Interaction between FtsW and penicillin-binding protein 3 (PBP3) directs PBP3 to mid-cell, controls...

Interaction between FtsW and penicillin-binding protein3 (PBP3) directs PBP3 to mid-cell, controls cellseptation and mediates the formation of a trimericcomplex involving FtsZ, FtsW and PBP3 inmycobacteria

Pratik Datta, Arunava Dasgupta,† Anil Kumar Singh,Partha Mukherjee, Manikuntala Kundu andJoyoti Basu*Department of Chemistry, Bose Institute, 93/1 AcharyaPrafulla Chandra Road, Kolkata 700009, India.

Summary

In bacteria, biogenesis of cell wall at the divisionsite requires penicillin-binding protein 3 (PBP3) (orFtsI). Using pull-down, bacterial two-hybrid, andpeptide-based interaction assays, we provide evi-dence that FtsW of Mycobacterium tuberculosis(FtsWMTB) interacts with PBP3 through two extracy-toplasmic loops. Pro306 in the larger loop and Pro386

in the smaller loop of FtsW are crucial for theseinteractions. Fluorescence microscopy shows thatconditional silencing of ftsW in Mycobacteriumsmegmatis prevents cell septation and positioningof PBP3 at mid-cell. Pull-down assays and condi-tional depletion of FtsW in M. smegmatis provideevidence that FtsZ, FtsW and PBP3 of mycobacteriaare capable of forming a ternary complex, with FtsWacting as a bridging molecule. Bacterial three-hybridanalysis suggests that in M. tuberculosis, the inter-action (unique to mycobacteria) of FtsZ with thecytosolic C-tail of FtsW strengthens the interactionof FtsW with PBP3. ftsW of M. smegmatis could bereplaced by ftsW of M. tuberculosis. FtsWMTB couldsupport formation of the FtsZ–FtsW–PBP3 ternarycomplex in M. smegmatis. Our findings raise thepossibility that in the genus Mycobacterium bindingof FtsZ to the C-tail of FtsW may modulate its inter-actions with PBP3, thereby potentially regulatingseptal peptidoglycan biogenesis.

Introduction

Cell division is a fundamental process central to bacterialpropagation. Understanding the mechanistic details of celldivision may therefore open hitherto unexplored possibili-ties of development of new antibacterials. A complex setof proteins likely to form a multiprotein complex is crucialto the formation of the septum during bacterial cell division(Donachie, 1993; Margolin, 2005; Rothfield et al., 2005).FtsZ (Erickson, 1997), a GTP-binding protein, is consid-ered to be the bacterial counterpart of eukaryotic tubulin(de Boer et al., 1992; RayChaudhuri and Park, 1992). Itmediates cell division by formation of the Z-ring (Bramhilland Thompson, 1994; Mukherjee and Lutkenhaus, 1994;Erickson et al., 1996). The Z-ring formed by FtsZ servesas a cytoskeletal scaffold for the recruitment of a numberof proteins in a sequential manner in Escherichia coli andin a cooperative fashion in Bacillus subtilis (Katis et al.,2000; DiLallo et al., 2003; Errington et al., 2003). Incertain instances, as in the case of FtsL, FtsB and FtsQ ofE. coli, evidence has been presented in favour of pre-assembly of these proteins before their localization tothe septal region (Buddelmeijer and Beckwith, 2004;Aarsman et al., 2005). This complex may also include FtsIand FtsW (Goehring et al., 2005).

In E. coli, FtsAand ZipAbind directly to the C-terminus ofFtsZ (Liu et al., 1999; Pichoff and Lutkenhaus, 2002) andare believed to stabilize the Z-rings at mid-cell. However,ZipA and FtsA are not ubiquitous, and counterparts havenot been recognized in the globally important pathogen,Mycobacterium tuberculosis, raising the question of howZ-rings may be stabilized in this case. Our previous studiessuggest that FtsW is a candidate protein which may beinvolved in stabilizing Z-rings. FtsZ and FtsW of M. tuber-culosis interact directly through oppositely charged resi-dues present in their C-tails (Datta et al., 2002).Rajagopalan and colleagues have recently providedmicroscopic evidence that in mycobacteria, FtsW colocal-izes with FtsZ to the mid-cell (Rajagopalan et al., 2005).

FtsW is a polytopic membrane protein that is present invirtually all bacteria that have a peptidoglycan cell wall

Accepted 20 October, 2006. *For correspondence. [email protected]; Tel. (+91) 332 350 6619; Fax (+91) 332 350 6790.†Present address: Institut Pasteur, Unite de Genetique Mycobacteri-enne, 25, rue du Dr Roux, Paris, France.

Molecular Microbiology (2006) 62(6), 1655–1673 doi:10.1111/j.1365-2958.2006.05491.xFirst published online 13 November 2006

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

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(Ikeda et al., 1989; Henriques et al., 1998). It is an essen-tial cell division gene in E. coli (Boyle et al., 1997). Inaddition to its role in stabilizing the FtsZ ring, FtsW hasbeen suggested to facilitate septal peptidoglycan synthe-sis by recruitment of penicillin-binding protein 3 (PBP3, orFtsI) to the division site (Weiss et al., 1997; Mercer andWeiss, 2002). PBP3 is the bacterial transpeptidase that isrequired specifically for cell division (Botta and Park,1981). Of all the likely components of the divisome, it isthe best characterized in terms of its enzymatic activity. Italso participates in protein–protein interactions critical forpeptidoglycan synthesis (Marrec-Farley et al., 2000;Mercer and Weiss, 2002; Piette et al., 2004). Indirect evi-dence has suggested that PBP3 and FtsW may interact.However, direct interactions between the two proteins hasnot yet been demonstrated biochemically. Using a varietyof biochemical approaches, we have investigated thispossibility using FtsW and the candidate PBP3 ofM. tuberculosis, expressed in E. coli. Our studies provideevidence that FtsW interacts directly with PBP3 throughtwo extracytoplasmic loops of FtsW spanning residues301–320 and 379–386. Pro306 and Pro386 which are con-served across bacterial species, are critical determinantsfor this interaction. In addition, we demonstrate that FtsZ,FtsW and PBP3 of M. tuberculosis form a ternary complexin vitro, with FtsW participating as a bridging molecule.Bacterial three-hybrid analysis supports the hypothesisthat binding of FtsZ to the C-tail of FtsW strengthensthe interaction of FtsW with PBP3 likely providing alink between cell division and septal peptidoglycanbiosynthesis. These views are strengthened by our obser-vations that ternary interactions among FtsZ, FtsW andPBP3 occur in vivo in Mycobacterium smegmatis andrequire Pro306 and Pro386 of FtsW. These interactionstherefore offer new avenues for exploring strategies fordevelopment of chemotherapeutic agents againstmycobacteria.

Results

Expression of PBP3 of M. tuberculosis andpenicillin-binding activity

The product of the gene Rv2163c present in cosmidMTCY270, one of the collection of clones representing thegenome of M. tuberculosis was predicted to represent thecounterpart of PBP3 (FtsI) of E. coli in M. tuberculosis,based on sequence similarity with the family of class Bhigh-molecular-mass penicillin-binding proteins (Goffinand Ghuysen, 2002) and the association of the gene witha cluster of genes such as ftsW and ftsZ comprising thedivision cell wall (dcw) cluster. The ORF Rv2163c desig-nated in the annotated genome of M. tuberculosis showsthe presence of a large N-terminal extension (with a likely

cytosolic disposition) absent in E. coli PBP3 (Fig. S1).However, we feel that pbp3 could arguably be encom-passed by nucleotides 2426805–2425046 of theM. tuberculosis H37Rv genome, based on sequencealignments (Fig. S1) and on the identification of a putativeribosome binding site upstream of the start site, whichwould give rise to a protein that would be shorter by 93amino acids than what is predicted now. Definitive proof ofthe translational site must await further investigation. Forthe purpose of this report, ‘PBP3’ refers to the 586 (ratherthan the 679) amino acid PBP3 encoded by nucleotides2426805–2425046 of the M. tuberculosis H37Rv genomeencompassing amino acid residues V94 to T679 (numberingbased on the TIGR sequence). The larger 679-amino-acidPBP3 could not be expressed and purified from E. coli.PBP3 and its C-terminal transpeptidase module(Goffin and Ghuysen, 2002) encompassing residues L314

to T679 were expressed in E. coli as 6¥ His-taggedproteins (Fig. 1A). The protein after purification bynickel-nitrilotriacetic acid (Ni2+-NTA) affinity chromato-graphy bound benzyl-[14C]-penicillin in a concentration-dependent manner (Fig. 1B), indicating that Rv2163cencodes a functional penicillin-binding protein. TheC-terminal domain encompassed by residues L314 to T679

bound penicillin with a similar affinity (Fig. 1B), supportingthe view that the C-terminal module of PBP3 ofM. tuberculosis functions as an independent penicillin-binding entity unlike its E. coli counterpart (Goffin et al.,1996).

