Disruption of divisome assembly rescued byFtsN–FtsA interaction in Escherichia coliSebastien Pichoffa,1,2, Shishen Dua,1, and Joe Lutkenhausa,2

aDepartment of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160

Contributed by Joe Lutkenhaus, June 8, 2018 (sent for review April 16, 2018; reviewed by William Margolin and David S. Weiss)

Cell division requires the assembly of a protein complex called thedivisome. The divisome assembles in a hierarchical manner, withFtsA functioning as a hub to connect the Z-ring with the rest of thedivisome and FtsN arriving last to activate themachine to synthesizepeptidoglycan. FtsEX arrives as the Z-ring forms and acts on FtsAto initiate recruitment of the other divisome components. In theabsence of FtsEX, recruitment is blocked; however, a multitude ofconditions allow FtsEX to be bypassed. Here, we find that all suchFtsEX bypass conditions, as well as the bypass of FtsK, depend uponthe interaction of FtsN with FtsA, which promotes the back-recruitment of the late components of the divisome. Furthermore,our results suggest that these bypass conditions enhance the weakinteraction of FtsN with FtsA and its periplasmic partners so that thedivisome proteins are brought to the Z-ring when the normalhierarchical pathway is disrupted.

FtsZ | FtsA | FtsN | divisome | Z-ring

Cell division in bacteria is carried out by a large protein machinecalled the divisome. In Escherichia coli, a dozen essential cell-

division proteins assemble into the divisome in two temporallydistinct steps (1). In the first step, the tubulin ancestor FtsZ poly-merizes into filaments that are anchored at the membrane by ZipAand FtsA to form the Z-ring (2–4). Assembly of this ring is spatiallyregulated, and once formed serves as a scaffold for recruitment ofthe late proteins during the second step, which occurs in a hier-archical sequence (5–8). What regulates this transition is not clear,but the actin homolog FtsA plays a crucial role in the process.In wild-type cells both FtsA and ZipA are required for this

second step; however, functional FtsA mutants that are specifi-cally impaired for self-interaction are able to bypass ZipA (9–11). This led to a model in which the multimeric status of FtsAregulates the recruitment of the late cell-division proteins (10).In the model, ZipA acts indirectly to promote FtsA’s monomericstatus at the Z-ring, in addition to anchoring FtsZ polymers.Furthermore, FtsA’s IC domain plays a critical role in recruitmentbecause it is involved in both FtsA’s self-interaction and the in-teraction with at least one of the late cell-division proteins (FtsN)(9, 12–15). These two interactions are mutually exclusive and theappearance of free IC domains at the Z-ring leads to the re-cruitment of the late proteins.Besides FtsA and ZipA, FtsEX, a pseudo ABC transporter,

localizes to the Z-ring as it forms and is also required for thesecond step of divisome assembly (16–18). FtsEX acts directly onFtsA to promote the recruitment, presumably by promoting theformation of monomeric FtsA (19). Furthermore, FtsEX interactswith FtsA in the cytoplasm to regulate the start of cell constriction(19) and with EnvC in the periplasm to coordinate cell wall syn-thesis and hydrolysis during division (20). Although the ATPaseactivity of FtsEX is not necessary for its role in recruitment, thelatter two functions require its ATPase activity. Thus, an FtsEXmutant unable to hydrolyze ATP promotes divisome assembly butis unable to initiate constriction (16).Despite its multiple functions in division, FtsEX is condi-

tionally essential. In medium of low osmolarity (LB with reducedNaCl, for example) or at temperatures ≥37 °C, cells lackingFtsEX display a lethal division defect and form smooth filaments

that assemble Z-rings but fail to recruit the late-division proteins(18). Increasing the osmolarity or lowering the growth temper-ature restores division to a ΔftsEX mutant (18, 21–23). This di-vision defect is also suppressed by overexpression of ftsQAZ,ftsN, ftsP, or dapE, or by mutations in ftsB, ftsL, and ftsW thatallow the bypass of ftsN (19, 21, 22). In addition, mutations in ftsAthat impair FtsA’s ability to self-interact are among the best sup-pressors of ΔftsEX (19, 21, 22). Under these suppressing condi-tions, a ΔftsEX mutant, like ΔenvC or ΔamiA/amiB mutants,displays a chaining phenotype, indicating that while its requirementfor recruitment and activation of the divisome are suppressed, itsrole in cell wall hydrolysis is not. Even though these suppressingconditions all promote the recruitment and activation of the late-division proteins (18), the mechanisms by which these many con-ditions suppress the requirement for FtsEX in cell division arenot known.FtsN is the last essential divisome protein to localize to the Z-

ring and its recruitment is complex, requiring FtsA, FtsQ, and FtsI(24, 25). However, once it arrives, it triggers the start of cellconstriction, likely by acting through the FtsQLB complex (26–28).FtsN was identified as a multicopy suppressor of a conditionalmutation in ftsA but suppresses conditional mutations in ftsI, ftsQ,or ftsK as well (29–31). FtsN has at least four functional domains:an N-terminal cytoplasmic domain (FtsNcyto), which contains aconserved motif responsible for interaction with FtsA; a trans-membrane domain; and two periplasmic domains, the E-domain(FtsNE), which triggers septal PG synthesis, and a C-terminalSPOR (septal peptidoglycan binding) domain, which interactswith peptidoglycan strands denuded by amidases (12, 27, 28, 32–36). FtsNE was designated as the essential domain because it can

Significance

Cell division in Escherichia coli requires 12 essential proteins thatassemble into the divisome in a sequential manner. Assemblystarts with formation of the Z-ring and culminates with the ar-rival of FtsN, which triggers septal peptidoglycan synthesis.Normally, deletion of any of these 12 proteins disrupts divisomeassembly and results in a division block. However, about half ofthese proteins can be bypassed under some conditions, raising aquestion of how the divisome assembles under such conditions.Here we show that these bypasses require the interaction ofFtsA with FtsN and that this normally weak interaction is en-hanced under these conditions, leading to the back-recruitmentof the other divisome proteins to the Z-ring.

