Molecular Microbiology (2004)

51

(3), 645–657 doi:10.1046/j.1365-2958.2003.03876.x

© 2003 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology1365-2958Blackwell Publishing Ltd, 200351

3645657

Original Article

Membrane interaction of FtsZC.-M. Koppelman

et al.

Accepted 10 October, 2003. *For correspondence. E-mail blaauwen@science.uva.nl; Tel. (+31) 20 525 5196; Fax (+31) 20 525 6271.Present addresses:

†

C.-M. Koppelman: Department of MolecularMicrobiology, Utrecht University, Padualaan 8, 3584 Utrecht, TheNetherlands.

‡

Sir William Dunn School of Pathology, University ofOxford, South Parks Road, Oxford OX1 3RE, UK.

R174 of

Escherichia coli

FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division

Cecile-Marie Koppelman,

1†

Mirjam E. G. Aarsman,

1

Jarne Postmus,

1

Evelien Pas,

1

Anton O. Muijsers,

2

Dirk-Jan Scheffers,

3‡

Nanne Nanninga

1

and Tanneke den Blaauwen

1

*

1

Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, Molecular Cytology, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.

2

Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, Biomolecular Mass Spectrometry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.

3

Molecular Microbiology, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands.

Summary

We investigated the interaction between FtsZ and thecytoplasmic membrane using inside-out vesicles.Comparison of the trypsin accessibility of purifiedFtsZ and cytoplasmic membrane-bound FtsZ revealedthat the protruding loop between helix 6 and helix 7is protected from trypsin digestion in the latter. Thishydrophobic loop contains an arginine residue atposition 174. To investigate the role of R174, this res-idue was replaced by an aspartic acid, and FtsZ-R174D was fused to green fluorescent protein (GFP).FtsZ-R174D-GFP could localize in an FtsZ and in anFtsZ84(Ts) background at both the permissive and thenon-permissive temperature, and it had a reducedaffinity for the cytoplasmic membrane compared withwild-type FtsZ. FtsZ-R174D could also localize in anFtsZ depletion strain. However, in contrast to wild-type FtsZ, FtsZ-R174D was not able to complementthe

ftsZ84

mutation or the depletion strain andinduced filamentation.

In vitro

polymerization experi-ments showed that FtsZ-R174D is able to polymerize,but that these polymers cannot form bundles in thepresence of 10 mM CaCl

2

. This is the first description

of an FtsZ mutant that has reduced affinity for thecytoplasmic membrane and does not support celldivision, but is still able to localize. The mutant is ableto form protofilaments

in vitro

but fails to bundle. Itsuggests that neither membrane interaction nor bun-dling is a requirement for initiation of cell division.

Introduction

Cell division in the rod-shaped bacterium

Escherichia coli

is initiated by FtsZ. FtsZ is a cytosolic protein that poly-merizes into a ring-like structure (Z-ring) underneath thecytoplasmic membrane at the middle of the cell (Bi andLutkenhaus, 1991; Ma

et al

., 1996; Den Blaauwen

et al

.,1999). Subsequently, other division proteins are recruitedto the Z-ring in the following order: ZipA, FtsA, FtsK, FtsQ,FtsL, FtsB (YgbQ), FtsW, FtsI and FtsN (for a review, seeRothfield

et al

., 1999; see also Chen and Beckwith, 2001;Buddelmeijer

et al

., 2002; Mercer and Weiss, 2002).Together with peptidoglycan-synthesizing enzymes, theseproteins form a complex, the divisome, which carries outthe process of cell division (Nanninga, 1998).

A poorly understood aspect of FtsZ is its initial interac-tion with the cytoplasmic membrane. As FtsZ does notcontain any large hydrophobic surface region(s), it isunlikely that it will insert into the membrane. Presumably,FtsZ interacts electrostatically with phospholipids and/orwith (a) membrane protein(s). To elucidate which domainof FtsZ is interacting with the cytoplasmic membrane, wecompared the trypsin accessibility of purified FtsZ withthat of membrane-bound FtsZ. We found that at least twospecific fragments do not occur when membrane-boundFtsZ is digested with trypsin. By N-terminal amino acidsequencing, it was found that one of these fragmentsstarts at amino acid G175, suggesting that the trypsincleavage site at residue R174 is located in a shieldeddomain of FtsZ. Comparing the position of residue R174with its corresponding residue in the three-dimensionalstructure of

Methanococcus jannaschii

FtsZ (Löwe andAmos, 1998; Nogales

et al

., 1998), it was revealed thatresidue R174 is located in a loop between helix 6 andhelix 7 (Fig. 2).

We investigated the role of residue R174 during celldivision by changing the positively charged arginine intoa negatively charged aspartic acid (FtsZ-R174D). FtsZ-

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,

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, 645–657

R174D was not able to complement the FtsZ84(Ts)mutant protein at the restrictive temperature or an FtsZ-depleted strain. It had reduced affinity for the cytoplasmicmembrane compared with that of the wild-type FtsZ. AnFtsZ-R174D-GFP fusion protein appeared to be able tolocalize in wild-type cells as well as in the thermosensitive

ftsZ84

(Ts) mutant strain at the restrictive temperature.The mutant could also localize in the FtsZ-depleted strain.Using

in vitro

polymerization studies, including polymersedimentation, light scattering and electron microscopy, itwas shown that, although FtsZ-R174D could still polymer-ize, the polymers were not able to bundle. Together, thedata have been interpreted to mean that altering the abilityof FtsZ to bind to the cytoplasmic membrane

in vivo

andto bundle

in vitro

abolishes its capability to support celldivision

in vivo.

Results

Trypsin digestion of purified FtsZ and membrane-bound FtsZ

To study the interaction between FtsZ and the

E. coli

cytoplasmic membrane, we isolated inside-out cytoplas-mic membrane vesicles (IMV). These IMV still contain afraction of the total amount of FtsZ present in the cell (datanot shown). To elucidate the domain of FtsZ involved inthe interaction with the cytoplasmic membrane, we useda trypsin digestion assay (see

Experimental procedures

).