Interaction of PBP3 with FtsW

In order to test the possibility that FtsW interacts withPBP3, we expressed the two proteins in tandem inE. coli, both under the control of the T7(lac) promoter,FtsW carrying a hexahistidine tag at its N-terminus, andPBP3 carrying a myc tag at its C-terminus. Both theproteins were expressed under the conditions described(Fig. 1C). Their expression was confirmed by Westernblotting using anti-His and anti-myc antibodies. His-FtsWwas pulled down from cell lysates using anti-His anti-body and protein A/G-agarose. The proteins immobilizedalong with His-FtsW were separated on SDS-PAGE,electrotransferred and probed with anti-myc antibody.The positive signal obtained after detection by chemilu-minescence (Fig. 1D), affirmed our view that PBP3 inter-acts with FtsW in vivo.

Analysis of the topology of FtsW

FtsW is a polytopic membrane protein. The topologies ofthe E. coli and Streptococcus pneumoniae FtsW proteinshave been determined experimentally (Gerard et al.,2002; Lara and Ayala, 2002). The topology of FtsW of

1656 P. Datta et al.

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 62, 1655–1673

M. tuberculosis was predicted using various availableonline programs such as TMHMM, SOSUI and HMMTOP. Inorder to validate the predicted topology, targeted fusionsof FtsW were generated with the N-terminal end ofthe TEM b-lactamase or the chloramphenicol acetyl-transferase (CAT) reporters, and susceptibilities of therespective E. coli transformants against ampicillin and/orchloramphenicol were determined (Table S1). E. coliJM105 (lacking chromosomal b-lactamase) had anminimum inhibitory concentration (MIC) of 4 mg ml-1

for ampicillin. The transformants FtsW–Xaa–TEMb-lactamase in which Xaa (the amino acid of FtsW at thefusion junction) was R82, D85, S89, P140, A146, F153, Q209,G224, R234, K301, G311, E320, P379 and P386 respectively, hadMICs of 200 mg ml-1 for ampicillin, consistent with the viewthat these fusion sites were each in the periplasm,

because b-lactamase fusion proteins can provide E. coliwith ampicillin resistance only if the b-lactamase moiety istranslocated to the periplasm (Broome-Smith et al.,1990). E. coli TG1 harbouring FtsW–Xaa–CAT fusions inwhich Xaa (the amino acid of FtsW at the junction offusion with CAT) was H58, V110, R120, A179, E190, G255, R257,D270, A281, K282, R342, R346 and R353 respectively, showedchloramphenicol resistance, consistent with the view thatthese fusion sites were each in the cytoplasm, becauseCAT-fusion proteins can provide E. coli with chloram-phenicol resistance only if CAT is present in the cytosol.Negative validation of the predicted topology was carriedout by analysing the b-lactamase constructs generated byfusion to transmembrane (V235, A208 and A300) or cytoplas-mic (H58, R257, R342 and R353) amino acids, as well as byanalysing CAT constructs fused to transmembrane

Fig. 1. Expression of different constructs of FtsW, PBP3 and analysis of their interactions.A. Coomassie blue-stained gels of uninduced and induced E. coli cells expressing His-PBP3 (last lane) and His-PBP3 (L314–T679) (middle lane).B. Fluorograms showing the binding of purified His-PBP3 (upper panel), and His-PBP3 (L314–T679) (lower panel) to different concentrations ofbenzyl-[14C]-penicillin.C. The panel on the left shows Amido Black-stained blots of uninduced and induced E. coli membranes coexpressing His-FtsW andmyc-PBP3. The panel on the right shows Western blots of membranes from induced cells probed with anti-His or anti-myc antibodies.D. Cell lysates obtained from E. coli coexpressing His-FtsW and myc-PBP3 (lanes 2, 6, 7) or His-FtsW alone (lane 3) or myc-PBP3 alone(lanes 4, 5) were incubated without (lanes 5, 6) or with anti-His antibody (lanes 2–4) or with an irrelevant antibody (lane 7). Proteins wereimmunoprecipitated with Protein A/G agarose. Immunoprecipitates were analysed by immunoblotting with anti-myc antibody. The arrowheadindicates the position of myc-PBP3. The first lane is a positive control of expressed myc-PBP3 only. The data shown in B and D arerepresentative of three separate experiments.

M. tuberculosis PBP3, FtsW and FtsZ form a complex 1657


(Y109, I122, L178, M191, A208, S253, A254, F283, N341 and L354) orperiplasmic (Q209, G224, R234 and G311) amino acids. Theexperimentally determined topology was closest to thatpredicted by HMMTOP, with 10 membrane spanningsegments. b-Lactamase fusions to cytoplasmic (H58, R257,R342 and R353) amino acids showed b-lactamase activitiesin crude extracts of cells comparable to that obtained fromE. coli JM105 cells expressing the TEM b-lactamasealone, confirming that the lack of ampicillin resistance wasnot due to improper folding of fusion proteins. The topol-ogy of FtsW derived from these results is presented inFig. S2.

Mapping of amino acid residues required for interactionof extracytoplasmic loops of FtsW with PBP3

Extracytosolic loops likely harbour the PBP3 binding inter-face of FtsW. In order to dissect the PBP3-binding ele-ments of FtsW, attempts were made to express several ofits extracytosolic loops as His-tagged proteins. Theseattempts were in several cases unsuccessful. Larger His-tagged constructs were therefore generated in order totest their ability to interact with PBP3. His-FtsW(1-300)was unable to pull down myc-PBP3 (Fig. 2A, lane 2),suggesting that extracytoplasmic loops, if any, encom-

Fig. 2. Interaction of different domains ofFtsW with PBP3. Lysates of E. coli expressingHis-tagged proteins derived from domains ofFtsW encompassing the indicated amino acidresidues (wt, or mutants) were incubatedwith Ni2+-NTA-agarose beads in order toimmobilize His-FtsW proteins. Lysates ofE. coli expressing myc-PBP3 (A) ofmyc-PBP3 (L314–T679) (B) were incubated withimmobilized His-FtsW constructs and theprecipitates containing proteins bound tothe agarose beads were analysed byimmunoblotting using anti-myc antibody.The first lanes of A and B represent positivecontrols with purified myc-PBP3 and -PBP3(L314–T679) respectively. The blot in A wasreprobed with anti-His antibody to ensureequal loading of all the His-FtsW proteins (C).(D) Incubations of Ni2+-NTA-agarose-boundHis-FtsW (G255–G524) with cell lysatesexpressing myc-PBP3 were carried out in theabsence or presence of peptide J4 or ascrambled (scr.) peptide (at the indicatedmolar ratios). The precipitates containingproteins bound to the beads were analysedby immunoblotting using anti-mycantibody. The first lane of D represents anegative control containing onlyNi2+-NTA-agarose-bound His-FtsW (G255–G524).(E) His-PBP3 or an irrelevant His-taggedprotein (encoded by the ORF Rv0129c) (ofsimilar pI) was allowed to bind biotinylatedpeptide J4 as described under Experimentalprocedures. Peptide-bound PBP3 wasdetected by pull-down withstreptavidin–agarose followed by Westernblotting with anti-His antibody. The datashown in A–E are representative of threeseparate experiments.



passed within this domain were not involved in interactionwith PBP3. His-FtsW (G255–G524) encompassing the pre-dicted extracytoplasmic loop amino acid residues 301 to320, and 379 to 386 was able to pull down myc-PBP3(Fig. 2A, lane 3) as well as its C-terminal module (Fig. 2B)from cell extracts expressing these proteins, suggestingthat this domain encompassed a PBP3-interacting regionof FtsW. The N-terminal module of PBP3 encompassingamino acid residues V94 to T313 was expressed in E. coli,but did not interact with FtsW (data not shown). PBP3could not be pulled down in a control tube containingNi2+-NTA agarose alone (data not shown) indicating thatthe interaction was specific. Attempts were made to mapthe amino acid residues within the two loops encom-passed by amino acid residues 301 to 320 and 379 to 386that were likely to be of importance in interaction withPBP3. When compared with E. coli FtsW, the regionencompassed by the amino acid residues 301 to 320showed stretches of conserved amino acid residues(Fig. S3). Derivatives of His-FtsW (G255–G524) lacking resi-dues F312 to F314 or G318 to E320 were capable of interactingwith PBP3, whereas interaction was abrogated by a dele-tion encompassing residues Y304 to P306 (data not shown),suggesting that amino acid(s) within the region encom-passing Y304 to P306 were necessary for interaction withPBP3. Each of the three residues within this region wasthen mutated successively to alanine. Only the P306Amutation resulted in a loss of binding of His-FtsW (G255–G524) to myc-PBP3 (Fig. 2A, lane 6). Comparison of thesequences of FtsW proteins available in the databaseshowed that the putative periplasmic loop spanning aminoacid residues P379 to P386 contains several conservedamino acid residues including P379 and P386. Proline resi-dues often occur near protein–protein interaction sites(Kini and Evans, 1995). His-FtsW (L361–G524) could bindmyc-PBP3 (Fig. 2A, lane 7). Mutation of P379 to Ala did notabrogate the interaction (data not shown). However,mutation of P386 to Ala led to an abrogation of interaction(Fig. 2A, lane 8), suggesting that it is one of the residuesinvolved in interactions with PBP3. In each case, expres-sion of the His-FtsW constructs being studied was verifiedby reprobing of blots with anti-His antibody (Fig. 2C).His-FtsW (G255–G524) bearing the double mutation P306A,P386A, did not bind myc-PBP3 (Fig. 2A, lane 4), confirm-ing the importance of these two proline residues in theinteraction.