Author contributions: S.P., S.D., and J.L. designed research; S.P. and S.D. performed re-search; S.P., S.D., and J.L. analyzed data; and S.P., S.D., and J.L. wrote the paper.

Reviewers: W.M., University of Texas Medical School; and D.S.W., University of Iowa.

The authors declare no conflict of interest.

Published under the PNAS license.1S.P. and S.D. contributed equally to this work.2To whom correspondence may be addressed. Email: spichoff@kumc.edu or jlutkenh@kumc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1806450115/-/DCSupplemental.

Published online July 2, 2018.

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complement an ftsN-deletion strain when it is overexpressed andexported to the periplasm (27). However, at the physiologicallevel, FtsNcyto is required to localize FtsNE (12).Overexpression of ftsN can bypass the requirement for ZipA,

FtsEX, or FtsK (21, 30, 31, 34). To bypass these genes, FtsNcyto

and presumably FtsNE are required and these two domains mustbe linked (30, 34). Pichoff et al. (34) explained the bypass ofZipA by proposing that overexpression of ftsN favors FtsA mono-mers at the Z-ring, resulting in the recruitment of the late cell-division proteins. For the suppression of ΔftsEX, in which thenormal hierarchical chain of recruitment is disrupted, it wassuggested that the FtsA–FtsN interaction allows FtsN to back-recruit the late proteins to the Z-ring.Here, we investigated whether the interaction between FtsA

and FtsN becomes essential for the multitude of conditions thatbypass FtsEX. The results demonstrate that this is the case andsuggest that there are two ways to link the late proteins of the divi-some to the Z-ring, both of which require FtsA. One is thephysiological pathway, in which FtsEX acts on FtsA to begin thehierarchical recruitment, and the other is a back-recruitment path-way that comes into play under bypass conditions and absolutelyrequires the interaction of FtsA with FtsN. Consistent with this, wefound that the FtsA–FtsN interaction is also critical for theoverexpression of ftsA* to bypass FtsK. This work also implies thatthe FtsA–FtsN interaction is promoted by the conditions that al-low suppression of ΔftsEX or ΔftsK.

ResultsSuppression of ΔftsEX and ΔzipA by ftsN Overexpression RequiresFtsNE and FtsNcyto. The interaction between FtsN and FtsA isnot essential because overexpression and export of FtsNE to theperiplasm complements ΔftsN (27, 33–35). Consistent with this,mutants impaired for this interaction (such as FtsND5N or aMalG–FtsN fusion) are able to complement ΔftsN as long as theyare expressed above the physiological level (33, 34) (SI Appendix,Fig. S1A). In contrast, the bypass of the essential genes ftsEX orzipA by overexpression of ftsN requires FtsN’s interaction withFtsA (34) (SI Appendix, Fig. S1 B and C). Furthermore, FtsNcyto,which interacts with FtsA, must be physically linked to FtsNE

because overexpression of these domains when they are unlinkeddoes not suppress ΔftsEX or ΔzipA (34). In addition, inactivationof FtsNE (by mutating three key residues) (28) (SI Appendix, Fig.S1A) in full-length FtsN revealed that a functional FtsNE mustbe physically linked to FtsNcyto to suppress ΔftsEX or ΔzipA (SIAppendix, Fig. S1 B and C). Thus, even though overexpression ofjust FtsNE is sufficient to complement ΔftsN, FtsNcyto must bephysically linked to FtsNE to bypass ftsEX or zipA. Thus, thebypass of ftsEX or zipA is more stringent than the complemen-tation of ΔftsN.

Suppression of ΔftsEX by Osmolarity, ftsB*, or ftsN OverexpressionAbsolutely Depends on the FtsN–FtsA Interaction. In addition to ftsNoverexpression, many conditions bypass ftsEX, including highosmolarity and ftsB* mutations that allow the bypass of FtsNE.Because the bypass by ftsN overexpression requires FtsNcyto to bephysically linked to FtsNE, we tested whether this was also truefor other conditions. To do this, we used ftsN alleles differingonly in their ability to interact with FtsA and tested whether theywould allow strains with deletions of both ftsEX and ftsN to growunder the bypass conditions. The alleles included ftsN, whichinteracts with FtsA, ftsND5N, which is strongly reduced for thisinteraction and a malG–ftsN fusion (MalG1–33

–FtsN46–319) inwhich the interaction is eliminated because the cytoplasmicand first transmembrane domains of FtsN are replaced with thecorresponding regions of MalG.We used a ΔftsN strain with ftsN provided by a plasmid

(pBL154) that is thermosensitive for replication (TB28 ftsN::kan/

pBL154). At nonpermissive temperature (≥37 °C) the plasmid islost, leading to FtsN depletion and resulting in cell filamentationand the inability to form colonies. We next deleted ftsEX byreplacing these genes with a gene encoding resistance to chlor-amphenicol (cm). To grow, these cells are now dependent on thepresence of both 0.2 M sucrose (increased osmolarity to suppressΔftsEX) and the thermosensitive replication plasmid providingFtsN. In addition, to avoid any recombination between the dif-ferent plasmids and the chromosome, the recA gene was inacti-vated (TB28 ftsN::kan ftsEX::cm recA::Tn10/pBL154). Plasmidsexpressing ftsN constructs capable or not of the FtsN–FtsA in-teraction [pSEB417 (Ptrc*::ftsN) or pSEB455 (Ptrc*::malG2–33