E. coli

FtsZ consists of 383 amino acids, of which 35 arepositively charged and can theoretically serve as trypsincleavage sites. However, because of the folding of theprotein and/or interaction with other molecules such asproteins or phospholipids, not all these potential sites willbe accessible to trypsin. We compared the accessibility ofthe trypsin digestion sites of purified FtsZ (free-FtsZ) withthat of cytoplasmic membrane-bound FtsZ (IMV-FtsZ).The trypsin fragments were analysed by SDS-PAGE andWestern blotting with monoclonal antibody F168-8 (mAb-8) and F168-12 (mAb-12), which recognize an epitope inthe amino acid sequences 144–158 and 337–383 of FtsZrespectively (Voskuil

et al

., 1994). The epitope of mAbF168-12 was reassigned to amino acid residues 371–383

of FtsZ. This was based on a comparison of the aminoacid sequence of

Pseudomonas putida

FtsZ and

E. coli

FtsZ, which are both recognized by mAb F168-12 (M.Cassanova and M. Vicente, unpublished results) and theobservation that a C-terminal fusion between FtsQ andamino acids 344–361 of FtsZ was not recognized by themAb (Table 1). As shown in Fig. 1, several digestion sitesof purified FtsZ were available for trypsin resulting in frag-ments that could be recognized with mAb-8 or mAb-12 orboth. Treatment of membrane-bound FtsZ resulted in apattern of protein fragments somewhat different from thedigestion pattern of purified FtsZ (Fig. 1). We selected twofragments of digested purified FtsZ that did not appearwhen IMV-FtsZ was digested (fragments A and B indi-cated by arrows in Fig. 1).

For further characterization of the two fragments, theirN-termini were sequenced according to the Edman deg-radation procedure. Fragment A starts with the aminoacids MFEPM, corresponding to the N-terminus of FtsZ.Based on its molecular mass of 25.8

±

0.4 kDa, as esti-mated from Western blotting, it is most likely a product ofcleavage at either residue R235 or residue R258. The firstamino acids of fragment B were GISLLD, starting at res-

Table 1.

Alignment of the C-termini of

Escheri-chia coli

and

Pseudomonas putida

FtsZ thatare both recognized by mAb F168-12.

Species Amino acid sequence (

E. coli

) 344–383

E. coli

MAPLTQE QKPVAKVVND NAPQTAKEPD YLDIPAFLRK QAD

P. putida

ERPTVMR NQAHAGAAAA AKLNPQDDLD YLDIPAFLRR QAD

Based on a reassignment of the molecular mass of the CNBr fragments described by Voskuil

et al

. (1994), the putative epitope was found to start at 345 instead of 337.

E. coli

FtsZ (Yi andLutkenhaus, 1985) and

P. putida

FtsZ (Yim and Vicente, 1998) C-termini have only the aminoacids 371–383 in common. In addition, a C-terminal fusion between

E. coli

FtsQ and aminoacids 344–361 of

E. coli

FtsZ was not recognized by mAb 12. Therefore, its epitope is tentativelyassigned to amino acids 371–383.

Fig. 1.

Comparison of trypsin digestion-derived FtsZ fragments of purified FtsZ (free FtsZ) and membrane-bound FtsZ (IMV-FtsZ). Sam-ples containing equal amounts of FtsZ were treated with trypsin and analysed by SDS-PAGE and Western blotting. Left, mAb-8. Right, mAb-12 (see

Experimental procedures

). Two of the digestion frag-ments that were not obtained with membrane-bound FtsZ are indi-cated by arrows.

Membrane interaction of FtsZ

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idue G175. This fragment thus arose after cleavage atresidue R174. As this fragment did not appear by digest-ing membrane-bound FtsZ, residue R174 is possibly pro-tected from trypsin digestion by the cytoplasmicmembrane. Because it was not unequivocally clear whichof the two putative residues of fragment A was shielded,we concentrated our efforts on R174 in fragment B.

Using the three-dimensional crystal structure of FtsZfrom

M. jannaschii

(Löwe and Amos, 1998) as a referenceand the alignment of the amino acid sequences of

E. coli

and

M. jannaschii

FtsZ, residue R174 of

E. coli

FtsZappeared to correspond to residue 201 of

M. jannaschii

FtsZ. This residue is located in a loop protruding betweenhelix 6 (H6) and the central helix 7 (H7) according to thenomenclature of Nogales

et al

. (1998; Fig. 2).

FtsZ-R174D is able to localize in an FtsZ84(Ts) strain at the non-permissive temperature

To investigate the putative function of the loop containingresidue R174, we replaced the positive residue R174 witha negatively charged aspartic acid residue, using poly-merase chain reaction (PCR)-based site-directedmutagenesis. To study the localization of the mutant, anFtsZ-R174D-GFP and an FtsZ-GFP fusion protein underthe control of an arabinose-inducible promoter wereconstructed.

Wild-type

E. coli

cells (MC4100

lysA

) were grown tosteady state in minimal glucose medium (GB1) at 28

∞

C.The expression level of the FtsZ-GFP fusion protein wasregulated in such a way that fluorescent rings could bedetected with only minor effects on cell length as a resultof overexpression of FtsZ (data not shown). In theseslowly growing cells (mass doubling time was 80 min),

ª

33.5% of the cells showed an FtsZ ring. Subsequently,these conditions were used for the expression of FtsZ-

R174D-GFP in wild-type cells. The cells contain a fluores-cent ring at the mid-cell position when grown in the pres-ence of 0.005% arabinose (results not shown). Thisindicates that the mutant protein is able to localize in thepresence of wild-type FtsZ.

As it is possible that FtsZ-R174D only localizes in thepresence of wild-type FtsZ, its localization was studied atthe permissive and non-permissive temperature in thetemperature-sensitive strain LMC509 [MC4100

lysA

carry-ing

ftsZ84

(

Ts

)]. Cells were grown in a low-salt glucoseminimal medium (1/2 GB1) to suppress partial functioningof FtsZ84(Ts) (Taschner

et al

., 1988). It has been foundbefore that, at the non-permissive temperature,FtsZ84(Ts) is not able to form rings under these condi-tions (Addinall

et al

., 1997). FtsZ-R174D-GFP was consti-tutively expressed in the presence of 0.005% arabinose.The localization of FtsZ-R174D-GFP in the LMC509 cellsgrown at the permissive temperature in the presence of0.005% arabinose (Fig. 3) was similar to the localizationin wild-type cells (not shown). After expression of FtsZ-R174D-GFP in LMC509 cells grown at the non-permissivetemperature (42

∞

C) for two mass doublings, fluorescentrings could still be observed (Fig. 3). A comparison of thedistribution of FtsZ-R174D-GFP and FtsZ-WT-GFP ringsin LMC509 cells grown for two mass doublings at 42

∞

Cshowed that, for each GFP fusion protein, rings areobserved at the mid-cell position in small cells, whereasin longer cells, rings could also be seen at the 1/4 and 3/4 position (Fig. 4). In a small population of long cells, evenat the 1/8 and 7/8 position, fluorescent rings could beobserved (Fig. 4). The number of FtsZ-R174D-GFP ringsper unit of cell length was found to be similar to that ofFtsZ-WT-GFP rings, indicating that FtsZ-R174D-GFPlocalizes similar to FtsZ-WT-GFP. We therefore infer thatFtsZ-R174D-GFP is able to localize in the absence ofwild-type FtsZ.