The putative extracytoplasmic loop encompassed byresidues 376–386 was too short to be tested for binding toPBP3 as an epitope-tagged protein. The role of this loopwas therefore confirmed by testing the ability of thepeptide J4 encompassing residues 376–386 of FtsW toinhibit the binding of FtsW (G255–G524) to myc-PBP3. J4could inhibit the interaction in a dose-dependent manner(Fig. 2D). No inhibition was obtained in the control tube

containing a scrambled sequence. The presence ofpeptide J4 did not affect the penicillin-binding ability ofPBP3 (data not shown). Alternatively, the biotinylatedpeptide J4 was incubated with His-PBP3 for 6 h at 25°Cand the association of the peptide with PBP3 was visual-ized by pulling down the complex with streptavidin–agarose, followed by electrophoretic separation anddetection with anti-His antibody (Fig. 2E). Peptide J4 wasfound to interact with PBP3. A variety of in vitro methodstherefore supported the view that the region encom-passed by residues 376–386 of protein FtsW represents asite of interaction of FtsW with PBP3. The residues 376–386 of FtsW therefore appeared likely to harbour deter-minants crucial to the FtsW-PBP3 binding interface.

Interactions between FtsZ, FtsW and PBP3

Given the fact that FtsW interacts with both FtsZ (Dattaet al., 2002) and PBP3, we tested the hypothesis thatthese proteins are capable of forming a ternary complex.We expressed S-tagged FtsZ, myc-tagged PBP3 and His-tagged FtsW (G255–G524). The expression of full-lengthFtsW was poor and FtsW was susceptible to proteolyticdegradation (Datta et al., 2002). We therefore expressedFtsW (G255–G524) which harbours both the FtsZ- and thePBP3-interacting determinants. Cells expressing the indi-vidual constructs were mixed prior to disruption, followedby immunoprecipitation of each of these proteinsseparately. The method was similar to the strategy usedby Noirclerc-Savoye et al. (2005) for studying the interac-tions between DivIB, DivIC and FtsL of S. pneumoniae.The identity of the co-immunoprecipitated proteins wasverified by Western blotting. Immunoprecipitation withanti-S-tag agarose, followed by immunoblotting with anti-His [for FtsW (G255–G524)] (Fig. 3, blot 1) or anti-myc (forPBP3) (Fig. 3, blot 3) showed that both FtsW and PBP3were part of a complex with FtsZ. When cells expressingFtsW (G255–G524) were omitted from the mixture prior tocell disruption, FtsZ could not pull down PBP3 from thelysates (blot 4). FtsW therefore appeared to be the bridg-ing partner in these interactions resulting in a trimericcomplex between the three proteins. Our earlier observa-tions have suggested that a stretch of aspartate residuesat the C-terminal end of FtsZ are crucial for its interactionswith FtsW of M. tuberculosis (Datta et al., 2002). Inharmony with this, the FtsZ (DD367–D370) mutant wasunable to co-immunoprecipitate FtsW (blot 1, last lane) orPBP3 (blot 3, last lane). In a similar manner, precipitationwith Ni2+-NTA agarose (Fig. 3, blots 5–7) followed byimmunoblotting with anti-myc (blot 5) or anti S-tag (blot 6)antibodies confirmed the formation of a trimeric complexbetween FtsW (G255–G524), FtsZ and PBP3. The interac-tion with PBP3 diminished upon mutation of either P306 toA or P386 to A (blot 5). The ability of the FtsW construct to



co-immunoprecipitate PBP3 was inhibited almost com-pletely in the case of the P306A, P386A double mutant(blot 5).

Bacterial two-hybrid analysis

The bacterial adenylate cyclase two-hybrid analysis(BACTH) based on the method of Karimova et al. (2005)was used to further analyse interactions between FtsW,FtsZ and PBP3. Pairwise interactions between FtsZ andFtsW, as well as between FtsW and PBP3 were analysedby co-transforming the E. coli cya strain DHM1 with pairsof recombinant plasmids expressing the T25 and T18hybrids. The efficiencies of functional complementationbetween the different hybrids were determined byb-galactosidase assays. FtsZ could interact with FtsW(Fig. 4A and B). This interaction was abrogated when

residues D367–D370 were deleted from FtsZ (Fig. 4B) orwhen residues R510–R514 were deleted from the FtsW con-struct (Fig. 4A), confirming the importance of thesestretches of oppositely charged residues in theinteraction. PBP3 could also interact with FtsW (Fig. 4C).However, this interaction was weakened twofold wheneither P306 or P386 of FtsW was individually mutated. Whenboth the residues were mutated, b-galactosidase activitywas reduced to the level of the negative control (Fig. 4C)suggesting the involvement of both these proline residuesof FtsW in its interaction with PBP3. In order to study thepossible ternary interaction between FtsZ, FtsW andPBP3, FtsZ (or its mutant) was coexpressed with FtsW inthe vector pKT25. FtsW, when coexpressed with FtsZ,showed a higher efficiency of interaction with PBP3expressed in pUT18C than FtsW alone with PBP3(Fig. 4D). However, FtsZ alone (in the absence of FtsW)

Fig. 3. Interactions among FtsZ, FtsW and PBP3. E. coli cells overexpressing individually, His-tagged FtsW (G255–G524), myc-tagged PBP3and S-tagged wild-type FtsZ (WT) [or its mutant (DD367–D370)] were mixed, lysed with Cell Lytic™ BII and solubilized cell lysate wasprecipitated with anti-S-tag agarose (blots 1–3), followed by Western analysis with the indicated antibodies. The fourth blot represents asimilar set of experiments, except that FtsW was omitted. Western analysis with anti-S tag antibody (second blot) confirmed equal loading ofFtsZ and its mutant. In a separate set of experiments, E. coli cells overexpressing individually, myc-tagged PBP3, S-tagged wild-type FtsZ(WT) and His-tagged FtsW [G255–G524 (WT)] (or its mutants as indicated in the figure), were mixed, lysed with Cell Lytic™ BII and solubilizedcell lysate was precipitated with Ni2+-NTA agarose (blots 5–7). The agarose-bound proteins were immunoblotted with anti-myc or anti-S tag oranti-His antibody to detect PBP3 or FtsZ or FtsW respectively. Western analysis with anti-His antibody (blot 7) confirmed equal loading ofFtsW and its mutants. E. coli cells expressing individual wild-type proteins (positive controls) were lysed and immunoblotted with theabove-mentioned antibodies separately in order to identify the position of PBP3, FtsZ and FtsW (indicated by arrows). Data shown arerepresentative of results obtained in three separate experiments.



did not interact with PBP3 (last bar in Fig. 4D). The speci-ficity of the effect of FtsZ in enhancing interaction betweenFtsW and PBP3 was borne out by the fact that the FtsZ(DD367–D370) mutant (which lacks the ability to interact withFtsW) was not able to enhance interaction when coex-pressed with FtsW (Fig. 4D). The knowledge that particu-lar deletions (or point mutations) selectively abolishedinteractions between specific partners, provide insightsinto critical determinants of intrinsic associations betweenthese molecules.

Prolines 306 and 386 of FtsW of M. tuberculosis arenecessary for viability

The preceding studies have focused primarily on in vitroassociations of FtsW, FtsZ and PBP3. We wanted to testthe relevance of these interactions in vivo. A pairwiseBLAST analysis (Altschul et al., 1997) showed thatM. smegmatis FtsZ, FtsW and PBP3 were 91%, 66% and78% identical; and 95%, 75% and 89% similar in aminoacid sequence to their M. tuberculosis counterparts(Figs S4–S6). The hydrophilic C-tails of FtsW and FtsZshown to mediate interaction between the two proteins ofM. tuberculosis were conserved in their M. smegmatiscounterparts. The two extracytoplasmic loops of FtsWpredicted to mediate interaction with PBP3 were alsoconserved in the M. tuberculosis and M. smegmatis

proteins. Taking these into consideration we chose therapidly growing mycobacterium, M. smegmatis as themodel system to test whether the associations are ofrelevance in vivo in mycobacteria.

In order to test whether FtsW of M. tuberculosis cansubstitute for FtsW of M. smegmatis, and to identify aminoacid residues critical for interaction of FtsW with PBP3, weused the two-step homologous recombination protocoldescribed by Rajagopalan et al. (2005) and Chauhanet al. (2006). Merodiploid single crossover (SCO) strainscarrying integrated copies of N-terminal, His-tagged wild-type ftsW of M. tuberculosis (ftsWMTB) or ftsWMTB genesencoding the P306A, or P386A or P306A, P386A mutants,were generated. The His-tag was incorporated in orderto facilitate detection as well as immunoprecipitation ofFtsWMTB. To test the consequences of mutating P306

and/or P386, we attempted to delete ftsW of M. smegmatisin the presence of an integrated copy of either wild-type ormutant ftsWMTB. Double crossovers (DCOs) were selectedand analysed by PCR using primers specific for either theintegrated copy of M. tuberculosis ftsW or the chromo-somal copy of M. smegmatis ftsW. DCOs carrying chro-mosomal M. smegmatis ftsW deletions were obtained incells expressing the integrated copy of either wild-typeftsWMTB or ftsWMTB encoding the P306A or the P386Amutations. By contrast only wild-type M. smegmatis ftsWpatterns were obtained in 30 DCOs carrying ftsWMTB

Fig. 4. Bacterial three- or two-hybrid analysisto study the interactions between FtsW,FtsZ and PBP3. E. coli DHM1 cells wereco-transformed with different constructs(or control vectors) as detailed below.A. pUT18C (vector, negative control) or FtsWin pUT18C or its mutant [FtsW (DR510–R514)] inpUT18C were co-transformed with FtsZ inpKNT25.B. pKNT25 (vector, negative control) or FtsZin pKNT25 or FtsZ (DD367–D370) in pKNT25was co-transformed with FtsW in pUT18C.C. PBP3 in pUT18C was co-transformed withpKT25 (vector, negative control) or FtsW (orits mutants as indicated) in pKT25.D. PBP3 in pUT18C was co-transformed withvector only (negative control) or FtsW, or FtsZor FtsW along with FtsZ [WT or (DD367–D370)]in pKT25 in order to study the interactionbetween FtsZ and PBP3. In each case,functional complementation between thehybrid proteins was quantified by measuringthe b-galactosidase activities in toluene-treated E. coli cells harbouring the plasmidsas indicated in the figure. Each bar representsthe mean value � SD from three independentexperiments.