–

ftsN46–319), respectively] or a control plasmid expressing ftsEX[pSEB428 (Ptrc*:: ftsEX)] were then introduced into thesestrains. Consistent with the FtsNE domain being the only es-sential domain, both ftsN plasmids complemented ΔftsN at 37 °C(Fig. 1, row B) and malG–ftsN required more isopropyl-β-D-thiogalactopyranoside (IPTG), as expected (Fig. 1, row C). Inaccordance with previous reports, the ftsN alleles are not re-quired for survival if the strain (TB28 ftsN::kan/pBL154) alsocarries the chromosomal mutation ftsBE56A (ftsB*), a knownsuppressor of ftsN or ftsEX deletions (19, 28) (Fig. 1, row D).Although plasmids carrying ftsEX or ftsN allow the ftsB* ΔftsNΔftsEX strain to grow at high temperature (Fig. 1, rows M andO), the vector does not (Fig. 1, rows L). Thus, although ftsB*suppresses the loss of ftsN or ftsEX, it is unable to suppress thesimultaneous loss of both. Note that we observe that FtsNoverexpression seems more toxic at a higher level of inducer inthe ftsB* ΔftsN ΔftsEX strain (Fig. 1, row M) than in the ftsB*ΔftsN strain (Fig. 1, row E). This could be related to the pre-viously observed toxicity of FtsN expressed in the ftsB* back-ground in low-osmolarity media (28), and because FtsEX has anessential role in low-osmolarity growth conditions (18), the tox-icity of FtsN in the ftsB* could be exacerbated in the ΔftsEXstrain for some unknown reason.Importantly, malG–ftsN does not allow survival of either the

ΔftsN ΔftsEX or the ftsB* ΔftsN ΔftsEX strain (Fig. 1, rows J andN), even though this construct complements the ΔftsN strain effi-ciently (Fig. 1, rows C and F). In addition, combining the ftsB*mutation with increased osmolarity, two conditions that on theirown suppress ΔftsEX, does not improve the ability of the strainexpressing malG-ftsN to form colonies at 37 °C. Thus, malG–ftsNcomplements ΔftsN in the presence of FtsEX; however, in theabsence of FtsEX, even in conditions where ΔftsEX is suppressedby the ftsB* mutation or increased osmolarity, it cannot. This resultstrongly indicates that the FtsN–FtsA interaction (due to FtsNcyto)becomes essential under these conditions that suppress ΔftsEX.

Suppression of ftsEX Deletion by FtsA* (FtsAR286W) also Depends onthe FtsN–FtsA Interaction. To confirm that the FtsN–FtsA in-teraction is a general requirement for suppression of ΔftsEX, wetested an additional suppressing condition (ftsAR286W, also knownas ftsA*) and in another strain background [W3110 instead ofMG1655 (TB28)]. Recently, Du et al. (19) reported that FtsA*, aswell as other FtsA*-like mutants impaired for self-interaction,suppress ΔftsEX. Therefore, ΔftsEX strains that were ftsA+ orftsAR286W, as well as controls, were transformed with plasmidsexpressing the various ftsN constructs under the control of an IPTG-inducible promoter [pSD268 (Ptac::ftsN), pSD268 (Ptac::ftsN

D5N), orpSD270 (Ptac::malG-ftsN)]. The resultant strains were thentested for their ability to be transduced to ftsN::kan using P1phage. Transductants were selected on plates with or withoutsucrose and with different IPTG concentrations. Note, the malG–

ftsN fusion in pSD270 is expressed at a higher level than wild-typeftsN from pSD268 due to altered translation signals. As a result,MalG–FtsN becomes toxic at higher induction levels (Fig. 2,compare rows A and B). Toxicity is also observed when FtsN isoverexpressed from a more-efficient expression vector (34).

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W3110 and W3110 ftsAR286W strains expressing the differentftsN constructs yielded many ftsN::kan transductants on all se-lection plates tested. This confirmed that our constructs com-plement ΔftsN in this strain background. On the other hand,W3110 ftsAR286W ΔftsEX and W3110 ΔftsEX only producedKanR transductants with ftsN or ftsND5N present and only onplates containing 1 mM IPTG for the latter. These ΔftsNtransductants were tested by spot assay on plates containing in-creasing IPTG concentrations (Fig. 2 and SI Appendix, Fig. S2for plates with 0.2 M sucrose). W3110 ftsAR286WΔftsEX or W3110ΔftsEX containing the MalG–FtsN fusion yielded no viable ftsN::kan transductants even on plates containing 0.2 M sucrose, whichsuppresses ΔftsEX (any colonies on the selections plates grewpoorly and would not regrow when restreaked). These resultsconfirm our previous observation in the TB28 background, thatan FtsN mutant unable to interact with FtsA cannot supportdeletions of both ftsEX and ftsN, even though malG–ftsN com-

plements ΔftsN and both 0.2 M sucrose and ftsAR286W cansuppress ΔftsEX.As seen in the spot assay (Fig. 2), and as previously reported

(34), ftsN is more efficient than ftsND5N at complementing ΔftsN(Fig. 2, compare rows B and C). This is consistent with FtsNcyto

being essential under physiological conditions (35). Consistentwith FtsND5N being impaired for interaction with FtsA, ftsND5N

has to be expressed at higher levels than ftsN to suppress theΔftsN ΔftsEX strain (Fig. 2, rows D and E). Even with this higherlevel of expression, colony formation and viability are not asrobust as with ftsN [Fig. 2 or SI Appendix, Fig. S2, compareW3110 ΔftsN ΔftsEX expressing ftsN (row D) or ftsND5N (rowE)]. Overexpression of ftsND5N in the ftsA* background allowsgrowth of the ΔftsN ΔftsEX strain (Fig. 2) (high IPTG) but it isnot as efficient as wild-type ftsN [Fig. 2, compare W3110ftsA*ΔftsN ΔftsEX expressing ftsN (row H) or ftsND5N (row I)].Suppression of ΔftsN ΔftsEX by ftsND5N in the ftsA* background

Fig. 2. Suppression of ΔftsN ΔftsEX by ftsA*requires the FtsN–FtsA interaction. W3110, W3110 ftsAR286W (ftsA*), W3110 ftsEX::cm, and W3110 ftsEX::cmftsAR286W were transformed with pSD268 (Ptac::ftsN ), pSD268-D5N (Ptac::ftsN

D5N), or pSD270 (Ptac::malG1–33–ftsN46–319). Then ftsN::kan was introduced by

P1 transduction by selecting at 30 °C on plates containing 0.2 M sucrose with chloramphenicol, kanamycin, spectinomycin, and variable IPTG concentrations.All but the ftsEX::cm strains expressing the MalG–FtsN fusion gave viable kanamycin-resistant transductants. Transductant colonies were resuspended in LBand serially diluted 10-fold, and spotted on selective plates containing increasing IPTG concentrations with or without 0.2 M sucrose at 30 °C. The proteinexpressed under IPTG control is indicated in the figure. The plates shown here are without sucrose; see SI Appendix, Fig. S2 for the plates containing sucrose.