Fig. 2.

Localization of the trypsin cleavage site at residue arginine174 in the loop connecting helix 6 and helix 7 on the structure of FtsZ. Residue R174 of

E. coli

FtsZ is projected in green CPK in the corresponding location on the molecular structure of

M. jannaschii

FtsZ (Löwe and Amos, 1998). GDP is also indicated in CPK. The rest of the molecule is depicted as ribbons. Helices H6 and H7 are indicated. The image was made using the program

MOLMOL

(Koradi

et al

., 1996).

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, 645–657

FtsZ-R174D is not able to complement FtsZ84(Ts)

Previously, it has been reported that, under finely tunedexpression conditions, FtsZ-GFP can complement thethermosensitive

ftsZ84

mutation at the restrictive temper-ature (Sun and Margolin, 1998). However, we were notable to reproduce this with our FtsZ-GFP construct (datanot shown). Therefore, we used the FtsZ-GFP fusion pro-teins exclusively for localization studies. To analysewhether FtsZ-R174D was able to complement the ther-mosensitive

ftsZ84

mutation at the restrictive tempera-ture, a stop codon was introduced between

ftsZ

/

ftsZ-R174D

and

gfp

in plasmids pMC209 and pMC376, result-ing in plasmids pMC377 and pMC378 respectively. Theseplasmids were subsequently used for complementationstudies.

LMC509 cells containing the plasmid carrying eitherFtsZ-WT or FtsZ-R174D were grown to steady state in 1/2 GB1 medium at 28

∞

C. FtsZ-WT expression was inducedusing 0.05% arabinose. After growth for two mass dou-blings at the non-permissive temperature, the cells had anaverage length of 2.12

±

0.49

m

m (

n

= 516), which is sim-ilar to the length of wild-type cells (2.24

±

0.51

m

m;

n

= 794), whereas the length of the thermosensitive cells(LMC509) without plasmid was 5.82

±

2.41

m

m (

n

= 502)at 28

∞

C. This indicates that expression of FtsZ-WT with0.05% arabinose is sufficient to complement the

ftsZ84

(Ts) mutation. However, when FtsZ-R174D wasexpressed under the same conditions, most cells occurredas long filaments. The length of these filaments(6.78

±

3.78

m

m,

n

= 507) was comparable to that of thethermosensitive cells without expression of FtsZ from aplasmid. This indicated that the FtsZ-R174D mutant is notable to complement

ftsZ84

(Ts). The lack of complemen-tation by the FtsZ-R174D mutant could be the result of adecrease in the affinity for the site of division. Therefore,the experiment was repeated with cells growing in thepresence of 0.1% arabinose. An eightfold increase in theconcentration of FtsZ-R174D, as measured by densitom-etry of the protein band on an immunoblot, did not result

Fig. 3.

Localization of FtsZ-WT-GFP and FtsZ-R174D-GFP in LMC509

E. coli

cells. Top, cells of LMC509 expressing FtsZ-WT-GFP for two mass doublings at 42

∞

C. Middle, cells of LMC509 expressing FtsZ-R174D-GFP for two mass doublings at 42

∞

C. Bottom, cells of LMC509 expressing FtsZ-R174D-GFP at 28

∞

C. LMC509 expressing either FtsZ-WT-GFP or FtsZ-R174D-GFP constitutively in the pres-ence of 0.005% arabinose were grown to steady state in 1/2 GB1 at 28

∞

C. Subsequently, the cultures were diluted into prewarmed medium and continued to grow for two mass doublings at 42

∞

C. Left, phase-contrast images; right, localization of GFP detecting fluores-cence. The bar is 1

m

m.

Fig. 4.

Distribution of fluorescent rings in LMC509. The distance of fluorescent rings from mid-cell position is indicated in relation to the cell length. Top, FtsZ-WT-GFP. Bottom, FtsZ-R174D-GFP. Negative values refer to the arbitrary left side of the cell; positive values refer to the arbitrary right side of the cell. Grey lines represent the one-quarter and three-quarter positions of the cell.

Membrane interaction of FtsZ

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in complementation of the FtsZ84(Ts) mutation at therestricted temperature (results not shown). From theseexperiments, it could be concluded that, although FtsZ-R174D is able to localize, it does not seem to be functionalin cell division.

A noteworthy difference between the filamentsobtained with the thermosensitive cells without and withexpression of FtsZ-R174D is their morphology. All fila-ments of LMC509 without plasmid were completelysmooth (Fig. 5, top) as found previously (Taschner

et al.,1988), whereas in all filaments expressing FtsZ-R174D,blunt constrictions could be observed (Fig. 5, bottom). Asdescribed before, smooth filaments are obtained either inthe absence of an FtsZ ring as in the case of theftsZ84(Ts) mutant at the non-permissive temperature(Addinall et al., 1997), or in the absence of FtsK (Wangand Lutkenhaus, 1998). Remarkably, in the latter case,FtsZ rings were present (Wang and Lutkenhaus, 1998).The presence of blunt constrictions in LMC509FtsZ-R174D at the non-permissive temperature therefore sug-gests that FtsZ-R174D at least initiated the formation ofa ring. In addition, it suggests that FtsK also localized, asthe absence of FtsK in the presence of FtsZ would haveresulted in smooth filaments, as shown by Wang andLutkenhaus (1998). Immunolabelling of FtsZ of these fila-ments showed the presence of multiple FtsZ rings(results not shown).