P306A, P386A (ftsW *). Failure to delete the chromo-somal ftsWSMEG in M. smegmatis carrying an integratedcopy of ftsW * supported our contention that both P306 andP386 are necessary for FtsW to fulfil its in vivo function.Mutation of either one of the two proline residues waslikely less deleterious for the cells than deletion of boththe proline residues. DCOs carrying ftsWMTB(WT), orftsWMTB(P306A) or ftsWMTB(P386A) were grown in Middlebrook7H9 broth. Growth was reduced twofold in strains carryingftsWMTB(P306A) or ftsWMTB(P386A) compared with the strainharbouring ftsWMTB (Fig. S7).

FtsW interacts with FtsZ and PBP3 in M. smegmatis

Double crossovers were used to analyse the interactionsbetween FtsZ, FtsW and PBP3 in vivo. Expression ofHis-FtsWMTB did not cause any dramatic change in mor-phology, and the viability of the strain was comparable to

that of the wild-type strain (data not shown), indicatingthat the production of His-FtsWMTB in a strain lacking thechromosomal copy of M. smegmatis FtsW was not toxic tothe cells. Lysates from cells expressing His-FtsWMTB (or itsmutants) were immunoprecipitated with anti-His antibody.Western analysis with anti-PBP3 antibody showed thatthe P306A or the P386A mutants of FtsWMTB were lessefficient in pulling down PBP3 than the wild-type FtsWMTB

(Fig. 5A). This supported the view that both P306 and P386

of FtsW play a role in defining the PBP3-interacting inter-face of FtsW. Reprobing with anti-His antibody confirmedthat the expression of His-FtsW was comparable in eachcase (Fig. 5A, lower blot). In the reverse experiment,lysates were immunoprecipitated with anti-PBP3 antibodyand probed with anti-His antibody. Western analysis againconfirmed that the P306A and the P386A mutants of FtsWwere less proficient in interacting with PBP3 than the wildtype (Fig. 5B). The specificity of the band visualized was

Fig. 5. Interaction of FtsZ and PBP3 with FtsW in M. smegmatis. M. smegmatis DCO strains (A, B, D and E, bearing His-tagged wild-type ormutated ftsW of M. tuberculosis as indicated in the figure) were grown, lysed and membranes were solubilized with Triton X-100 as describedunder Experimental procedures. Solubilized membranes were immunoprecipitated with anti-His (A) or anti-PBP3 antibody (B), followed byimmunoblotting with anti-PBP3 (A) or anti-His (B) antibody. The lower blot represents reprobing with anti-His (A) or anti-PBP3 (B) antibody.The first lane of panel A represents immunoprecipitation from the solubilized membranes of DCO (wild type) with anti-His antibody followed byimmunoblotting with pre-immune sera. The last lane of panel B represents immunoprecipitation from the solubilized membranes of DCO(wild type) with pre-immune sera followed by immunoblotting with anti-His antibody. Panel C (negative control) represents immunoprecipitationfrom the solubilized membranes of untransformed M. smegmatis mc2 155 only. For D and E, immunoprecipitation was carried out withanti-PBP3 or anti-FtsZ antibody followed by immunoblotting with anti-FtsZ or anti-PBP3 respectively. The first lane of each blot representsimmunoprecipitation with pre-immune sera only. The lower blots of D and E represent reprobing with anti-PBP3 or anti-FtsZ respectively.



confirmed by the fact that immunoprecipitation of lysatesfrom wild-type M. smegmatis mc2 155 with anti-PBP3 anti-body followed by Western analysis with anti-His antibodydid not give any band (Fig. 5C). The role of P306 and P386

in regulating ternary interactions between FtsZ, FtsW andPBP3 was then evaluated using anti-PBP3 and anti-FtsZantibodies which did not cross-react (data not shown).Immunoprecipitation with anti-PBP3 followed by Westernanalysis with anti-FtsZ (Fig. 5D) and vice versa (Fig. 5E)showed weakened FtsZ–PBP3 interaction in DCOs car-rying ftsWMTB encoding the P306A or P386A mutants com-pared with the wild type. The expression of PBP3 andFtsZ in cell lysates was comparable in all the DCOs (lowerblots of panels D and E respectively).

Depletion of FtsW of M. smegmatis by antisensing

In order to analyse whether FtsW acts as a bridgingmolecule within a ternary complex involving FtsZ, FtsWand PBP3, in vivo, it was necessary to develop an FtsW-depleted system. Construction of an ftsW knockout wasnot possible because ftsW is an essential gene. An anti-sense construct of FtsW in pMIND, pJB223, wasdesigned. pJB223 carried the whole of the ftsW gene ofM. smegmatis in antisense orientation under the control ofthe tetRO region. Conditional depletion of ftsW fromM. smegmatis harbouring pJB223 was achieved by theaddition of tetracycline (Blokpoel et al., 2005). Growth inthe presence of tetracycline was followed up to 36 h. Celllysis was recorded by a decrease in absorbance at600 nm beginning 24 h after growth (data not shown). Nolysis was observed at this time point when cells weregrown in the absence of tetracycline. We chose to recordthe consequences of FtsW depletion 16 h after growth inthe presence of tetracycline. At this time point, Northernanalysis (as described by Ji et al., 1999) using both senseand antisense probes designed on the ftsW sequence,confirmed the presence of antisense RNA and theabsence of sense RNA (data not shown). Cells wereelongated, but no lysis had occurred. No change in mor-phology was observed in control cultures of M. smegmatis(without the inducible construct) before and after the addi-tion of tetracycline (data not shown), confirming that thealtered morphology was not a consequence of exposureto tetracycline.

M. smegmatis mc2 155 harbouring pJB223 was grownin the absence or in the presence of tetracycline. Wheatgerm agglutinin (WGA)-Alexa 488 staining was done inorder to visualize cell wall and septa. WGA binds specifi-cally to N-acetylglucosamine in the outer peptidoglycanlayer for Gram-positive bacteria (Sizemore et al., 1990)including M. smegmatis (Dasgupta et al., 2006).Tetracycline-induced FtsW depletion was associated withimpaired septum formation inferred from the inability to

detect WGA-Alexa 488 staining between nucleoids(Fig. 6). 85 � 5% (n = 250) of the cells after ftsW silencingcontained more than one nucleoid per cell as observed byDAPI staining. On an average, each cell contained 2.8nucleoids after FtsW depletion.

Localization of FtsZ and PBP3 in FtsW-depletedM. smegmatis

In order to study the septation defect in more detail, immu-nofluorescence microscopy was performed to localizeFtsZ and PBP3. FtsZ localized at mid-cell in uninducedcells (Fig. 7A). When FtsW was depleted (as describedabove), FtsZ was still observed between nucleoids(Fig. 7B). However, cell septation was not complete(Fig. 7B). In the wild-type or –tet cells, one FtsZ ring percell was observed. When FtsW was depleted (+tet), weobserved one to six rings per cell, with the majority of thefilaments containing three rings per filament. In uninducedcells, PBP3 localized to mid-cell as well as at the poles(Fig. 8). Staining at the poles was visible in small cells,suggesting that the new cell pole had likely retained somePBP3 from the previous division. This finding was similarto that observed in the case of E. coli PBP3 (Weiss et al.,1997). When FtsW was depleted from M. smegmatis/pJB223 by the addition of tetracycline, PBP3 could nolonger be visualized by immunofluorescence microscopy(data not shown). DAPI staining (Fig. 7B, top panel) veri-fied that the filaments exhibited proper nucleoid segrega-tion even though PBP3 failed to localize, indicating thatthe cells were healthy. Control experiments using pre-immune sera of FtsZ or PBP3 did not show any staining ofcells (data not shown).