Fig. 1. Suppression of ΔftsN ΔftsEX by osmolarity and ftsB* requires FtsN–FtsA interaction. TB28 ftsN::kan recA::Tn10, TB28 ftsN::kan ftsEX::cm recA::Tn10,and isogenic variants carrying the ftsBE56A (i.e., ftsB*) mutation and pBL154 (pSC101 SpecR RepTs ftsN) to provide ftsN at 30 °C were grown with 0.2 M sucroseto suppress ΔftsEX. The strains were transformed with pDSW208 (Ptrc*::gfp) or derivatives pSEB417 (Ptrc*::ftsN ), pSEB455 (Ptrc*::malG2–33

– ftsN46–319) orpSEB428 (Ptrc*::ftsEX) by selecting on plates containing 0.2 M sucrose and antibiotics at 30 °C. To test for complementation of ΔftsN, colonies of each strainwere resuspended in LB + 0.2 M sucrose, serially diluted 10-fold, spotted onto ampicillin plates containing 0.2 M sucrose with increasing IPTG concentrations,and incubated at 37 °C (nonpermissive for the replication of pBL154). The protein expressed under IPTG control is indicated for each strain.

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(Fig. 2, row I) is slightly better than in the ftsA wild-type back-ground (Fig. 2, row E) and this is consistent with the observationthat ftsA mutants impaired for self-interaction require less FtsNthan an isogenic wild-type ftsA strain (19). Similarly, it takes lessftsN to suppress ΔftsN ΔftsEX in the ftsA* strain than in the ftsA+

background [Fig. 2, compare ftsN expression in W3110 ΔftsNΔftsEX (row D) vs. W3110 ftsA* ΔftsN ΔftsEX (row H)]. Likewise,the complementation of ΔftsN requires less expression of ftsND5N

in the ftsA* background than in the wild-type background [Fig. 2,compare ftsND5N expression in W3110 ΔftsN (row C) vs. W3110ΔftsN ftsA* (row G)]. The fact that a ftsA* strain requires lessFtsN to grow suggests that FtsA* interacts better with FtsN. Thiswould also explain why the ftsA* strains are less affected by theftsND5N mutation, which impairs (but does not abolish) theFtsN–FtsA interaction (34). However, with MalG–FtsN, there isno FtsN–FtsA interaction and this fusion cannot suppress ΔftsNΔftsEX, even in the ftsA* background, as no viable ftsN::kantransductants were recovered, as described above.In this set of experiments, we combined up to three known con-

ditions (overexpression of ftsN, ftsA*, and increased osmolarity),each of which can suppress ΔftsEX. Despite this, the MalG–FtsNfusion never supported the growth of the ΔftsN ΔftsEX strain.Furthermore, specifically weakening the FtsN–FtsA interaction withthe FtsND5N mutant led to a reduced ability to suppress ΔftsEX.Taken together, these results confirm the conclusion from Fig. 1:FtsN must interact with FtsA to suppress the simultaneous loss offtsN and ftsEX, even in conditions where ΔftsEX is usually sup-pressed and the essential function of FtsN (FtsNE) is providedabove the level required to complement ΔftsN. This require-ment for the FtsN–FtsA interaction is independent of the strainbackground and the way ΔftsEX is suppressed.

Suppression of ΔftsEX by ftsP Overexpression also Depends on theFtsN–FtsA Interaction. FtsP (also known as SufI) is a nonessentialperiplasmic protein recruited to the septum in an FtsN-dependent manner (37). However, under conditions of stress,such as low osmolarity, oxidative stress, or DNA damage, itappears critical for division (21, 22). In addition, deletion of ftsPis synthetic-lethal with ΔftsEX and this synthetic lethality is notsuppressed by the usual suppressors of ΔftsEX (such as increasedosmolarity, increased ftsQAZ, or ftsN expression), even thoughthey can also suppress ΔftsP (in low-osmolarity media) (21, 22).ftsP is a very good multicopy suppressor of ΔftsEX so it is thoughtthat FtsP and FtsEX may perform an essential but redundantfunction during cell division (22). Because FtsP is in the peri-plasm and lacks a direct link to FtsA in the cytoplasm, and ap-pears to have a somewhat redundant function with FtsEX, wetested whether the suppression of ΔftsEX by multicopy ftsP alsorequired the FtsN–FtsA interaction.The multicopy plasmid pSD277 (pBR322 ftsP) suppressed

ΔftsEX because W3110 ftsEX::cm carrying the plasmid no longerrequired 0.2 M sucrose to grow (Fig. 3A). To test whether theFtsN–FtsA interaction was critical for ftsP overexpression tosuppress ΔftsEX, we constructed ftsN::kan strains expressing eitherftsN (pSD268), ftsND5N (pSD268-D5N), or malG-ftsN (pSD270)and also carrying the compatible ftsP multicopy plasmid pSD277,and then introduced ftsEX::cm by P1 transduction by selecting forCmR at different IPTG concentrations in the presence or absenceof 0.2 M sucrose. The strain expressing wild-type ftsN gave colonieson plates with all concentrations of IPTG and with or without su-crose, while the strain expressing ftsND5N only gave small colonieson sucrose plates with 1 mM IPTG. However, as shown in the spotassay of these transductants (Fig. 3B), only cells expressing wild-type ftsN were able to grow well. Transductants expressing ftsND5N

were not able to grow even at the highest IPTG concentrationtested. Note that at this IPTG concentration, the FtsND5N level ishigh enough to fully complement ftsN::kan (Fig. 2). Further-more, we were unable to obtain viable transductants in the strain

expressing malG–ftsN under any condition, indicating that theinteraction between FtsN and FtsA is required for ftsP over-expression to suppress ΔftsEX.