FtsZ-R174D is able to localize in FtsZ-depleted cells

To exclude the possibility that FtsZ-R174D could localizein LMC509 because of co-localization with FtsZ84(Ts) atthe restrictive temperature, we studied the localization ofthe mutant in the FtsZ depletion strain VIP205. Thisstrain contains the chromosomal structural ftsZ geneunder the control of the tac promoter (Garrido et al.,1993). VIP205 and VIP205 expressing the wild-type

FtsZ protein from plasmid pMC205 or the FtsZR74Dmutant from plasmid pMC378 were grown in TY at 37∞Csupplemented with kanamycin, ampicillin and 30 mMIPTG. At an optical density (OD) of 0.3, the culture wasdiluted 20 times in prewarmed medium containing 0.2%arabinose instead of IPTG, and growth was continuedfor 3.5 mass doublings (MDs). Subsequently, the cellswere harvested, fixed and labelled with Alexa 546-conju-gated FAb F168-12 against FtsZ for immunofluores-cence microscopy. The average FtsZ concentration inthe cells of the culture was determined by immunoblot-ting and densitometry. After 3.5 MDs in the absence ofIPTG, VIP205 cells contained ª11% of the amount ofFtsZ in the cells in the presence of IPTG. This was asufficient reduction in the FtsZ concentration to causefilamentation (average cell length 19.4 ± 8.9 mm,n = 250), and it abolished any FtsZ ring localization(Fig. 6A). VIP205pMC209 complemented the FtsZdepletion by expressing wild-type FtsZ and showed nor-mal average cell length (5.0 ± 1.35 mm, n = 587), and89% of the cells showed a central FtsZ ring (on average5.56 mm cell length/ring, Fig. 6B). Although the VIP205expressing FtsZ-R174D filamented (average cell length37.6 ± 14.8 mm, n = 206) and could not complement theFtsZ depletion, the cells contained FtsZ rings at regularpositions in most filaments (on average 9.66 mm celllength/ring, Fig. 6C). This indicates either that FtsZ-R174D was able to form a ring itself but is not able toproceed to division or that the residual wild-type FtsZwas sufficient to enable localization of the mutant pro-tein. In the latter case, one out of 10 FtsZ molecules inthe ring could be a wild-type molecule.

FtsZ-R174D has decreased affinity for the cytoplasmic membrane

If the protection of R174 in trypsin treated IMVs was trulydue to its association with the membrane and not a resultof being polymerized at the membrane, the mutant FtsZ-R174D should have a lower affinity for the membranescompared with the wild type. Therefore, LMC509 cells andLMC509 expressing wild-type FtsZ or FtsZ-R174D fromplasmid were grown to steady state in 1/2 GB1 in thepresence of 0.05% arabinose at 28∞C and subsequentlyshifted to 42∞C for two mass doublings. The cells wereharvested and vesicles isolated. The amount of FtsZ wasanalysed by immunoblot, and the protein bands werequantified by densitometry (Fig. 7). The FtsZ-R174D ves-icles contained only 5% of membrane-associated FtsZcompared with the wild-type FtsZ vesicles, whereas thetotal synthesized cellular amount of both proteins was thesame. This indicates that FtsZ-R174D has a reducedaffinity for the cytoplasmic membrane.

Fig. 5. Morphology of filaments of LMC509 and LMC509 expressing FtsZ-R174D. Cells were grown to steady state at 28∞C and subse-quently diluted into prewarmed medium and grown for three mass doublings at 42∞C. Top, LMC509 cells. Bottom, expression of FtsZ-R174D from plasmid in LMC509. Blunt constrictions are indicated by arrows. The bar is 2.5 mm.

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FtsZ-R174D is able to form polymers, but these cannot bundle

To be able to detect any difference in the polymerizationor depolymerization behaviour of FtsZ-WT and FtsZ-R174D, both proteins were purified, and polymerizationwas studied by sedimentation as well as by light scatter-ing. Polymerization was initiated by the addition of GTPto FtsZ in polymerization buffer containing MgCl2, supple-mented with CaCl2 depending on the sample (see figurelegends). Neither FtsZ-WT nor FtsZ-R174D polymerscould be detected in the pellets obtained from the samples

without GTP or without CaCl2 (Fig. 8A, lanes 2, 4, 8 and10). In the samples containing GTP and CaCl2, about 64%of the total amount of FtsZ-WT was found in the pellet,whereas 25% of FtsZ-R174D was sedimented. This indi-cated that FtsZ-R174D was able to polymerize, althoughless efficiently than FtsZ-WT.

The intensity increase in scattered light was used as ameasure for the polymerization rate and the amount ofFtsZ polymers. FtsZ (12.5 mM) was incubated in pre-warmed polymerization buffer supplemented with MgCl2and CaCl2. After 2 min collection of baseline data, GTPwas added to initiate polymerization. As seen in Fig. 8B,the light scattering signal increased immediately afterGTP addition, indicating rapid polymerization. Dependingon the GTP concentration, the light scattering signalremained stable for 0.5–10 min, representing a balancebetween polymerization and depolymerization. Subse-quently, the stable light scattering signal decreased, indi-cating complete hydrolysis of the available GTP and theresulting depolymerization of FtsZ (Mukherjee and Lut-kenhaus, 1999). Independent of the GTP concentration,the light scattering signal obtained from polymerization ofFtsZ-R174D (Fig. 8B, dotted lines) was lower than thesignal from FtsZ-WT (Fig. 8B, solid lines). This is in agree-ment with the results obtained from the sedimentationassay (see above). Nevertheless, the polymerization anddepolymerization rates of FtsZ-WT and FtsZ-R174D weresimilar, although the amount of scattering material pro-duced was less. A possible explanation might be a differ-ence in the arrangement of the wild-type polymerscompared with the FtsZ-R174D polymers.

Electron microscopy of FtsZ- and FtsZ-R174D polymers

In vitro, FtsZ polymers can form bundles in the presenceof Ca2+ (Yu and Margolin, 1997; Mukherjee and Lutken-haus, 1999). Bundling as such would increase the lightscattering signal. To test whether the relatively low lightscattering signal of FtsZ-R174D might be a result of lessefficient bundling of polymers rather than less efficientpolymerization activity, we examined FtsZ-WT and FtsZ-R174D polymers by electron microscopy. Polymerizationof FtsZ-WT in the absence of CaCl2 resulted in thin

Fig. 6. Localization of FtsZ and FtsZ-R174D in FtsZ-depleted VIP205 E. coli cells.A. Cells of VIP205B. Cells of VIP205 expressing FtsZ.C. Cells of VIP205 expressing FtsZ-R174D.The cultures were grown in TY at 37∞C to an OD600 of 0.3 in the presence of 30 mM IPTG to induce expression from the endogenous FtsZ. Subsequently, the cultures were diluted 20 times in medium without IPTG, but with 0.2% arabinose to induce the plasmid-encoded ftsZ genes. The cells were harvested and fixed for immunofluores-cence microscopy after 3.5 MDs. The bar is 1 mm.

Fig. 7. Immunoblot of vesicles obtained from LMC509 cells and LMC509 cells expressing wild-type FtsZ or FtsZ-R174D from plasmid. The cells were grown in 1/2 GB1 at 28∞C in the presence of 0.005% arabinose and subsequently shifted to 42∞C for two mass doublings before harvesting. The blot was incubated with F168-12 mAb against FtsZ and developed by a chemoluminescence substrate.

