The cell division complex (divisome) is assembled at mid-cell to synthesize and cleave the septum and to separate the cell into two daughters. Herein we consider a protein as a divisome component if it localizes to mid-cell in an FtsZ-dependent manner and if it participates in the process of cell division, whether or not it is essential. The assembly of the divisome is initiated by the GTP-dependent polymerization of the tubulin-like FtsZ in a head-to-tail association.66–68 Individual subunits form filaments and arches that combine to a ring-structure—termed Z-ring—proximal to the inner face of the cytoplasmic membrane at the prospective division site.69,70 The Z-ring is highly dynamic and FtsZ subunits constantly exchange with free, cytosolic FtsZ molecules in a time scale of a few seconds.71 In vitro, rings of FtsZ can form and spontaneously open and depolymerize, as observed by atomic force microscopy.72 FtsZ is stabilized at mid-cell position by several positive regulators (see below). In addition, there are at least two mechanisms that, together, prevent Z-ring formation and cell division away from mid-cell. The MinC/MinD/MinE proteins prevent Z-ring assembly near the poles and their absence causes polar divisions, leading to DNA-free minicells. In E. coli the MinC/MinD inhibitor complex of Z-ring formation oscillates from pole-to-pole, driven by the membrane-bound ATPase MinE, resulting in the average concentration of MinC/MinD being smallest at mid-cell, thus allowing Z-ring assembly.73,74 The crystal structure of MinD–MinE complexes are consistent with a model of MinE moving between membrane-bound MinD molecules driven by induced conformational changes.75
Cell division in Gram-negative bacteria
The divisome proteins of E. coli (Table 1) assemble at mid-cell in two steps (Fig. 3):85 FtsZ, FtsA, ZipA, ZapA, ZapB, ZapC, ZapD, and FtsEX assemble early at the future division site and well before a constriction is visible. Their localization coincides with the phase of preseptal elongation, and it has been suggested that once assembled the early cell division proteins control cell wall elongation complexes containing PBP1A and PBP2.61 Immediately before constriction the divisome matures by incorporating FtsK, FtsQ, FtsL, FtsB, FtsW, PBP3 (FtsI)-PBP1B, and FtsN, while PBP2 leaves the cell division site.55,85 How the steps in divisome assembly are temporally controlled is largely unknown but likely involves multiple protein–protein interactions between its components. The interactions of relevant divisome proteins are summarized in Figure 4. The timing of mid-cell arrival of cell division proteins is similar in C. crescentus, with some differences, for example the relatively late arrival of FtsW and FtsB.86
Table 1. Divisome proteins of E. coli
|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 FtsZ polymers (FtsA, ZipA)|
| || || • Stabilization of Z-ring and regulation of its dynamics (ZapA, ZapB, ZapC, ZapD)|
|Divisome maturation and stability, PG-binding|| • FtsK (ftsK), FtsQ (ftsQ), FtsL (ftsL), FtsB (ftsB)|| • Recruitment of downstream divisome 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 cell separation (AmiA, AmiB, AmiC)|
| || • FtsE (ftsEc), FtsX (ftsXc), 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 stability during division (TolA,TolB, TolQ, TolR, Pal)|
| || || • PG binding (Pal)|
Figure 3. Hierarchical recruitment of cell division proteins. The divisome is build from inside the cell, with FtsZ and the early cell division proteins localizing well before septation starts. The black arrows indicate dependency on mid-cell localization mediated in most cases by direct protein–protein interaction. The gray arrows show further direct interactions involving peptidoglycan enzymes. The late cell division proteins include the lipid II flippase FtsW (green), peptidoglycan synthases (blue), and peptidoglycan hydrolases (red). PBP1B-LpoB are not essential for cell division, as their function can be taken over by PBP1A-LpoA. Peptidoglycan hydrolases including the amidases are not required for cell division but for separation of daughter cells.
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Figure 4. Protein–protein interactions of divisome proteins. Interactions are shown individually. Solid black lines represent direct interactions 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 the reference list. The referencing is not exhaustive for well-studied interactions (like FtsZ-FtsZ or FtsZ-FtsA).
