, but an overexpression of lppcannot rescue pal deletion strains, indicating thatPal has amore specific role than simply tethering theOM to the peptidoglycan.195 Indeed, Pal has beenimplicated with members of the TolPal system inaiding outer membrane constriction during cell di-vision. Cells deficient in this function typically formshort chains in low osmotic growth medium andhave a reduced outer membrane stability, leadingto periplasmic leakage and the formation of outermembrane blebs at division sites.196

The TolPal system is conserved in Gram-negative species and consists of the integral cyto-plasmic membrane proteins TolQ, TolR, and TolA,the soluble periplasmic protein TolB, and the outermembrane protein Pal.197 Each of these proteinslocalize to mid-cell during division dependent onFtsN, with TolQ and TolA localizing independentlyfrom the other TolPal components.196 TolA is ableto localize to mid-cell in ftsA mutant cells that di-vide without FtsN, indicating that ongoing sep-tation might be sufficient for its localization.137

Proteinprotein interactions between TolPal pro-teins can potentially connect the cytoplasmic mem-brane, peptidoglycan, andoutermembrane.196 TolQcontains two larger cytoplasmic and three trans-membrane regions; TolR and TolA are both an-chored in the cytoplasmic membrane by a singletransmembrane region near the N-terminus with

Figure 6. Cartoon of the divisome. The proteins of the divi-some are shown as ovals, spheres, or as filament (FtsZ). Theirlocalization in the cytoplasmic membrane (CM), periplasm, oroutermembrane (OM) are indicated. Interactions between pro-teinswithin thecomplexare roughly represented. It isnotknownif MipAMltA, which interact with PBP1B, localize to mid-cell.

most of the protein locating in the periplasm.198 Theperiplasmic part of TolR includes a C-terminal am-phiphatic helix that is proposed to associate with theperiplasmic leaflet of the cytoplasmicmembrane.199

The periplasmic region of TolA contains the elon-gated, mainly -helical, domain II and the globularC-terminal domain III.200,201 The crystal structureof TolB shows a C-terminal six-bladed -propellerdomain, along with a globular N-terminal domainthat features a flexible 12 amino acid N-terminusthat binds into a cleft between theN- andC-terminaldomains when TolB interacts with Pal.202,203

TolQ, TolR, and TolA form a cytoplasmic mem-brane complex through interactions via their trans-membrane regions.204,205 Domain II of TolA alsointeracts with YbgF, a nonessential protein ofunknown function encoded by the last gene inthe tolpal operon.206 Presumably, TolA undergoesconformational changes in its periplasmic domainsdriven by TolQ/TolR, changes that are energized bythe membranes proton motive force (pmf). Themechanism is thought to provide the energy for



outer membrane constriction during cell division,but most details of the process are not understood.Likely, there are different states of interactions be-tween the TolPal proteins in the cell envelope(Fig. 5).196,207 TolA was cross-linked to Pal viadomain III, and subsequent in vivo cross-linkingmapped separate interaction sites of Pal with TolBand TolA.208,209 However, studies with the purifiedproteins could not confirm an interaction betweenTolAandPal or the formationof a ternaryTolAPalTolB complex.203 Gerding et al. proposed that pmf-dependent conformational changes inTolAproducecycles of Pal-binding and -dissociation and, conse-quently, facilitate transient interactions between theouter membrane and PG and the outer membraneand the inner membrane, respectively.196 In addi-tion, TolB might cycle between a Pal-bound andPal-unbound form whereby only the latter allowsfor interaction with TolA via its flexible 12 aminoacid N-terminus (Fig. 5).203

The PBP1BLpoB complex spans from the cyto-plasmicmembrane to the outermembrane and thuscould contribute to the constriction of the outermembrane during cell division. This hypothesis issupported by the observation that cells lacking botha functional TolPal system (pal) and LpoB show asevere lysis phenotype, while the single mutants areviable and do not lyse.24 In a pal background, LpoBmislocalization to the inner membrane is as detri-mental to the cell as is the deletion of lpoB.24 There-fore, correct outer membrane localization of LpoBis required to support outermembrane constrictionin the absence of a functional TolPal system. Forunknown reasons PBP1ALpoA cannot take overthis specific function of PBP1BLpoB. In summary,in E. coli it is presumably the combined activitiesof the TolPal system and the peptidoglycan syn-thesis complex for cell division (with PBP1B andLpoB) that facilitate outer membrane constrictionduring cell division. TolPal proteins are essentialin C. crescentus, which lacks PBP1B and LpoB.210

In C. crescentus, the LytM-domain protein DipM isrequired for constriction. A dipM mutant also hasthicker peptidoglycan and loses the integrity of theouter membrane, presumably by mal-functioningof the TolPal system.211213

Concluding remarksMuch progress has been made over the last decadestoward understanding the physiology of bacterial

cell division. The divisome consists of more than20 proteins, including novel ones recently identified(Fig. 6). The crystal structures of some of the keyproteins and some important interactions are nowknown, as are their interdependency for localiza-tion and the timing of their arrival at the divisionsite. However, the molecular mechanisms and dy-namics underlying the synthesis and cleavage of thedivision septum have remained largely unknown.Many cell division proteins lack an enzymatic ac-tivity and might act in coordinating cytokinesis,peptidoglycan synthesis and cleavage, and outermembrane invagination. We expect that recenttechnological advances, including in vitro recon-struction of peptidoglycan synthesis and divisomalprotein assembly, superresolution microscopy, andhigh-throughput chemical genomics,will helpbringa more complete understanding of bacterial celldivision.

AcknowledgmentsWe thank Jacob Biboy for critical reading ofthe manuscript. This work was supported by theBiotechnology and Biological Sciences ResearchCouncil, UK, and the European Commission (DI-VINOCELL HEALTH-F3-2009-223431).

Conflicts of interestThe authors declare no conflicts of interest.

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