In the Absence of FtsEX and the FtsN–FtsA Interaction the Late-Division Proteins Are Not Recruited to the Z-Ring. When ΔftsEXcells are suppressed by high osmolarity, ftsA*, or other sup-pressors, the late-divisome proteins localize at the Z-ring andseptation ensues (18). This indicates that FtsEX’s involvement inthe recruitment of these late-divisome proteins can be bypassed.In the absence of FtsN the remainder of the divisome assembles(24), indicating that when overexpressed FtsNE complementsΔftsN it acts on a preassembled divisome (27). Based upon theseobservations, divisome proteins should be recruited when ΔftsEXis suppressed by sucrose or ftsA* and overexpression of FtsNE

(provided in our case by the MalG–FtsN fusion) should allowcells to divide. However, as shown in Figs. 1–3, ΔftsN ΔftsEX cellsexpressing MalG–FtsN underwent extensive filamentation andcould not form colonies, even under conditions where the loss ofFtsEX is normally suppressed (Fig. 4 C, F, and I). To determine thecause of this filamentation we investigated the localization of someof the late essential division proteins (FtsI and FtsN in Fig. 4, andFtsK in SI Appendix, Fig. S3) by immunofluorescence microscopy.When both FtsEX and FtsN are absent and ftsN is not expressed,

cells become filamentous and contain multiple Z-rings (Fig. 4A).However, none of the cell-division proteins that should localizeafter FtsA are present at the Z-ring, even though the media contain0.2 M sucrose to suppress ΔftsEX (this condition serves as a controlto verify FtsN is depleted after the loss of the repTs plasmid) (Fig. 4D and G, and SI Appendix, Fig. S3 and Table S1). The lack oflocalization of FtsK and FtsI in cells lacking FtsN and FtsEX is alittle unexpected, because as discussed above, these proteins havebeen shown previously to localize in the absence of FtsN or inΔftsEX cells grown in 0.2 M sucrose. This localization defect mustbe due to the absence of FtsN in the ΔftsEX background becauseexpressing ftsN from an IPTG-inducible promoter results in effi-cient localization of these divisome proteins at the septum (Fig. 4B, E, and H and SI Appendix, Fig. S3 and Table S1). Moreover,these cells display the previously reported chaining phenotype of aΔftsEX strain (18). In contrast, when cells lacking ftsEX and de-pleted for ftsN were grown in 0.2 M sucrose and malG–ftsNexpressed at a level sufficient to complement the isogenic strainlacking only ftsN (Fig. 1), the late cell-division proteins (FtsI, FtsK,and MalG–FtsN were tested) failed to localize even though multiple

Fig. 3. Suppression of ΔftsN ΔftsEX by multicopy ftsP requires the FtsN–FtsAinteraction. (A) Multicopy ftsP suppresses ΔftsEX. W3110 ΔftsEX::cm con-taining pSD277 (pBR322 ftsP) or the vector were spotted on ampicillin platesat 30 °C with or without 0.2 M sucrose. (B) In W3110 ΔftsN::kan/pSD277transformed with pSD268 (Ptac::ftsN ), pSD268-D5N (Ptac::ftsN

D5N), or pSD270(Ptac::malG1–33-ftsN46–319), deletion of the chromosomal ftsEX was attemptedby P1 transduction of ftsEX::cm and selection on 0.2-M sucrose platescontaining antibiotics and variable concentrations of IPTG at 30 °C. No vi-able transductants were obtained with strains expressing malG–ftsN at anyIPTG concentration, while colonies obtained with cells expressing ftsN orftsND5N were picked, serially diluted 10-fold, and spotted onto the sameselection plates.

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Z-rings were observed (Fig. 4 C, F, and I and SI Appendix, Fig.S3 and Table S1). This observation demonstrates that in theftsEX-null background, and in the absence of the interaction be-tween FtsA and FtsN (when MalG–FtsN is expressed), the late-division proteins are not localized. In summary, in the absence ofFtsEX the recruitment of the late cell-division proteins to the Z-ring is dependent on the ability of FtsN to interact with FtsA.

FtsN–FtsA Interaction Is Critical for Bypass of FtsK. FtsK localizes tothe Z-ring after FtsEX and is required for the recruitment of thedownstream division proteins (18, 38, 39). However, FtsK, likeZipA and FtsEX, is also dispensable for division under certainconditions, including multicopy ftsQAZ and overexpression offtsN, ftsQ, or ftsA* (30, 31). How the proteins downstream ofFtsK (FtsQLB, FtsWI, and FtsN) localize to the Z-ring in itsabsence is not clear. However, suppression of ΔftsK by over-expression of ftsN requires FtsNcyto, indicating that the FtsN–FtsAinteraction is critical (30). We confirmed this finding by P1 trans-duction of an ftsK::kan allele into strains overexpressing ftsN orftsND5N. A wild-type strain containing a plasmid expressing ftsK1–210,which complements ΔftsK (40), was used as a control for the ef-ficiency of P1 transduction of ftsK::kan. As shown in SI Appendix,Fig. S4, ftsK::kan was readily introduced into strains expressingftsK1–210 or overexpressing ftsN. However, only a backgroundnumber of nonviable transductants was obtained with FtsND5N.Consistent with previous findings that FtsNE is critical for FtsN tosuppress many division defects, we found that FtsNE was alsocritical for the bypass of FtsK (FtsNWY/AA, an FtsNE domainmutant, did not allow the introduction of ftsK::kan).We next tested whether the FtsN–FtsA interaction was critical

for bypassing ftsK under other suppressing conditions [over-expression of ftsQ and overexpression of ftsA* (31)]. We firsttried to confirm that overexpression of ftsQ or ftsA* bypasses ftsKby using P1 transduction of the ftsK::kan allele into cells with an