Complex formation involving FtsZ and PBP3 inFtsW-depleted M. smegmatis

We designed experiments to test the status of thecomplex between FtsW, PBP3 and FtsZ in FtsW-depleted M. smegmatis. In the presence of tetracycline,PBP3 could pull down FtsZ in cells carrying the controlvector (pMIND) only (Fig. 9A, lane 3) but not in cellsfrom which FtsW had been depleted by antisensing(pMIND-FtsW) (Fig. 9A, lane 2). Conversely, anti-FtsZantibody could not pull down PBP3 in FtsW-depletedcells (Fig. 9B, lane 2). These results clearly suggestedthat the in vivo complex formation between FtsZ andPBP3 was specific. Immunoblotting of the cell lysateobtained after FtsW depletion did not show any alter-ation in the content of either FtsZ or PBP3 (Fig. S8) indi-cating that the failure to detect interaction between FtsZand PBP3 in the absence of FtsW was due to theabsence of neither FtsZ nor PBP3.



Discussion

Cell division and the formation of the wall peptidoglycanare steps which are critical to the propagation and survival

of most bacterial species. A multicomponent proteinmachinery has been postulated to assemble in an orderedfashion in E. coli to co-ordinate cell septation at mid-cell.The GTP-binding tubulin-like protein FtsZ serves as a

Fig. 6. Fluorescence microscopy of M. smegmatis mc2 155 before and after depletion of FtsW. M. smegmatis mc2 155 was transformed withpMIND carrying ftsW. Cells were grown without (–TET) or with (+TET) tetracycline. Upper micrographs: DAPI staining; middle micrograph:WGA-Alexa 488 (green) staining; lower micrographs: overlay of DAPI (blue) and WGA-Alexa 488 (green) staining. Each bar represents 2 mm.The cell in the inset in each panel is shown after magnification.



cytoskeletal scaffold for the recruitment of a battery ofproteins to the division site. FtsW is believed to stabilizethe Z ring at the membrane. Our own studies have shownthat M. tuberculosis FtsW interacts directly with FtsZthrough strings of oppositely charged residues at theirC-termini (Datta et al., 2002). In this study, we have analy-sed the interaction between FtsW and its likely bindingpartner PBP3 on the extracytoplasmic side of themembrane. We also designed experiments to test thehypothesis that the polytopic membrane protein FtsW is abridging molecule linking cytokinesis with septal cell wallbiosynthesis.

Using the algorithm HMMTOP (Tusnady and Simon,1998; 2001), FtsW was predicted to span the membrane10 times with both N- and C-termini in the cytoplasm. Thepredicted topology was confirmed by generating a seriesof b-lactamase and CAT fusions. Our results did notsupport a model predicting a large extracytosolic loopbetween transmembrane segments 7 and 8 proposed inthe case of FtsW of E. coli (Lara and Ayala, 2002) andS. pneumoniae (Gerard et al., 2002). In vitro pull-down,competition and peptide binding assays suggested thatthe two loops spanning residues 301 to 320 and 379 to386 interact directly with PBP3 with the conserved resi-

dues P306 and P386 being critical for the interaction withPBP3. Pastoret et al. (2004) have shown that simulta-neous mutation of both P368 and P375 of E. coli FtsW (thecounterparts of P379 and P386 of M. tuberculosis FtsWrespectively) prevents the recruitment of PBP3, suggest-ing that this short extracytoplasmic loop is likely to beimportant for FtsW–PBP3 interactions in vivo in E. coli aswell. However, in mycobacteria, the larger loop carryingP306 also appeared likely to be involved in FtsW–PBP3interaction, whereas no reports suggest that this loop isnecessary for FtsW to fulfil its function in E. coli. Our viewswere further strengthened by the following observations.ftsWMTB could replace ftsWSMEG without retardation ofgrowth or loss of viability. DCOs could still be obtainedwhen the chromosomal copy of ftsWSMEG was inactivatedin the presence of an integrated copy of ftsWMTB encodingpoint mutations either at P306 or at P386, whereas ftsWMTB

encoding the P306A, P386A double mutant could notreplace ftsWSMEG.

Earlier work conducted in several laboratories, mostlywith E. coli, has relied on mutating a particular componentof the divisome and analysing the ability of the mutant torecruit other components, as the preferred approach tostudying interactions between proteins of the multicompo-

Fig. 7. Visualization of FtsZ in M. smegmatis by fluorescence microscopy. M. smegmatis mc2 155 was transformed with pMIND carrying ftsWfollowed by induction with tetracycline in order to inactivate ftsW conditionally. Transformed cells before (A) or after (B) induction were stainedwith DAPI (blue, left panel) for visualizing nucleoids. Immunolocalization of FtsZ was carried out by incubation with anti-FtsZ antibody asdescribed under Experimental procedures followed by staining with Alexa 488-conjugated rabbit IgG (green, middle panel). The panel on theright is a merge of the first two panels showing the presence of FtsZ between nucleoids. The failure of cells to separate after silencing ftsW isvisible (B, merged image). Each bar represents 2 mm. The cell in the inset is shown after magnification.



nent cell division machinery. Direct biochemical evidenceof interaction between divisome-associated proteins hasbeen shown only recently in the case of FtsQ, FtsB andFtsL in E. coli (Buddelmeijer and Beckwith, 2004), andmore recently between DivIC, DivIB and FtsL ofS. pneumoniae (Noirclerc-Savoye et al., 2005). In spite ofits limitations, in vitro analyses of protein–protein interac-tions offer a method of validating whether interacting part-ners are by themselves sufficient for an interaction tooccur, or whether additional players are required. Ourdetailed in vitro studies have provided insight into howdirect interactions between FtsZ and FtsW; and FtsW andPBP3 occur. Our studies are the first to demonstratebiochemically, direct protein–protein interactions betweenFtsW and PBP3. We observed that in in vitro assays theC-terminal module of PBP3 interacts with FtsW, whereasthe N-terminal module encompassing residues V94 to T313

(harbouring several amino acid residues conserved inE. coli PBP3 and predicted to be important for localization

Fig. 8. Visualization of PBP3 in M. smegmatis by fluorescencemicroscopy. M. smegmatis mc2 155 was transformed with pMINDcarrying ftsW. Transformed cells before induction with tetracyclinewere stained with DAPI (top panel) for visualizing nucleoids orAlexa 488 (green, middle panel). Immunolocalization of PBP3 wascarried out by incubation with anti-PBP3 antibody as describedunder Experimental procedures followed by staining with Alexa488-conjugated rabbit IgG (green, middle panel). The bottom panelis a merge of the first two panels showing the presence of PBP3 atthe division site between nucleoids. PBP3 could not be visualizedby immunostaining after induction with tetracycline to silence ftsW.No panel has therefore been provided for cells after induction withtetracycline. Each bar represents 2 mm. The white and orangearrows indicate the positioning of PBP3 at poles and mid-cellrespectively.

Fig. 9. Complex formation between FtsZ, FtsW and PBP3 inM. smegmatis. M. smegmatis mc2 155 was transformed with pMINDalone (lane 3 of A and B) or pMIND carrying ftsW (lanes 1, 2 and 5of A and B). Transformed cells were left uninduced (–), or induced(+) with tetracycline to induce conditional inactivation of ftsW.Membranes were prepared from M. smegmatis (lane 4 of A and B)as well as from transformed cells before and after induction withtetracycline. Solubilized membranes were immunoprecipitated withanti-PBP3 (lanes 1–4 of A) or anti-FtsZ (lanes 1–4 of B). In controlexperiments, solubilized membranes obtained from transformedcells before induction (–) with tetracycline were immunoprecipitatedwith pre-immune sera of PBP3 (lane 5, A) or FtsZ (lane 5, B).Immunoprecipitates were blotted with anti-FtsZ (A) or anti-PBP3 (B)antibody. Lane 6 of A and B represents Western analysis ofrecombinant, purified FtsZ and PBP3 with their respectiveantibodies.



of E. coli PBP3 to mid-cell) did not interact with FtsW.However, as we could not express any construct harbour-ing the first 93 amino acids of PBP3, a definitive answerregarding the role of the N-terminal 93 amino acids inmediating interaction of PBP3 with FtsW must awaitfurther experimentation.

In order to establish the physiological significance of theinteraction between FtsW and PBP3 in mycobacteria, weused M. smegmatis as a model system. The relevance ofthe FtsZ–FtsW and the FtsW–PBP3 interaction wasestablished by silencing ftsW in M. smegmatis. Underthese conditions, PBP3 failed to localize to mid-cell.Although FtsZ could still be observed at mid-cell, cellseptation was compromised. Localization of FtsZ at mid-cell was therefore not sufficient for cell division.

We propose that in mycobacteria, FtsZ, FtsW andPBP3 form a complex. The following lines of evidencesupport this view. (i) In vitro reconstitution experimentsshowed that FtsW, FtsZ and PBP3 form a ternarycomplex. FtsZ and PBP3 were not capable of interactingin the absence of FtsW, suggesting that FtsW is a bridgingmolecule. (ii) Bacterial two/three-hybrid analyses sug-gested the likely existence of FtsZ–FtsW–PBP3interactions. FtsW could interact with PBP3 as deter-mined by b-galactosidase assays. Coexpression of FtsZwith FtsW enhanced b-galactosidase activity, suggestingstrengthening of the FtsW-PBP3 interactions by the pres-ence of FtsZ. (iii) Expression of a chromosomal copy ofHis-tagged ftsWMTB in M. smegmatis (inactivated in itschromosomal ftsW gene), followed by immunoprecipita-tion with anti-His antibody and Western blotting with anti-FtsZ and anti-PBP3 antibody, confirmed that the FtsWimmunoprecipitate contained both FtsZ and PBP3. Thefact that anti-PBP3 could pull down FtsZ and vice versafrom extracts of these cells strengthened the view thatternary interactions among FtsZ, FtsW and PBP3 occurin vivo. The weakening of the band intensities in cellscarrying ftsWMTB(P306A) or ftsWMTB(P386A), suggested thatboth these proline residues of FtsW were important forternary complex formation. (iv) ftsW was silenced inM. smegmatis using an antisensing approach. In controlcells (where ftsW had not been silenced), FtsZ couldpull down PBP3 and vice versa, whereas the results ofpull-down experiments were negative when ftsW wassilenced. These results confirmed the likely existence ofan FtsZ–FtsW–PBP3 ternary complex in mycobacteria,and suggested that the interactions are of physiologicalrelevance.