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© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 645–657

protofilaments (Fig. 9B). The mean thickness of these fil-aments was 8.1 ± 1.5 nm, which is similar to publishedmeasurements (Mukherjee and Lutkenhaus, 1999). Afterpolymerization of FtsZ-WT with CaCl2, in addition to thinfilaments, bundles of protofilaments could be observed(Fig. 9A), with a mean thickness 37.8 ± 10.9 nm. Thisassembly of filaments into bundles in the presence of Ca2+

is in agreement with previous reports (Yu and Margolin,1997; Löwe and Amos, 1999; Mukherjee and Lutkenhaus,1999). Remarkably, when FtsZ-R174D was polymerizedin the presence of CaCl2, only protofilaments could beobserved (Fig. 9C). The average thickness of these fila-ments was 8.8 ± 1.3 nm, which is similar to the thicknessof the FtsZ-WT protofilaments obtained without CaCl2.When FtsZ-R174D was polymerized in buffer withoutCaCl2, in addition to short protofilaments, small ring-likestructures were formed (not shown). We suggest that theinability of FtsZ-R174D protofilaments to assemble intobundles might have caused the lower light scattering sig-nal of FtsZ-R174D in comparison with FtsZ-WT. Presum-

ably, the same holds for the lower yield of FtsZ -R174D inthe sedimentation assay.

Discussion

The initial aim of this study was to elucidate which part ofFtsZ is involved in the interaction with a membrane com-ponent. When comparing the accessibility of purified FtsZand membrane-bound FtsZ to trypsin, we found specificprotein fragments that did not appear when membrane-bound FtsZ was digested. From one of these fragments,N-terminal sequencing revealed that the trypsin cleavagesite between R174 and G175 is shielded. Using the three-

Fig. 8. Polymerization of FtsZ and FtsZ-R174D.A. Polymerization of FtsZ-WT and FtsZ-R174D as determined by a sedimentation assay (see Experimental procedures). The pellet (p) contains polymerized material, and the supernatant (s) contains the unpolymerized material. Samples were incubated with (+) or without (–) GTP and/or CaCl2.B. Light scattering of FtsZ polymers. FtsZ (continuous lines) and FtsZ-R174D (dotted lines) were incubated in the presence of 5 mM MgCl2 and 10 mM CaCl2. After 300 s (to obtain a stable baseline signal), 200 mM (lines 1), 100 mM (lines 2) or 50 mM GTP was added to induce polymerization.

Fig. 9. Electron microscopy of negatively stained FtsZ polymers.A. FtsZ-WT polymerized with 20 mM GTP in the presence of Ca2+ forms bundles with a mean thickness of 37.8 ± 10.9 nm.B. FtsZ-WT polymerized with 20 mM GTP in the absence of Ca2+ forms protofilaments with a mean thickness of 8.1 ± 1.5 nm, and sometimes protofilaments that are intertwined (Lu et al., 2001)C. FtsZ-R174D polymerized with 20 mM GTP in the presence of Ca2+ forms protofilaments with a mean thickness of 8.8 ± 13 nm, but not bundles.The bar is 0.1 mm.

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dimensional structure of M. jannaschii FtsZ as a refer-ence, it could be inferred, according to the secondarystructure assignment of Nogales et al. (1998), that residueR174 is located in a loop between helix H6 and helix H7(cf. Fig. 2) This loop protrudes from the surface and con-tains mainly hydrophobic residues, suggesting that thisloop might be involved in an interaction.

To study the function of this loop, we replaced the argi-nine by an aspartic acid (FtsZ-R174D). FtsZ-R174D wasable to localize in wild-type E. coli and in the ftsZ84(Ts)mutant strain at both the permissive and the non-permis-sive temperature (Fig. 3). However, at the non-permissivetemperature, long filaments appeared, whereas under thesame conditions, expression of wild-type FtsZ was suffi-cient to carry out normal division. Vesicles isolated at thenon-permissive temperature retained only 5% of the FtsZ-R174D mutant protein in comparison with the wild-typeFtsZ, indicating a decreased affinity of this protein for amembrane component. Despite this decreased affinity forthe membrane, the mutant protein was able to localize,even in an FtsZ-depleted strain. Apparently, the block incell division involving R174 might occur at a stage laterthan its initial localization. Alternatively, the mutant fluo-rescent rings might lack the correct spatial organization ofwild-type FtsZ polymers.

Is protection against trypsin digestion in membrane-bound FtsZ caused by polymerization?

The inaccessibility of residue R174 to trypsin in mem-brane-bound FtsZ might be the result of the presence ofFtsZ polymers in the cytoplasmic membrane preparation.Three arguments make this unlikely. First, whereas a con-centration of at least 2 mM FtsZ is required for polymer-ization (Yu and Margolin, 1997; Mukherjee andLutkenhaus, 1998; 1999), the final FtsZ concentration inthe trypsin digestion assays was 0.1 mM, which is farbelow the concentration required for polymerization. Sec-ondly, it has been shown that the stability of FtsZ polymersis regulated by GTP hydrolysis, whereas complete con-sumption of the available GTP results in depolymerization(Mukherjee and Lutkenhaus, 1998; 1999). In our case, noGTP or Mg2+ was added during the isolation of the IMV orduring the trypsin treatment. Thirdly, amino acid R174 isnot part of the protofilament polymerization interface(Löwe and Amos, 1998; 1999) and, therefore, will not beprotected from trypsin in a protofilament. These threearguments suggest that it is not likely that the differencesfound between free FtsZ and membrane-bound FtsZ arethe result of the presence of FtsZ polymers in the IMVfraction. However, it cannot be excluded that the disrup-tion of the cells by French pressure cell shears the divi-somal complex in such a way that the isolated membranevesicles contain divisome fragments and possibly FtsZ

bundles (see below). In this case, R174 is either trypsinprotected because it is concealed by lateral interactionsor because the residue is concealed by the interactionwith one of the other divisomal proteins.

FtsZ-R174D can form protofilaments but does not bundle

The observation of fluorescent rings after expression ofFtsZ-R174D indicates that it is able to polymerize. Fromin vitro polymerization experiments, we concluded that theFtsZ-R174D (de)polymerization rate is similar to that ofFtsZ-WT. However, as visualized by electron microscopy(Fig. 9), FtsZ-R174D polymerizes into single protofila-ments in the presence of Ca2+, whereas FtsZ-WT protofil-aments form bundles under the same conditions. The lackof ability to form bundles could explain the reducedamounts of polymerized material of FtsZ-R174D, as deter-mined by the sedimentation assay and by light scattering(Fig. 8).