increased level of FtsQ or FtsA*. While ftsK::kan was readilyintroduced into a strain overexpressing ftsA*, only a backgroundnumber of transductants was obtained in a strain overexpressingftsQ, suggesting that overexpressing ftsQ cannot bypass ftsK inour system (SI Appendix, Fig. S4). Nonetheless, the successfuldeletion of ftsK in the presence of an increased level of FtsA*allowed us to test the importance of FtsNcyto in this bypass. Wefirst introduced ftsN::cat into the ftsA* strain in the presence of aplasmid expressing wild-type ftsN or ftsND5N inducible by IPTG.The resulting strains were then transformed with a plasmidconstitutively expressing ftsA* to increase the level of FtsA* andthen tested for the bypass of ftsK by P1 transduction. As shown inFig. 5A, the ftsK::kan allele could be introduced into both strains;however, the strain expressing ftsND5N only gave transductants atthe higher IPTG concentration. These transductants could notgrow when restreaked on the same selection plates, whereasthose obtained in the strain expressing ftsN could (Fig. 5B). Thisresult demonstrates that reducing the interaction between FtsAand FtsN eliminates the ability of FtsA* to bypass ftsK, sup-porting our idea that the FtsN–FtsA interaction is critical for thebypass of ftsK.

Increasing ftsN Expression or Mutating ftsA to Impair FtsA’s Ability toSelf-Interact Enhances the FtsN–FtsA Interaction. The results pre-sented above indicate an essential role for the FtsN–FtsA in-teraction in the assembly of the divisome when the loss of ftsEXor ftsK disrupts the hierarchical recruitment pathway. In otherwords, the conditions that suppress ΔftsEX or ΔftsK may favorthe FtsN–FtsA interaction to allow the back-recruitment ofdivisome proteins to the Z-ring. Consistent with this idea, and asexplained above, FtsA mutants impaired for their ability to self-interact require less FtsN to complement an ftsN-deletion strainthan the same strain with a wild-type ftsA allele (19). Many ofthese FtsA mutants also seem to be more efficient than wild-type

Fig. 4. Immunolocalization of FtsZ, FtsI, and FtsN in a ΔftsN ΔftsEX strain expressing different FtsN constructs. TB28 ftsN::kan ftsEX::cm recA::Tn10/pBL154(pSC101 SpecR RepTs ftsN) was transformed with pDSW208 (colE1, Ptrc*::gfp, lacI

Q, AmpR) (A, D, and G) or its derivatives pSEB417 (Ptrc*::ftsN) (B, E, and H) orpSEB455 (Ptrc*::malG2–33

–ftsN46–319) (C, F, and I). Transformants were grown at 30 °C for several generations and then shifted to 37 °C for 240 min (to diluteFtsN provided by pBL154) in 0.2 M sucrose liquid media containing ampicillin and 0.1 mM IPTG, which allows suppression by the Ptrc*::ftsN plasmid (Fig. 1).Samples were then taken, fixed, and processed for fluorescence microscopy, as described in Materials and Methods. The different panels are photographs ofcells (combination of phase and fluorescence) stained with anti‐FtsZ (A–C), anti‐FtsN (D–F), and anti‐FtsI (G–I) antisera as indicated. White arrows show septallocalizations. (Magnification: 1,000×.)

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FtsA in division because they complement ΔftsA at a lower levelof expression (9). Based on these observations, it was proposedthat monomeric FtsA is more effective at recruiting late cell-division proteins, such as FtsN, because the IC domain (whichis essential for the recruitment) is exposed (9, 12–15). Similarlyto these FtsA mutants (9, 11, 19, 22), overexpression of ftsN alsosuppresses ΔftsEX (21) and ΔzipA (34). Taken together, theseobservations suggest that increasing the FtsN level might reducethe amount of FtsA necessary for cells to grow, in a sense makingFtsA behave like a mutant impaired for self-interaction.To test this, we checked whether cells require less FtsA for

division in the presence of an increased level of FtsN. We used aΔftsA strain with ftsA expressed from a plasmid under the controlof an IPTG-inducible promoter and introduced pKD140 (pBR322,ftsN), which causes a 10-fold increase in FtsN (34). If the strainrequires less FtsA for division in the presence of increased FtsN,less IPTG should be required for growth. We also complementedthe ftsA-null cells with FtsAR286W, a well-characterized mutant ofFtsA impaired for self-interaction, to see how it responded to FtsN.As shown in Fig. 6, the ΔftsA strain without the ftsN plasmid

required ≥125 μM IPTG to induce sufficient FtsA for growth,whereas the strain with the multicopy ftsN plasmid requiredmuch less IPTG (30 μM). The minimum IPTG level required forgrowth with excess FtsN is similar or even slightly less than thatrequired for complementation by FtsAR286W (FtsA*). In con-trast, increasing the cellular level of FtsN only marginally im-

proves the ability of ftsAR286W to complement the ftsA-depletedstrain, probably because the less-oligomeric FtsA*’s ability to interactwith FtsN is already optimal (reflecting a higher number of FtsA’s ICdomains available to interact with FtsN). Furthermore, ftsAR286W andmost of the other ftsA self-interaction mutants are more efficient thanwild-type ftsA in complementing an ftsA-depletion strain, as seenpreviously (9). Thus, the results demonstrate that increasing the levelof FtsN dramatically reduces the amount of FtsA required for di-vision similar to the ftsAR286W mutation.