The present findings emphasize how little we under-stand of cell division in mycobacteria. In recent years theformation of protein complexes involving several divisomeproteins has been demonstrated in E. coli (Goehringet al., 2005). FtsQ, FtsL and FtsB form complexes (Bud-delmeijer and Beckwith, 2004), and a subassembly of

FtsA, PBP3 and FtsN has also been suggested (Corbinet al., 2004). In mycobacteria, it is possible that some ofthe divisome proteins may exist in the cell as multiproteincomplexes. We observe that FtsZ is recruited to mid-celleven when FtsW is depleted. PBP3, on the other hand,localizes to mid-cell only in the presence of FtsW. Thepresent study suggests that binding of FtsZ to FtsW couldpotentially modulate the association of FtsW with PBP3.While initial targeting of FtsZ to the predivisional site isprobably FtsW-independent, the interaction betweenFtsW and FtsZ occurring through their C-tails (unique tomycobacteria) is of likely physiological relevance. Thereport by Rajagopalan et al. (2005) that ftsZ * encodingFtsZ with mutated C-terminal residues needed for inter-action with FtsW did not complement, supports this view.This suggests that a complex network of interactionsbetween the components of the divisome likelyco-ordinates cell division with biogenesis of septalpeptidoglycan. The current hypothesis is that PBPs local-ize at the division site mainly through multienzymecomplexes. Cytoskeletal proteins have been speculatedto be part of such complexes (Cabeen and Jacobs-Wagner, 2005). RodA and FtsW have been projected ascandidate bridging molecules in the two complexesinvolved in cell elongation and cell division respectively.Evidence of the existence of the FtsZ–FtsW–PBP3ternary complex in mycobacteria makes it tempting tospeculate that the formation of this complex imparts FtsZwith the ability to regulate the activity of PBP3 (which inE. coli, is to modify the existing peptidoglycan to an inertpolar peptidoglycan). Rigorous experimentation is neces-sary to elucidate the precise function of PBP3 in pepti-doglycan remodelling in mycobacteria; and to elucidatehow peptidoglycan is generated. The present findingsencourage further investigation into how the componentsof the cell division machinery assemble in mycobacteriaand how multiprotein complexes regulate septal pepti-doglycan biosynthesis.

Experimental procedures

Molecular biological procedures

Standard procedures for cloning and analysis of DNA, PCR,electroporation and transformation were used (Sambrooket al., 1989). Enzymes used to manipulate DNA were fromRoche Applied Science, Mannheim, Germany. All constructsmade by PCR were sequenced to verify their integrity. E. colistrains were routinely grown in Luria broth (LB). All constructswere verified by sequencing.

The pbp3 (Rv2163 c) gene of M. tuberculosis (encodingamino acids V94 to T679, TIGR numbering) was amplified fromcosmid MTCY270 and cloned in pET28a (Novagen) usingasymmetric NcoI and XhoI sites or in pBAD-Myc/HisB(Invitrogen) (between BglII and EcoRI) to generate plasmidspJB301 and pJB302 respectively. A construct for expression



of PBP3 (L314–T679) was generated by cloning an appropriateshorter amplicon between the NdeI and EcoRI sites ofpET28a to generate plasmid pJB303. In order to obtain myc-tagged PBP3 (L314–T679) under the control of the T7 promoter,the gene was amplified using pJB301 as template and theproduct was cloned between the asymmetric BglII and EcoRIsites in pBAD-Myc/HisB to generate plasmid pJB304. Next,the gene was amplified using pJB304 as template and clonedbetween the NdeI and EcoRV sites of pET29a(+) to generateplasmid pJB305 for expression of myc-tagged PBP3(L314–T679). A construct for dual expression of FtsW and PBP3was generated in the vector pET-DUET (Novagen). The1.6 kb NcoI-EcoRI fragment encoding the ftsW gene wasexcised from pJB201 (Datta et al., 2002) and cloned in pET-DUET to give plasmid pJB306. The pbp3 gene was amplifiedfrom plasmid pJB302 and cloned between the BglII andEcoRV sites of the plasmid pJB306 to generate plasmidpJB307. The primers are given in Table S2.

The constructs for expression of His-tagged proteins ofdifferent domains of FtsW were generated using the primerpairs depicted in Table S3 and pJB201 as template. Theamplified PCR products were cloned in pET28a.

Mutants within the derivatives of FtsW were generated byoverlap extension PCR. The primers used are depicted inTable S4. The construct for expression of FtsZ bearing aC-terminal S-tag was generated by amplification of the ftsZgene from plasmid pJB101 (Datta et al., 2002) using theprimer pair 5′-TATGGATCCATATGACCCCCCCGCACAACTA-3′ (sense) and 5′-TTTGGTACCGCGGCGCATGAAG-3′(antisense), and cloning between the NdeI and KpnI sites (inbold) of the vector pACYC-DUET (Novagen) to generateplasmid pJB102. The mutant of FtsZ deleted in residuesD367–D370 was generated by overlap extension PCR asdescribed earlier (Datta et al., 2002) and cloned into pACYC-DUET as described above to generate pJB103.

Construction of b-lactamase fusions with truncated FtsWderivatives

PCR was performed using the sense primer5′-ATCGGATCCATATGCTAACCCGGTTGCTGC-3′, anti-sense primers carrying portions of the ftsW gene (seeTable 5) and pJB201 as template. The amplified productswere cloned between the BamHI (in bold) and PvuII sites ofpJBS633 which carries the mature TEM b-lactamase encod-ing blaM (Broome-Smith and Spratt, 1986). E. coli JM105was transformed with the ligation mixture, and transformantsgrowing on LB agar plates containing 50 mg ml-1 kanamycinwere chosen for further analysis. Transformants containingin-frame FtsW-b-lactamase fusions were detected by theirability to grow when patched with toothpicks on to agar con-taining 200 mg ml-1 ampicillin (Broome-Smith and Spratt,1986). The nucleotide sequences across the b-lactamasefusion junctions were determined. The MICs of ampicillin forE. coli JM105 containing FtsW–Xaa–TEM b-lactamasefusions were determined by spotting 4 ml of a 1:105 dilution ofan overnight culture (approximately 40 bacteria) on LB agarplates containing a range of doubling concentrations of ampi-cillin (Broome-Smith and Spratt, 1986). The MIC was thelowest concentration of ampicillin that prevented the growthof bacterial colonies.

Construction and analysis of targeted CAT fusions

The cat gene was PCR-amplified from pACYC184 usingthe forward primer 5′-AGGGTACCAAAAAAATCACTGGATATA-3′ (KpnI site in bold) and reverse primer5′-ATAAAGCTTCGCCCCGCCCTGCCACTC-3′ (Hind III sitein bold) and inserted between the KpnI and HindIII sites ofpBADMycHisA generating pBAD-CAT. CAT constructionswere created by targeted PCR fusion with derivatives ofFtsW, using the forward primer 5′-ATAGATCTGTGCTAACCCGGTTGCTG-3′ containing a BglII site (in bold) andreverse primers encoding portions of the ftsW gene(Table S6). The constructs were transformed in E. coli TG1containing 100 mg ml-1 ampicillin and tested for resistance tochloramphenicol as follows. An overnight culture in LBmedium was diluted 10-fold with fresh medium and allowed togrow for 3 h. Induction was then carried out by the addition of0.05% L-arabinose for 1 h, and 4 ml of a 104 dilution of thecultures was spotted on plates containing different concen-trations of chloramphenicol in combination with 0.2%L-arabinose. Growth was observed after 16 h.

Growth and assay of b-lactamase activity of E. colitransformants carrying in-frame b-lactamase fusionproteins

Escherichia coli JM105 lacking chromosomal b-lactamasewas transformed with plasmids carrying in-frame b-lactamasefusions of FtsW domains, and grown at 37°C in LB containing50 mg ml-1 kanamycin. b-Lactamase activity in the differentcellular fractions was assayed using nitrocefin as substrate(O’Callaghan et al., 1972).

Polyclonal anti-FtsZ and anti-PBP3 antibodies

Polyclonal antibodies against purified recombinant FtsZ andPBP3 of M. tuberculosis were raised in rabbits by Imgenex,Bhubaneswar, India.