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ZipA and the actin-like FtsA are essential for cell division. They bind to the same C-terminal region in FtsZ, stabilize the Z-ring, and anchor it to the cytoplasmic membrane.87,88 FtsA polymerizes bi-directionally and forms membrane-attached protofilaments.89,90 The membrane potential stimulates the attachment of FtsA and other cell morphogenesis proteins like MreB and MinD to the membranes, which explains why compounds affecting the membrane potential delocalize these proteins and cause growth and cell division defects.91 Other cytoplasmic Z-ring associated proteins, ZapA, ZapB, ZapC, and ZapD, are dispensable for cell division. ZapA and ZapD interact with FtsZ, stimulating protofilament association and stabilizing the Z-ring.92–94 Cells with reduced FtsZ level cannot divide when they lack ZapA, and a double mutant lacking zapA and zapD has an increased cell length, consistent with a slight division defect.92,93 ZapB interacts with ZapA and forms spontaneous filaments in vitro.95,96 ZapB-mCherry localizes inside the Z-ring and presumably stabilizes the Z-ring via ZapA.97 ZapA and ZapB are required for mid-cell anchoring of MatP, a protein that structures the chromosomal terminus region into a macrodomain.98 The relocation of the terminus macrodomain from the cell pole to the division site via MatP–ZapA/ZapB interactions occurs prior to the replication of the terminus region and is required for proper nucleoid segregation.98 ZapC interacts with FtsZ and localizes to mid-cell where it stabilizes the Z-ring by promoting FtsZ polymer bundling and by suppressing the GTPase activity of FtsZ.99,100 In bacteria other than E. coli there are different combinations of FtsZ-ring stabilizing proteins.101
FtsEX is an ATP-binding cassette (ABC) transporter homolog that binds to FtsZ via the ATP-binding protein FtsE.102–104 Mid-cell localization of FtsE depends on the presence of FtsZ and ZipA. FtsE has been shown to be essential for cell division when cells grow in low-osmolarity growth medium;105,106 its function in cell division is to recruit and regulate peptidoglycan hydrolases (see below).
In order to start septation, a number of essential “late” cell division proteins, FtsK, FtsQ, FtsL, FtsB, FtsW, PBP3, and FtsN, assemble to the Z-ring almost simultaneously and in an interdependent fashion (Fig. 3).85,107 Several other proteins not essential for septation also localize to mid-cell depending on Fts proteins, including the peptidoglycan synthase PBP1B63 and the hydrolases AmiB and AmiC108,109 and their regulators LpoB,24 EnvC, and NlpD.110 Presumably, these peptidoglycan enzymes are not essential for cell division/cell separation because their function can be taken over by redundant enzymes. For example, in the absence of the bi-functional peptidolgycan synthase PBP1B, the homologous PBP1A becomes essential and shows enhanced localization at mid-cell, where it presumably takes over PBP1B's role in septal peptidoglycan synthesis.51
FtsK is a multifunctional protein involved in both chromosome segregation and cell division. In bacterial two hybrid assays FtsK interacts with FtsZ, FtsQ, FtsL, and PBP3.111–113 The cytoplasmic domain of FtsK forms hexamers to directionally transport DNA, required for decatenation of sister chromosomes, together with the Xer recombinases.114,115 This function of FtsK is only required in the fraction of cells with catenated chromosomes. The membrane/periplasmic part of FtsK is essential for cell division, presumably by stabilizing late-cell division proteins at the septum, which explains the existence of mutations in ftsA and ftsQ that allow for septation in the absence of FtsK.112,116,117