DiscussionThe E. coli divisome contains a dozen essential proteins thatassemble into a large protein complex that carries out PG syn-thesis at the division site. Assembly of the divisome starts withformation of a Z-ring (formed by FtsZ, FtsA, and ZipA) followedby the hierarchical recruitment of the downstream proteins. Be-cause this is a hierarchical pathway, the recruitment of a givenprotein requires the presence of all upstream proteins and in turn isneeded for the localization of any downstream proteins. Therefore,upon deletion of a protein in the pathway, the Z-ring still forms,but the hierarchical assembly pathway is disrupted. Surprisingly,a number of these division proteins—such as ZipA, FtsEX, andFtsK—can be bypassed by an assortment of conditions. How thenis the divisome assembled in these bypassing conditions?In this study we show that many conditions that suppress

ΔftsEX, even if they seem to be acting at different stages ofdivisome maturation, absolutely depend on the ability of FtsN tointeract with FtsA. Some of the suppressors tested here act early(FtsA*); some later (such as overexpression of FtsN or the ftsB*mutation); or for some, such as increased osmolarity or over-expression of FtsP, it is not clear. Nevertheless, all of them re-quire FtsNcyto, which interacts with FtsA, to be linked with afunctional FtsNE. In addition, when cells lacking FtsEX aregrown in the presence of increased osmolarity, a conditionknown to efficiently suppress ΔftsEX and promote formation ofthe divisome, the late-division proteins are not recruited to the Z-ring in the absence of this FtsN–FtsA interaction. Taken together,these results strongly support a model (Fig. 7A) in which the FtsN–FtsA interaction becomes critical to link the late cell-divisionproteins to the Z-ring in the absence of FtsEX. In addition, ourresults imply that the different conditions that bypass FtsEX pro-mote the FtsN–FtsA interaction.FtsN overexpression suppresses many temperature-sensitive

mutants and also suppresses the loss of FtsEX, ZipA, and FtsK(21, 29–31). In each case, FtsNcyto is essential but not sufficientbecause overexpression of FtsNE or FtsNcyto, or of both, whenunlinked does not suppress even in the presence of wild-type ftsNon the chromosome (34). On the other hand, FtsNSPOR, which isresponsible for septal murein binding (27, 32, 36, 41) and self-enhancement of FtsN recruitment at the septum once constriction

Fig. 5. The FtsN–FtsA interaction is required for the bypass of ftsK by ftsA*.(A) Deletion of chromosomal ftsK was attempted by P1 transduction offtsK::kan into W3110 ftsN::cm ftsAR286W (ftsA*) transformed with pSD266*(ftsA*expressed from its native promoter) and pSD268 (Ptac::ftsN) or pSD268-D5N (Ptac::ftsN

D5N). Selection was on plates containing kanamycin and vari-able concentrations of IPTG incubated at 30 °C for 36 h. FtsN expressing cellsgave transductants at IPTG concentration of 0.01 mM and above, while cellsexpressing FtsND5N only produced colonies at 0.1 mM IPTG and above. (B)Only colonies from strains expressing FtsN (a and b) but not FtsND5N (c and d)can grow when picked from the transduction plates and restreaked ontoidentical fresh selection plates, as illustrated here.

Fig. 6. Increasing FtsN allows cells to divide with less FtsA. CH2 (ftsA-null)/pSEB452 (ftsA, SpecR, repts) was transformed with a combination of compatibleplasmids to modulate the expression of ftsA, ftsAR286W, or ftsN. pKD140 (pBR322 ftsN, AmpR) increases the cellular concentration of FtsN ∼10 times, which issufficient to bypass ZipA (34). pSEB439 (pACYC184, Ptrc*::gfp, lacI

Q, CmR) and its derivatives with ftsA alleles allow modulation of ftsA expression with IPTG.Complementation of FtsA depletion in the different situations was assessed by a spot assay at 42 °C.

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starts, is dispensable for suppression (19, 30, 34). Earlier worksuggested that FtsNcyto along with the transmembrane domain maystabilize the divisome, allowing the bypass (30, 31). The workreported here details what “stabilization of the divisome” means inthese situations. FtsNcyto interacting with FtsA in the cytoplasmmust be linked to FtsNE interacting with periplasmic proteins torecruit and link the late cell-division proteins to the Z-ring when thenormal hierarchical chain of recruitment is broken (for exampleΔftsEX) (Fig. 7A).

Under physiological conditions, the hierarchical recruitmentof divisome proteins operates and FtsN’s essential role is to ac-tivate PG synthesis through its E domain (27, 28) (Fig. 7B). Inthat case, the interaction between FtsA and FtsN may just act tolocalize FtsNE. In the absence of this interaction, FtsNE does notlocalize well and is therefore less efficient at activating PG syn-thesis and needs to be overexpressed to fulfill its role (27). Thisfits well with the current model, suggesting that the FtsA mole-cule involved in the recruitment of the divisome is not fully in-volved in self-interaction and has its IC domain available tointeract with FtsN. Furthermore, recruitment of FtsN to thedivisome is complex and is dependent on FtsQ, FtsI, and FtsA(12, 24, 25, 35, 42). By interacting with FtsA, but also beingdependent upon FtsQ and FtsI, FtsN is put in contact withFtsQLB and FtsWI complexes that are already attached to FtsA,and which it needs to activate. Hence, a role for the FtsN–FtsAinteraction is to position FtsN at a mature divisome enhancingthe efficiency of the activation of FtsQLB and, therefore, FtsWIso that PG synthesis occurs (Fig. 7B).When FtsN does not interact with FtsA, FtsNE has to be present

at higher levels to activate PG synthesis, because it localizes poorly(27, 35). However, when FtsEX is also missing our results showthat strains cannot survive with just FtsNE, despite trying differentconditions that suppress ΔftsEX (Fig. 7C). The cells filament be-cause the late cell-division proteins are not recruited to the Z-ring.This observation clearly suggests that the FtsN–FtsA interaction iscritical because it appears to be the only way to link the late-divisome proteins and the Z-ring when the hierarchical pathwayis disrupted.One possible role of the FtsN–FtsA interaction that we cannot

totally rule out is that it causes FtsA to interact directly withFtsQ or FtsK to recruit other divisome components and allowthe bypass of FtsEX. In support of this hypothesis, FtsQ interactswith FtsA in the bacterial two-hybrid assay (43). Importantlythough, overexpressing FtsNcyto linked to its transmembrane do-main (FtsN1–54) but not physically linked with FtsNE (exported tothe periplasm at a level sufficient to complement ΔftsN) or over-expressing a full-length FtsN mutant with the E domain inacti-vated, cannot suppress ΔftsEX or ΔzipA (34). This stresses anessential role for FtsNE in the periplasm, in addition to FtsNinteracting with FtsA to bypass FtsEX. The role of FtsNE is notlimited to simply activating the FtsQLB complex because, asshown in Fig. 1, FtsB*—a mutant of FtsB already activated for PGsynthesis and which can bypass either FtsN or FtsEX—does notallow growth in the absence of these proteins. FtsNE appears tohave a more direct role in linking the divisome to the Z-ring and itis dependent on its ability to be localized at the Z-ring throughFtsA (FtsND5N does not suppress ΔftsEX). Therefore, we do notfavor the FtsN–FtsA interaction causing FtsA to interact directlywith some other late-division protein to allow the bypass, but thinkthat FtsN directly back-recruits the late cell-division proteins tothe Z-ring through its interaction with FtsA in the cytoplasm andits E domain interacting with other proteins in the periplasm.The existence of back-recruitment was demonstrated by the