Whereas FtsZ protofilaments can assemble into poly-mer sheets or bundles in vitro (Stricker et al., 2002 andreferences therein), nothing is known about their arrange-ment in vivo. One possibility is that the Z ring is composedof continuous protofilaments, which cover the whole cir-cumference of the cell. Another possibility is that overlap-ping short FtsZ protofilaments might form the Z ring,whether or not arranged in bundles (Stricker et al., 2002).The advantage of this last model is its flexibility during cellconstriction. Presumably, depolymerization of short fila-ments will not lead to breakdown of the complete ringstructure, whereas this might be the case in the firstmodel. Thus, by exchanging longer filaments for shorterones, the circumference of the Z ring could easilydecrease. In fact, it has been shown recently by in vivofluorescence recovery after photobleaching experimentsthat the FtsZ ring is a highly dynamic polymer (Strickeret al., 2002). How do short filaments form a flexible yetstable structure? Perhaps the overlapping filaments arekept together via interprotofilament interactions, similar tothose occurring during bundling in vitro. Possibly, R174 isinvolved in bundling in vivo (see also below). Alternatively,the overlapping filaments are kept together by other divi-some proteins. In this case, residue R174 could beinvolved in the interaction with one of the other divisomeproteins.

Interactions between FtsZ and other cell division proteins

Are there potential protein candidates that interact withresidue R174? Direct interactions between FtsZ and thecell division proteins FtsA and ZipA have been reported.In both cases, it was revealed that the interacting domainof FtsZ is located at the extreme C-terminus (Ma andMargolin, 1999; Hale et al., 2000). The C-terminal mutants

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of Ma and Margolin (1999) are, like FtsZ-R174D, able tolocalize and are not functional in cell division. BecauseFtsA did not bind to the majority of the C-terminal mutants,it was proposed that the FtsZ rings were not sufficientlystabilized to support division (Ma and Margolin, 1999).Similarly, the decrease in membrane affinity of the FtsZ-R174D mutant might decrease the stability of the Z ring.However, it should be noted that the C-terminal mutantsof Ma and Margolin (1999) have a smooth morphology,whereas the FtsZ-R174D has blunt constrictions. Thiscould indicate a block in cell division at a later stage as aresult of decreased affinity for divisomal proteins otherthan FtsA or ZipA.

Comparison of FtsZ-R174D with a Caulobacter crescentus mutant

In a recent study, several clustered charged amino acidresidues to alanine mutants of C. crescentus FtsZ weretested for their ability to support cell division in an FtsZ-depleted strain (Wang et al., 2001). Interestingly, one ofthose mutants, ER178AA, was located in the loopbetween helix H6 and H7, at the site corresponding toresidue 174 and 175 of E. coli FtsZ. However, this muta-tion resulted in a phenotype indistinguishable from thewild type, indicating that the mutation was not critical forcell division. Our results, however, indicate that residueR174 of E. coli FtsZ plays an essential role in cell division.Alignment of protein sequence of these loops of E. coliand C. crescentus FtsZ revealed that they are quite differ-ent (not shown). Whereas the E. coli loop consists mainlyof hydrophobic residues with the central positivelycharged R, the C. crescentus loop contains a mixture ofcharged, uncharged and hydrophobic residues. In fact,this loop is not at all conserved in the presently knownsequences of FtsZ (results not shown). This suggests thatthe loop in E. coli FtsZ might be involved in an interactionspecific for this organism.

Protection against trypsin digestion and lack of bundling

Above, we have argued that protection of residue R174 isnot due to its shielding in polymerized FtsZ, but to itsinteraction with the cytoplasmic membrane. Although itwas identified as a shielded site, FtsZ-R174D was stillable to localize as a fluorescent band at the cytoplasmicmembrane. It is feasible that the FtsZ-R174D mutant co-localized with the residual 10% wild-type FtsZ protein inthe FtsZ-depleted strain VIP205 after 3.5 MDs. We werenot able to reduce the amount of endogenous FtsZ back-ground further as the filaments started to die. If theobserved rings in the VIP205 expressing the FtsZ-R174Dmutant for 3.5 MDs were indeed a mixture of wild-type andmutant molecules, only one wild type per 10 FtsZ-R174D

molecules would be possible. It cannot be excluded thatthese wild-type FtsZ proteins are the membrane-interact-ing ones in the ring. An argument against this possibilityis that the FtsZ84(Ts) protein of LMC509 also has a verylow affinity for the cytoplasmic membrane at the restrictivetemperature, whereas the FtsZ-R174D mutant still local-ized in this strain. Whether this band represents anauthentic ring structure cannot be judged. The finding thatthe FtsZ-R174D mutant shows blunt constrictions con-trasts with the smooth filament phenotype of classic FtsZmutants that do not localize. It suggests that residue R174binds to a component involved in a later stage of thedivision process and that membrane interaction is not arequirement for the initiation of cell division. In addition,the FtsZ-R174D mutant is affected in bundling of protofil-aments in vitro.

We speculate that residue R174 has a dual function.This dual function could be performed because there is alarge excess of the number of FtsZ molecules comparedwith the number of its membrane partners (ZipA, FtsK,FtsI, etc.) present in the divisome. We thus suggest thata fraction of the FtsZ molecules is involved in membraneinteraction via residue R174, whereas this residue in thelarger fraction of the FtsZ molecules plays a role inbundling.

Experimental procedures

Bacterial strains and growth conditions

Escherichia coli K-12 MC4100lysA was used as wild-typestrain (Table 2). The thermosensitive LMC509 strain isisogenic to MC4100lysA apart from ftsZ84(Ts) (Taschneret al., 1988). MC4100lysA was grown at 28∞C to steadystate in glucose minimal medium (GB1) containing 6.33 g ofK2HPO4·3H2O, 2.95 g of KH2PO4, 1.05 g of (NH4)2SO4,0.10 g of MgSO4·7H2O, 0.28 mg of FeSO4·7H2O, 7.1 mg ofCa(NO3)2·4H2O, 4 mg of thiamine, 4 g of glucose and 50 mgof lysine per litre, pH 7.0, at 28∞C. LMC509 was grown in alow-salt glucose minimal medium (1/2 GB1), containing1.33 g of K2HPO4·3H2O and 1.48 g of KH2PO4 per litre,pH 7.0, but otherwise identical to GB1 medium. The mediawere supplemented with ampicillin (100 mg ml-1) or kanamy-cin (50 mg ml-1) when needed. Cell growth was followed bymeasuring the optical density (OD) at 450 nm, while cellnumbers were monitored using an electronic particlecounter (orifice 30 mm). Cultures were considered to be insteady-state growth if the ratio between OD and the numberof cells remained constant over time (Fishov et al., 1995).VIP205 (Table 2) contains the chromosomal structural ftsZgene under the control of the tac promoter (Garrido et al.,1993). As no other copy of the ftsZ gene is present, thestrain depends for growth on externally added IPTG(30 mM). VIP205 was grown at 37∞C in rich medium (TY):10 g of bacto tryptone, 5 g of yeast extract, 5 g of NaCl and3 ml of 1 N NaOH per litre. The cells were never grownbeyond an optical density of 0.3, which was monitored at600 nm.