Tandem expression of FtsW and PBP3 andco-immunoprecipitation

Escherichia coli BL21 (DE3)/pJB307 cells (coexpressing His-FtsW and myc-PBP3) was induced with 25 mM IPTG at 25°Cfor 6 h. Cells were lysed with Cell Lytic™ B-II (Sigma Chemi-cal, St Louis, MO, USA). The cell-free supernatant was incu-bated overnight with monoclonal anti-His antibody (1:100)(Roche Applied Science, Mannheim, Germany) at 4°C fol-lowed by addition of 5 ml of protein A/G-agarose. After incu-bation for 3 h at 4°C, the beads were pelleted, washed oncein lysis buffer, boiled in SDS gel denaturing buffer, and sepa-rated on SDS-polyacrylamide gels prior to Western blottingwith anti-myc antibody (Roche Applied Science).

Co-immunoprecipitation of FtsW, FtsZ and PBP3

Escherichia coli BL21(DE3) cells harbouring plasmidsexpressing S-tagged FtsZ (or its mutant) or myc-taggedPBP3 or His-tagged FtsW (G255–G524) separately, were



induced with 100 mM IPTG for 3 h at 37°C. Cells obtainedfrom 60 ml cultures were mixed, treated with 500 ml of CellLytic™ BII reagent for 15 min at room temperature. Threehundred microlitres of soluble cell lysate was precipitatedwith either S-protein agarose (10 ml) or with Ni2+-NTA agarose(10 ml) for 2 h at 30°C. The agarose-bound protein complexeswere washed with 50 mM Na-phosphate, pH 7.5, 0.5 M NaCland the protein samples were boiled with 20 ml of 2¥ Laemmlisample buffer for 10 min. Ten microlitres of sample wasloaded on each lane and proteins were separated on SDS-PAGE followed by electrophoretic transfer to PVDFmembrane. The blots were probed with monoclonal anti-His(1:1000) or anti-myc (Upstate, Charlottesville, VA, USA)(1:1000) or HRP-conjugated anti-S tag (EMD Biosciences,Novagen, Madison, WI, USA) (1:5000) antibody. Blots weresubsequently incubated with HRP-conjugated rabbit anti-mouse IgG (1:1000) (Cell Signaling Technology, Beverly, MA,USA) in the case of probing with anti-His and anti-myc anti-body and developed with Lumiglo (Cell Signaling Technology)chemiluminescence reagent.

Expression and purification of PBP3 and PBP3(L314–T679) in E. coli BL21 (DE3)

Cells harbouring pJB301 or pJB303 were grown to an OD600

of 0.6. IPTG was added to a final concentration of 0.1 mMand growth was continued at 37°C with shaking for 4 h. Cellswere lysed with Cell Lytic™ B-II (Sigma). His-PBP3 and-PBP3 (L314–T679) were purified from cell lysates by chroma-tography on Ni2+-NTA agarose.

Penicillin-binding assays

Purified His-PBP3 or His-PBP3 (L314–T679) was labelled withbenzyl-[14C]-penicillin for 30 min at 37°C (Granier et al., 1994)and analysed by SDS-PAGE, Coomassie Blue staining andfluorography of the gels.

Pull-down assay

His-tagged proteins of different domains of FtsW wereallowed to bind to Ni2+-NTA agarose. The soluble fractions ofthe cell lysate from E. coli cells expressing PBP3 (or itsC-terminal domain) carrying a myc tag were incubated ineach tube containing Ni2+-NTA agarose-bound FtsW-derivedproteins for 1 h at 30°C. In each case, the slurry was washedthoroughly with PBS, boiled in SDS gel sample denaturingbuffer, and loaded on SDS-polyacrylamide gels. The sepa-rated proteins were electroblotted onto nitrocellulose,blocked in blocking buffer [5% non-fat dry milk in 1¥TBS containing 0.05% (v/v) Tween 20], and probed withanti-myc antibody. Detection was carried out by incubationwith anti-mouse IgG-HRP-conjugate and enhancedchemiluminescence. In each case, blots were reprobed withanti-His antibody (Santa Cruz Biotechnology, Santa Cruz,CA, USA) to ensure equal loading in all lanes.

Competitive inhibition of the association of protein PBP3with His-FtsW (L361–A406) by peptide J4

This was studied by allowing myc-PBP3 to interact with His-FtsW (G255–G524) in 100 ml of buffer A [5 mM sodium phos-

phate buffer (pH 8), containing 120 mM KCl, 20 mg ml-1

phenylmethylsulphonylfluoride, 1 mg ml-1 gelatin] for 6 h at25°C in the absence or presence of different concentrationsof the peptide H2N-GLLPVTGLQLP-COOH (designatedpeptide J4) corresponding to the sequence of amino acidresidues G376 to P386 of FtsW. A control reaction was set upwith a scrambled peptide (H2N-LPQTGLPLGLV-COOH). Ni2+-NTA agarose was added to each tube, followed byincubation at 25°C. The suspension was centrifuged at2000 g, the pellet was washed twice with 100 ml of buffer A,boiled for 5 min in SDS/PAGE-denaturing buffer and sub-jected to SDS/PAGE, followed by immunoblotting with anti-His antibody.

Analysis of the interaction between FtsW-derivedpeptide and PBP3 in solution

Biotinylated peptide J4 was incubated with PBP3 in buffer Afor 6 h at 25°C in a volume of 100 ml. Control reactions wereset up with an irrelevant (encoded by Rv0129c) His-taggedprotein with pI similar to that of PBP3. After incubation, 10 ml(50% slurry) of streptavidin–agarose was added to each tube,followed by incubation for 30 min at 25°C. The suspensionwas centrifuged, the pellet was subjected to SDS-PAGE andimmunoblotting as described above.

Plasmid constructions for the BACTH and BACTHcomplementation assays

Recombinant plasmids used in the BACTH complementationassays (Karimova et al., 2005) were generated by PCRamplification of the genes coding for the different Fts proteinsusing appropriate primers. A construct for expression ofPBP3 was generated from pJB301, using the sense andantisense primers 5′-ATGGATCCCGGAAACGCGGTCATCTTGGTG-3′ (BamHI site in bold) and 5′-TATGAATTCTAGGTGGCCTGCAAGACCAAAGG-3′ (EcoRI site inbold) respectively, and subcloned into the correspondingsites of the pUT18C to generate pUT18C-PBP3. The con-struct for expression of FtsW was generated from pJB201,using the sense and antisense primers 5′-TCCATGGATCTAGAGTGCTAACCCGGTTGCTG-3′ (XbaI site in bold)(primer 1) and 5′-ATGGTACCACCCGTAACGCTGACCTT-3′(KpnI site in bold) (primer 2) respectively, and cloningbetween the same sites of pUT18C and pKT25 to generatepUT18C-FtsW and pKT25-FtsW respectively. Mutants withinthe derivatives of FtsW were generated by overlap extensionPCR using appropriate primers. The final round of PCR wasperformed using the sense and antisense primers 1 and 2respectively. The products were cloned between the XbaI andKpnI sites (in bold) of pKT25 to generate mutants of FtsW inpKT25.

The resulting recombinant plasmids expressed hybrid pro-teins in which the polypeptides of interest were fused to the Ctermini of two fragments T25 (for FtsW or its mutants) andT18 (for FtsW and PBP3) of the catalytic domain of Bordetellapertussis adenylate cyclase (AC).

Full-length ftsZ gene was PCR-amplified from pJB101using the primer pair 5′-TTTTAAGCTTTATGACCCCCCGCACAACTA-3′ (sense) and 5′-ATGGATCCAGATATCGCG



GCGCATGAAGGGCGGCA-3′ (antisense) (primer 3) andcloned between the HindIII and BamHI (sites in bold) ofpKNT25 generating pKNT25-FtsZ, where FtsZ is fused to theN-terminal end of the T25 fragment of AC. The mutant of FtsZdeleted in residues D367 to D370 was generated by overlapextension PCR essentially as described before (Datta et al.,2002) and the final product was cloned between the HindIIIand BamHI sites of pKNT25.

For coexpression of FtsW and FtsZ, the ftsW and ftsZgenes were cloned in the plasmid pCKB110 (Choudhuri et al.,2002). Briefly, pJB101 was digested with NdeI and HindIII,the fragment encoding ftsZ (or its mutant) gene was excisedand recloned into the same sites downstream of the Shine–Dalgarno sequence present in pCKB110 to generate pJB111.The ftsW gene was amplified from pJB201 using the primers1 and 2, and cloned between the NcoI and KpnI sites (in bold)upstream of the Shine–Dalgarno sequence in pJB111, togenerate pJB112 harbouring the ftsW and ftsZ genes with theartificial ribosome binding site AGGA upstream of the ftsZgene. Subsequently the cassette containing the ftsW and ftsZgenes was amplified from pJB111 by PCR using the senseand antisense primers 1 and 3 respectively, and clonedbetween the XbaI (in bold) and SmaI sites of pKT25 to gen-erate the pKT25-ftsW/ftsZ plasmid containing the ftsW andftsZ genes fused to the T18 gene of AC. The mutated ver-sions of the ftsW or ftsZ genes were generated by overlapextension PCR as described earlier.

For BACTH complementation assays, recombinant pKT25(or pKNT25) and pUT18C carrying fts genes were used invarious combinations to co-transform E. coli DHM1 cells(Karimova et al., 2005). b-Galactosidase activities of thetransformants were determined as described by Karimovaet al. (2005).