Beckwith laboratory by using premature targeting in which a latecell-division protein, such as FtsQ, FtsW, or FtsL, was fused tothe FtsZ-binding protein ZapA (25, 42). Because these ZapAfusions localize directly to the Z-ring, they bypass the normalhierarchical localization requirements. Furthermore, these ZapAfusions are able to recruit all other late cell-division proteins,upstream and downstream of the ZapA fusion, with the excep-tion of FtsN. FtsN’s recruitment to the Z-ring requires not onlyFtsQ and FtsI but also FtsA. Interestingly, in cells depleted forFtsK, FtsN localizes and cells divide if it is overexpressed. In adifferent study, the Beckwith laboratory also showed that FtsNcyto isrequired for the bypass of FtsK (30). In this study, we confirmedthat suppression of ΔftsK by overexpression of ftsN or ftsA* requiresa functional FtsNcyto. Furthermore, we show that suppression of

Fig. 7. Model for the role of FtsN–FtsA interaction in the recruitment of thelate-divisome proteins. FtsA links the PG synthesis machinery with the Z-ringthrough FtsEX or FtsN. This figure is adapted from a previous published model(19). For simplicity, only some of the core division proteins are represented (Zrepresents FtsZ, and so forth). The black arrows symbolize the recruitmentpathway and the green arrows indicate the activation of septal PG synthesis.The hierarchical dependencies of the recruitment of proteins or protein sub-complexes into the divisome are based on previous studies (see text). In thefirst step, ZipA (not depicted here) and FtsEX promote and stabilize FtsAmonomers to expose the IC domain (not involved in self-interaction anymore)to interact with the late-division proteins (5, 10, 19, 34). FtsN’s cytoplasmic(FtsNcyto) and essential (FtsNE) domains are colored red and yellow, re-spectively, and FtsNS represents the SPOR domain. (A) Divisome assembly inΔftsEX. In all conditions that suppress the ΔftsEX deletion [which disrupts (redcross) the “normal” hierarchical recruitment pathway], the interaction be-tween FtsA and FtsNcyto (red arrow) becomes essential. This interaction and theperiplasmic interactions involving FtsNE back-recruit the late cell-division pro-teins to form a functional divisome and activate septal PG synthesis. (B) Sup-pression of ΔftsN requires overexpression of MalG–FtsN. When ftsN is deletedand complemented by MalG–FtsN, the recruitment of the divisome proteinsfollows the classic hierarchical pathway, which depends upon FtsEX. FtsNE

activates septal PG synthesis (unless FtsQLB complex is already activated by amutation in ftsB or ftsL). The loss of FtsN–FtsA interaction (red cross) leads to aless-efficient septal localization of the FtsNE domain which is compensated forby the overexpression of MalG–FtsN (double black arrows). (C) MalG–FtsNcannot rescue ΔftsEX ΔftsN. In the absence of both the FtsN–FtsA interactionand FtsEX the late cell-division proteins are not recruited to the Z-ring, so thedivisome cannot mature and no septal PG synthesis occurs.

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an ftsK deletion also requires a functional FtsNE, similar to thebypass of ftsEX or zipA. Together, these results indicate that theFtsN–FtsA interaction is essential in all situations where there is abreak in the hierarchical recruitment pathway. In these situations,the FtsN–FtsA interaction seems to be the only way to back-recruitthe other cell-division proteins to the Z-ring. Therefore, we proposethat there are only two ways to link the septal PG machinery to theZ-ring: one is the normal physiological pathway (through FtsEX,FtsK, and so forth) and the other is dependent upon the FtsN–FtsAinteraction and only evident when there is a “break” in the hier-archical recruitment. This latter pathway also requires a suppres-sive condition that enhances the FtsN–FtsA interaction.In addition to revealing the FtsN-mediated back-recruitment

pathway for divisome assembly, this study also demonstrates thatoverexpression of ftsN enhances the ability of FtsA to comple-ment, mimicking FtsA mutants impaired for self-interaction.This may explain why many FtsA mutants impaired for self-interaction and overexpression of FtsN are similar in their abil-ity to suppress division defects. By promoting the interactionbetween FtsN and FtsA, either by using FtsA mutants impairedfor self-interaction or by increasing the level of FtsN, the efficiency

of forming the divisome is enhanced. In the literature the bypassof one cell-division protein in the hierarchical pathway by theoverexpression of another one, such as FtsN or the use of ftsA*,has been generally referred to as enhancing divisome stability.This increased “divisome stability” is due to the enhancement ofthe FtsN–FtsA interaction. In this regard, it would be interestingto determine whether the other conditions that bypass ftsEX—such as high osmolarity and overexpression of ftsP and ftsB*—enhance the FtsN–FtsA interaction, and if so, how they improvethe interaction.

Materials and MethodsDescriptions of strains, plasmids and details of their constructions as well asgrowth conditions can be found in the SI Appendix and are also summarizedin SI Appendix, Table S2.

Procedures for P1 transductions and immunofluorescencemicroscopy havebeen done according to protocols previously published (4, 19) and on samplestaken in the conditions described in the text or SI Appendix.

ACKNOWLEDGMENTS. This study was supported by the NIH Grant GM29746(to J.L.).

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