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Isolation of E. coli cytoplasmic membrane vesicles

Escherichia coli cytoplasmic membrane vesicles (IMV) wereisolated from a steady-state grown culture of MC4100lysAessentially as described previously (De Vrije et al., 1987).The cytoplasmic and outer membrane vesicles were sepa-rated according to the method of Osborn and Munson (1979).The membrane vesicles were resuspended in buffer [50 mMtriethanolamine-HAc, pH 7.5, 250 mM sucrose, 1 mM dithio-threitol (DTT) and 0.5 mM Pefablock (Roche Diagnostics)],frozen in liquid nitrogen and stored at -70∞C.

Trypsin digestion of purified FtsZ and membrane-bound FtsZ

Samples containing 10 mg of purified FtsZ were incubated onice with 10 U ml-1 (final concentration) trypsin in a total vol-ume of 50 ml of 50 mM Hepes, pH 7.5, 0.1 mM EDTA. To stopthe digestion, 12.5 ml of five times concentrated SDS samplebuffer (250 mM Tris-HCl, pH 8.3, 10% SDS, 10% DTT, 50%glycerol, 0.05% bromophenol blue) was added, and the sam-ples were immediately incubated for 5 min at 100∞C.

For the digestion of membrane-bound FtsZ, 40 mg of IMV(based on protein concentration) per sample were used.These isolated vesicles still contained 2.5 mg of FtsZ per mgof protein. The IMV suspension was diluted to a final volumeof 50 ml in buffer L [50 mM triethanolamine-HAc, pH 7.5,250 mM sucrose, 1 mM DTT and 0.5 mM Pefablock (RocheDiagnostics)], containing sucrose to prevent osmotic shock.The samples were incubated with 10 U ml-1 (final concentra-tion) trypsin for 60 min on ice. The digestion was stopped bythe addition of four volumes of ice-cold acetone, and theproteins were precipitated by incubation for 30 min at -70∞C.The samples were centrifuged for 15 min at 20000 g at 4∞C.The pellets were resuspended in SDS sample buffer andincubated for 5 min at 100∞C.

Samples were analysed by SDS–PAGE on a 12% poly-acrylamide gel and subsequently submitted to Western blot-ting. Depending on the experiment, blots were incubated withthe monoclonal antibodies F168-8 (mAb-8) and F168-12

(mAb-12) against FtsZ (Voskuil et al., 1994) or a polyclonalantibody against FtsZ and a secondary antibody coupled toa peroxidase. Protein bands were detected using the Piercechemoluminescence method as described by themanufacturer.

For N-terminal sequencing, the proteins were transferredto polyvinylidene difluoride (PVDF; Millipore) membrane afterSDS-PAGE. Subsequently, the membrane was stained for5 min with 0.25% Coomassie brilliant blue G-250 (ServaFeinbiochemica) in an aqueous solution of 40% methanoland 5% acetic acid, and destained with 40% methanol inwater. Bands of interest were cut out and submitted to auto-matic Edman sequencing on an Applied Biosystems 494protein sequencer.

Plasmid construction

For the construction of an ftsZ-c3gfp translational fusion, aplasmid from our laboratory stock containing the ftsZ geneincluding its own Shine–Dalgarno sequence was used. Thestop codon of the ftsZ gene was changed into a leucine. TheftsZ-non-stop (ns) gene was cloned into pUC18, resulting inpMC207. Subsequently, a c3gfp fragment encoding anenhanced version of green fluorescent protein (GFP; Crameriet al., 1996) (obtained from P. Stemmer, Affymax ResearchInstitute, CA, USA) was ligated into this vector yieldingpMC208. The ftsZ-ns-c3gfp gene was recloned into a pBAD-MycHisA vector, which provides regulated expression of pro-teins (Invitrogen) to obtain pMC209. This vector was used forsite-directed mutagenesis using the Stratagene Quick-Change mutagenesis method to change residue arginine 174into aspartic acid, resulting in plasmid pMC376. The samemutagenesis was performed on an IPTG-inducible expres-sion plasmid containing the ftsZ gene (pRRE6; from ourlaboratory stock), resulting in plasmid pMC379. The followingprimers were used: FWFtsZ-R174D (5¢-CTGCTGAAAGTTCTGGGCGACGGTATCTCCCTGCTGG-3¢) as forwardprimer and REVFtsZ-R174D (5¢-GCACCAGCAGGGAGATACCGTCGCCCAGAACTTTCAGCAG-3¢) as reverseprimer.

Table 2. Bacterial strains and plasmids.

Strain or plasmid Genotype and/or features Source

MC4100lysA F–, araD139, D(argF-lac)U169, deoC1, flbB5301, Taschner et al. (1988)ptsF25, rbsR, relA1, rpsL150, lysA1

LMC509 F–, araD139, D(argF-lac)U169, deoC1, flbB5301, ptsF25, Taschner et al. (1988)rbsR, relA1, rpsL150, lysA1, ftsZ84(Ts)

BL21(DE3) F–, dcm, ompT, hsdS (rB– mB

–), gal (DE3) StratageneBL21(DE3)-pLysS BL21(DE3) + pLysS StratageneVIP205 AraD139, D(ara-leu)7697, D(lac)X74, galU, Garrido et al. (1993)

galK, rpsL, ftsA::kan-T4-LacIq-ptac-ftsZpRRE6 pET11b-ftsZ stop R. van EijsdenpRRE7 pET26b-ftsZ R. van EijsdenpMC206 pRRE7-ftsZ-non-stop This studypMC207 pUC18-ftsZ-non-stop This studypMC208 pUC18-ftsZ-non-stop-c3gfp This studypMC209 pBadMycHisA-ftsZ-non-stop-c3gfp This studypMC376 pBadMycHisA-ftsZ-R174D-non-stop-c3gfp This studypMC377 pBadMycHisA-ftsZ-stop This studypMC378 pBadMycHisA-ftsZ-R174D-stop This studypMC379 pRRE6-ftsZ-R174D This study

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To perform studies with FtsZ-WT and FtsZ-R174D withoutGFP on an adjustable plasmid such as pBADMycHisA vector,a stop codon was introduced, using the Quick-Changemutagenesis method, between ftsZ and gfp in plasmidspMC209 and pMC376, resulting in plasmids pMC377 andpMC378 respectively. The following primers were used:FtsZFWstop (5¢-CCCAGCATTCCTGCGTAAGCAAGCTGATTAAGCTTGCTCTAGAATGGCTAGCAAAGGAG-3¢) as for-ward and FtsZREVstop (5¢-CTCCTTTGCTAGCCATTCTAGAGCAAGCTTAATCAGCTTGCTTACGCAGGAATGCTGGG-3¢) as reverse.