Construction of FtsW replacement vectors inM. smegmatis

A two-step homologous recombination strategy as describedby Parish and Stoker (2000) was used to disruptM. smegmatis ftsW at its native locus in the presence of anintegrated copy of either wild-type or mutant ftsW ofM. tuberculosis. Two vectors p2NIL, the gene manipulatingvector without any mycobacterial origin of replication andpGOAL17, the marker gene cassette-containing vector wereused for this purpose. A suicide plasmid with an 840 bpinternal deletion in the ftsW gene was constructed in twosteps. In the first step, a 779 bp DNA fragment encompassingthe 5′ end of ftsW and its upstream flanking region was PCRamplified using the primer pair 5′-AAAAAAGCTTACGCGTTATCGCGACACG-3′ (sense) and 5′-TAGGTACCAGCCGCGTGAACCGTTGG-3′ (antisense). The amplicon wascloned in p2NIL between the asymmetric HindIII and KpnIsites (bold) to give rise to pJB398. In the next step, 736 bpof DNA fragment bearing the 3′ end of ftsW and its down-stream flanking region was amplified using the primerpair 5′-ATGGATCCTCGCAGGCGACCAC-3′ (sense) and5′-TGGTACCAGTTCCAGGTCATAGCCG-3′ (antisense).The amplicon was cloned in pJB398 between the BamHI andKpnI sites (bold) to give rise to pJB399. Finally, a 6.1 kb PacIfragment carrying the lacZ, aph and sacB genes was isolated

from pGOAL17 and inserted into pJB399 to generate thesuicide plasmid pJB401.

Isolation of the strains carrying wild-type ormutated ftsWMTB

Mycobacterium smegmatis mc2 155 was electroporated withdenatured pJB401 DNA. SCOs were selected on agar platescontaining kanamycin and Xgal. Kanamycin-resistant, blue,SCO were isolated. In the next step, His-tagged ftsWMTB (orits mutants) were cloned under the control of the hsp60promoter as described earlier (Dasgupta et al., 2006) andhsp60-ftsW was cloned in pUC19. A 3.757 kb Hyg-integrasecassette was excised from pUC-HY-INT (Mahenthiralingamet al., 1998) and inserted into the recombinant pUC constructto generate an integrative vector. In order to inactivate ftsW atits native location, ftsWMTB or its mutants was first cloned inthe integrative vector as described above, and electroporatedinto the SCO. The resultant merodiploid strains werescreened for DCOs as described by Chauhan et al. (2006).White, Kans, Hygr and sucrose-resistant DCO colonieswere analysed by PCR as well as by Southern blottingusing primers selective for ftsW of M. tuberculosis orM. smegmatis. The expression of FtsWMTB or its mutants inDCO was also confirmed by Western blotting using anti-His-antibody. The viability as well as growth kinetics of DCOswere analysed as described earlier (Dasgupta et al., 2006).

Analysis of interactions among FtsZ, FtsW and PBP3 inmycobacteria

Mycobacterium smegmatis mc2 155 or its transformants werelysed using 0.1 mm zirconia glass beads and a Mini Beadbeater (Biospec Products, Oklahoma) (Yamamoto et al.,2001) and centrifuged at 10 000 g for 15 min. Membraneswere pelleted from the supernatant by centrifugation at100 000 g for 1 h. Membranes were solubilized with 2%Triton X-100 in 10 mM Tris-HCl buffer, pH 8.0 at 4°C. Solu-bilized membranes based on equivalent cell number orprotein concentration were immunoprecipitated with eitheranti-His, or anti-PBP3, or anti-FtsZ antibody or with pre-immune sera followed by Western blotting with appropriateantibodies.

Conditional inactivation of ftsW in mycobacteria

For conditional inactivation of ftsW, antisensing of ftsW wasperformed by PCR amplification of the ftsW gene using theprimer pair 5′-ATGGATCCTCACCCGTAACGCTGACCTTC-3′ and 5′-ATGAATTCGTGGGCAGCATCCTGACC-3′, fol-lowed by cloning of the PCR product in reverse orientation inthe vector pMIND (Blokpoel et al., 2005) between the BamHI(in bold) and EcoRV sites to generate pJB223.

In order to conditionally inactivate ftsW, M. smegmatis mc2

155 was electroporated with either pJB223 or the emptyvector. Transformants were grown up to mid log phase andinduced with tetracycline (20 ng ml-1) for different periods oftime up to 24 h. Northern analysis was performed asdescribed by Ji et al. (1999) to confirm antisensing of ftsW.



Briefly, M. smegmatis was grown in the absence or in thepresence of tetracycline, and total RNA was extracted byusing a Qiagen RNeasy mini protocol kit (Qiagen GmbH,Hilden, Germany). Ten microgram aliquots of RNA wereseparated by electrophoresis on a 1.2% agarose-1.8% form-aldehyde gel and blotted onto a nylon membrane (AmershamBiosciences, a division of GE Healthcare, Buckinghamshire,UK). RNA was cross-linked to the membrane by UV irradia-tion by using a UV Stratalinker (Stratagene, Germany).Single-stranded DNA oligonucleotides probes for eithersense ftsW RNA, 5′-CGAGGATCCACCGGCAGAGATGAGCGGCAGCTACAG-3′) or antisense ftsW RNA, 5′-TGATCATGGTGCTCTCGGCGTCGGGCG-3′ or 16s RNA, 5′-GCGATTACTAGCGACGCCGACTT-3′ were labelled using T4polynucleotide kinase according to standard procedures.Blots were pre-hybridized and then hybridized with [32P]-labelled single-stranded DNA oligonucleotides (100 pmol).The DNA–RNA hybridization was detected after exposure toX-ray film.

Fluorescence microscopy

Immunostaining was adapted from the method of Harry et al.(1995) with some modifications. Cells were fixed by incubationfor 15 min at room temperature followed by 45 min on ice in2.5% (v/v) paraformaldehyde, 0.04% (v/v) glutaraldehyde,30 mM sodium phosphate (pH 7.5). After washing in PBS, thecells were permeabilized by exposing to 2% toluene for 2 min,and immediately transferred to slides. The slides were washedwith PBS, air-dried, dipped in methanol (-20°C) for 5 min andthen in acetone (-20°C) for 30 s and allowed to dry. Afterrehydration with PBS, the slides were blocked for 2 h at roomtemperature with 2% (w/v) BSA-PBS and incubated for 1 hwith appropriate dilutions of primary antibody in BSA-PBS.The slides were washed extensively with PBS and then incu-bated with a 1:1000 dilution of Alexa 488-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) in BSA-PBS. For staining of DNA, 2 mg ml-1 of 4′,6-diamino-2-phenylindole (DAPI) was included with the secondaryantibody. WGA-Alexa 488 (Molecular Probes) 2 mg ml-1

was used for staining of cell walls (Sizemore et al.,1990). WGA is a lectin that binds to oligomers ofN-acetylglucosamine and N-acetylmuramic acid. After exten-sive washing with PBS, the slides were mounted using 50%glycerol. In controls for assessing specificity of the primaryantibodies, the incubation with the pre-immune sera wasincluded.

Acknowledgements

This work was supported in part by grants from the IndianCouncil of Medical Research and the Department of Biotech-nology to J.B. The authors would like to thank Dr StewartCole, Institut Pasteur, Paris for the cosmid MTCY270, DrDaniel Ladant and Dr Gouzel Karimova, Institut Pasteur,Paris for the reagents for the BACTH assays, Dr Brian Rob-ertson, Imperial College, London for the vector pMIND, DrRichard Stokes, University of British Columbia, Vancouver forthe vector pUC-HY-INT, Neil Stoker, Royal VeterinaryCollege, London, and Tanya Parish, Queen Mary’s School of

Medicine and Dentistry, London, for the vectors p2NIL andpGOAL17, Dr Anuradha Lohia, Bose Institute for fluores-cence microscopy and Dr Martine Nguyen-Disteche, Univer-sity of Liege, Belgium, for helpful discussions.

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Supplementary material

The following supplementary material is available for thisarticle online:Fig. S1. Alignment of the amino acid sequences of PBP3 ofM. tuberculosis (Mtb) and E. coli (Ec).Fig. S2. Membrane topology of M. tuberculosis (Mtb)FtsW.Fig. S3. Alignment of the amino acid sequences of FtsW ofM. tuberculosis (ftsw_mtb) and E. coli (ftsw_ec).Fig. S4. Pairwise BLAST analysis of FtsZ of M. tuberculosis(Mtb) and M. smegmatis (Msmeg).Fig. S5. Pairwise BLAST analysis of FtsW of M. tuberculosis(Mtb) and M. smegmatis (Msmeg).Fig. S6. Pairwise BLAST analysis of PBP3 of M. tuberculosis(Mtb) and M. smegmatis (Msmeg).Fig. S7. Growth kinetics of M. smegmatis DCOs.Fig. S8. M. smegmatis mc2 155 was transformed withpMIND alone or pMIND carrying ftsW.Table S1. Resistance of E. coli transformed with the ftsW-bla or ftsW-CAT fusion constructs to ampicillin and chloram-phenicol respectively.Table S2. Primers for constructs of PBP3 and its domains.Table S3. Primers for constructs of His-tagged proteins ofdifferent domains of FtsW.Table S4. Primers for generation of FtsW mutants.Table S5. Primers for generating C-terminal fusions of TEMb-lactamase to FtsW derivatives.Table S6. Primers for generating C-terminal fusions of CATto FtsW derivatives.

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