FtsK is required for septal recruitment of a preformed FtsQ–FtsL–FtsB complex.107,118 Each of these proteins is a bitopic membrane protein with a small cytoplasmic part and periplasmic domains. The crystal structure of the periplasmic domains of FtsQ identified two distinct regions: (i) a POTRA (polypeptide transport associated) domain, of which the second β-strand is essential for FtsQ mid-cell localization, and (ii) the C-terminal β-domain of FtsQ that is essential for the recruitment of FtsL, FtsB, and FtsW.119,120 The C-terminal domain of the periplasmic part of FtsL is required for its mid-cell localization via interaction with FtsQ.121 FtsQ-FtsL-FtsB 1:1:1 or 2:2:2 complexes have been modeled,122 the latter being consistent with the crystal structure of an FtsQ dimer.120 Small angle X-ray scattering of a homologous DivIB(FtsQ)-FtsL-DivIC(FtsB) complex from the Gram-positive Streptococcus pneumoniae is consistent with a 1:1:1 complex.123 In E. coli FtsQ has been shown to interact with FtsW and PBP3 by bacterial two-hybrid analysis.111,113 The requirement for FtsQ can be bypassed by expressing fusion proteins of ZapA-FtsL and ZapA-FtsB, either of which is sufficient for the recruitment of FtsW and PBP3.124
FtsW is member of the SEDS (shape, elongation, division, and sporulation) family of integral membrane proteins that also includes the essential cell elongation protein RodA and the sporulation protein SpoVE of the Gram-positive B. subtilis.125 FtsW, an integral transmembrane protein with 10 transmembrane regions, has recently been identified as the long-searched transporter (flippase) for the lipid II precursor.32 Consistent with this function, FtsW interacts with both PG synthases, PBP3 and PBP1B, and is required for recruiting PBP3 to the divisome, presumably together with PBP1B.63,126–128 The TPase PBP3 also interacts with FtsL,113 FtsQ,113 PBP1B,63 and FtsN.129 PBP3 can be recruited to the divisome independently of the FtsQLB complex via an FtsW-ZapA fusion, and targeting a FtsW-PBP3 fusion to mid-cell restored the recruitment of FtsQLB in cells depleted of FtsA.124 These data suggest that FtsW-PBP3 and FtsQLB complexes form independently of one another before recruitment to the divisome, which is consistent with interaction studies using Förster Resonance Energy Transfer (FRET) and co-immunoprecipitation.126,127 In C. crescentus, PBP3's early localization to the Z-ring relies largely on a functional TPase domain, indicating that TPase substrates, or an active state of the enzyme, might be required.130
FtsN is an essential cell division protein that has been thought to be restricted to enterobacteria. However, recent work in C. crescentus showed that FtsN-like proteins are more widely distributed among the proteobacteria.131 In E. coli, FtsN interacts with both early and late cell division proteins—FtsA, ZapA, PBP3, FtsW, and PBP1B—as shown by various in vitro (surface plasmon resonance, affinity chromatography) and in vivo (co-immunoprecipitation, FRET) techniques.126,127,129,132 Bacterial two-hybrid analysis shows additional interactions with FtsQ.111,113 FtsN is a bitopic membrane protein with a short cytoplasmic part and one transmembrane region. The periplasmic part contains three α-helices followed by a proline/glutamate-rich unstructured region and a globular C-terminal SPOR peptidoglycan binding domain.133 While the SPOR domain has been shown to interact with peptidoglycan in vitro it is not essential for cell division.134 Presumably, the peptidoglycan-binding function of FtsN's SPOR domain can be taken over by the SPOR domain-containing proteins DamX, DedD, and RlpA, which localize to cell division site, as mutants lacking multiple SPOR domain proteins show cell division defects.135,136 The precise role of FtsN during cell division is not known. Its periplasmic region, comprising the three partially formed α-helices (amino acids 62–67, 80–93, and 117–123), is essential for cell division for unknown reasons. FtsN interacts with both PBP1B and PBP363,129 and stimulates the activity of PBP1B, presumably by stabilizing the dimeric form of PBP1B129 consistent with the hypothesis that a main functional role for FtsN in cell division is to coordinate the peptidoglycan synthases active during septation. Additional functions of FtsN have also been suggested. For example, FtsN could provide a signal for completion of divisome assembly to the cytoskeletal components, a role supported by its cytoplasmic interaction with the 1c subdomain of FtsA132 and the existence of mutants with altered FtsA that can divide without FtsN.137 FtsN has also been implicated in a divisome stabilizing function, which is supported by the observation that FtsN-depletion leads to the disassembly of the already assembled divisome components, including the early proteins.138