Primers for the mutagenesis were obtained from MWG-Biotech. The mutations were confirmed by DNA sequencing(BaseClear). DNA manipulation techniques were essentiallyperformed as described by Sambrook et al. (1989).

Preparation of cells for microscopy

Cells were fixed in 2.8% formaldehyde (FA) and 0.04% glut-araldehyde (GA) in growth medium. To prevent repolymer-ization of FtsZ84(Ts) into the ring (Addinall et al., 1997), inthe case of growth at 42∞C, a mixture of FA and GA wasprewarmed before the cells were added. After incubation for15 min at room temperature, the cells were collected by cen-trifugation at 8000 g for 5 min, washed once in PBS(140 mM NaCl, 27 mM KCl, 10 mM Na2HPO4·2H2O and2 mM KH2PO4 per litre, pH 7.2) and stored in PBS at 4∞Cuntil analysis.

Microscopy and image analysis

Cells were immobilized on 1% agarose in water slab-coated object glasses based on the method described byVan Helvoort and Woldringh (1994) with the following modi-fications. An agarose surface was prepared by adding 1%agarose on an object slide, which was kept at 65∞C on aheating block. The agarose layer was streaked out anddried completely at room temperature to ensure the attach-ment of a second layer. Subsequently, a drop of 80 ml of1% agarose (65∞C) was placed on the preheated objectslide and covered with a siliconized coverslip. After 10 minat room temperature, the coverslip was carefully removed,and the fresh agarose ‘microslab’ was air dried for another5 min. A drop of 3 ml of cell suspension was added andcovered with a clean coverslip, which was sealed with nailpolish.

The cells were photographed with a cooled Princeton CCDcamera or a Coolsnap-fx camera mounted on an OlympusBX-60 fluorescence microscope through a UPLANFl 100¥NA 1.3 oil objective. Images were taken using the publicdomain program OBJECT-IMAGE2.08 by Norbert Vischer(University of Amsterdam; http://simon.bio.uva.nl/object-image.html), which is based on NIH IMAGE by Wayne Ras-band. In all experiments, the cells were first photographed inthe phase-contrast mode and subsequently, when required,with a GFP fluorescence filter (U-MNB, excitation 470–490 nm). The two photographs were stacked, and the lengthof each cell was measured in the phase-contrast image,whereas the number of FtsZ rings was determined in theGFP image.

Overproduction and purification of FtsZ

FtsZ-WT and FtsZ-R174D were purified as described forFtsZ-WT (Scheffers et al., 2000). FtsZ-WT was isolated fromE. coli BL21 (DE3) transformed with pRRE6, whereas for theisolation of FtsZ-R174D, E. coli BL21 (DE3) pLysS (Strat-agene) transformed with pRRE6-FtsZ-R174D was used. Thepurified proteins were dialysed against 50 mM Hepes-NaOH,pH 7.2, 0.1 mM EDTA, 0.1 mM phenylmethylsulphonyl fluo-ride (PMSF) and 10% glycerol, aliquoted, frozen in liquidnitrogen and stored at -70∞C.

Polymerization studies

To investigate the polymerization activity of FtsZ, a sedimen-tation assay and a light scattering assay were performed. Thesedimentation of FtsZ polymers was carried out as describedpreviously (Scheffers et al., 2000). A sample of 13.3 mM FtsZ(40 mg) was incubated at 30∞C in 75 ml of polymerizationbuffer (50 mM MES/NaOH, pH 6.5, and 50 mM KCl) supple-mented with 3.5 mM MgCl2. Dependent on the sample,10 mM CaCl2 and/or 0.2 mM GTP (after 2 min prewarming)were added, and the samples were incubated for 5 min at30∞C. Polymers were sedimented by centrifugation using aBeckman airfuge (A-100 18∞ rotor) at 28 p.s.i. for 10 min. Theamount of FtsZ in both pellets and supernatants was analy-sed by SDS-PAGE (12% gel), Coomassie brilliant blue stain-ing and quantified by densitometry.

Light scattering was performed essentially as describedbefore (Mukherjee and Lutkenhaus, 1999). Samples contain-ing 12.5 mM FtsZ were incubated at 30∞C in 2.3 ml of poly-merization buffer supplemented with 5 mM MgCl2 and 10 mMCaCl2. After 300 s of baseline data collection, polymerizationwas induced with 20–200 mM GTP. Light scattering at 90∞was measured on a QuantaMaster 2000-4 fluorescencespectrophotometer (Photon Technology International), withthe excitation and emission wavelength set at 350 nm.

Electron microscopy

FtsZ (12.5 mM) was polymerized with 20 mM GTP at 30∞C in50 ml of prewarmed polymerization buffer, supplemented with5 mM MgCl2 and, depending on the sample, with 10 mMCaCl2. After 2 min, 3 ml of the polymerization reaction wasapplied to a 400-mesh carbon-coated grid for 5 min andblotted dry by placement of a filter paper to the side of thegrid. The grid was subsequently negatively stained for 1 minby 3 ml of 1% aqueous solution of uranyl acetate and blotteddry. Finally, the grids were dried for 1 h at 60∞C. Grids wereviewed in a Philips 400T transmission electron microscope(a generous gift from A. Knoester, Shell Research and Tech-nology Centre, Amsterdam). Images were taken with acooled Princeton CCD camera, and measurements weremade using the public domain program OBJECT-IMAGE2.08.

Acknowledgements

We thank Mercedes Casanova and Miguel Vicente for thedata on the recognition of the Pseudomonas putida FtsZ bymAb F168-12, and Pilar Palacios and Miguel Vicente for the

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VIP205 strain. We also thank Daniele Carretoni for the poly-clonal antibodies against FtsZ. This work was supported bythe Netherlands Organisation for Scientific Research (NWO)via its life science branch NWO-ALW (C.-M. Koppelman andD.-J. Scheffers, grant no. 805.33.221-P) and a Vernieuwing-simpuls NWO Grant (T. den Blaauwen) The proteinsequencer was largely funded by NWO via its medicalresearch branch NWO-MW.

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