FtsP (SufI) is a recently characterized soluble periplasmic protein involved in cell division, although its precise role is unknown. It localizes to the division sites dependent on the presence of FtsZ, FtsQ, FtsL, and FtsN.139 FtsP is dispensable under normal growth conditions but is required for divisome stability when cells grow at various stress conditions, including oxidative stress and DNA damage.140 The crystal structure of FtsP shows structural similarity to the multicopper oxidase protein family, but does not bind the metal ion.139
Cell division proteins specific to Gram-positive bacteria
In this section we highlight several recent findings specific to Gram-positive bacteria. These have a thick septum peptidoglycan with distinct zones of high and low electron densities, as visualized by cryo-electrontomography,141,142 and they complete septal cross-wall synthesis before daughter cell separation (Fig. 1). Gram-positive species contain most of the essential division proteins discussed above, with a few exceptions such as FtsK, FtsP, and FtsN.
Conserved eukaryotic-type Ser/Thr protein kinases regulate various cellular processes including cell division in Gram-positive species. The Streptococcus pneumoniae protein kinase StkP is required for proper septal cell wall synthesis by yet unknown mechanisms.143,144 It contains several PASTA domains, which are also present in some PBPs and have been suggested to bind to peptidoglycan fragments and β-lactams.145 StkP phosphorylates the cell division protein DivIVA, which is found in many Gram-positive species that undergo elongation and division modes of PG synthesis, and both proteins localize to mid-cell and the poles. In Bacillus subtilis DivIVA regulates the septation site by positioning and stabilizing the FtsZ inhibitor MinC/MinD at the cell poles. Mutants lacking DivIVA lose the topological control over MinC/MinD and form minicells derived from aberrant septation events at the poles.146 DivIVA localizes to the new poles immediately after daughter cell separation and independent of divisome proteins to prevent aberrant Z-ring assembly after division.147,148 Interestingly, DivIVA has an amphiphatic alpha-helix for membrane attachment, and finds the new cell pole localization by virtue of its affinity to curved membranes, a feature that explains its polar localization in a number of heterologous organisms like E. coli and yeast.149
The cytoplasmic cell division protein EzrA is present in B. subtilis and S. aureus, but EzrA depletion causes a severe cell division phenotype and an increase in cell diameter only in the latter spherical-shaped species.150–152 EzrA has a N-terminal membrane anchor and interacts with FtsZ. It acts as a negative regulator of Z-ring assembly by decreasing GTP binding affinity and increasing the GTPase activity of FtsZ, thus increasing FtsZ-depolymerization.151,153,154 Its localization to the divisome during division suggested that it regulates Z-ring dynamics during constriction.153 EzrA has also been shown to play a role in the switch from lateral to septal cell wall synthesis in B. subtilis, together with GpsB.155 This is achieved by positioning the class A PBP1 (encoded by the ponA gene), the major PG synthase of B. subtilis, through direct protein–protein interactions. EzrA and GpsB recruit PBP1 to the divisome, and GpsB is responsible for the removal of PBP1 from the mature cell pole after division making it available for cell elongation.155
Another recently identified member of the B. subtilis divisome, not found in Gram-negative bacteria, is SepF. Cells lacking SepF form grossly distorted division septa.156 SepF is recruited to the divisome by direct interaction with FtsZ. It negatively regulates the GTPase activity of FtsZ and thus stabilizes FtsZ filaments.156,157 Interestingly, in vitro SepF forms relatively large rings with a diameter of 50 nm and it is able to bundle FtsZ filaments into long, regular structures which resemble microtubules. Therefore, it has been proposed that SepF is required for the regular arrangement and bundling of FtsZ filaments for proper septum placement.158