Vol. 174, No. 1

Roles of MinC and MinD in the Site-Specific Septation BlockMediated by the MinCDE System of Escherichia coli

PIET A. J. DE BOER,* ROBIN E. CROSSLEY, AND LAWRENCE I. ROTHFIELD

Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030

Received 26 August 1991/Accepted 21 October 1991

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation ofpotential division sites at the ceil poles in a process that requires the coordinate action of the MinC, MinD, andMinE proteins. In the absence of MinE, the coordinate expression of MinC and MinD leads to a generalinhibition of cell division. MinE gives topological specificity to the division inhibition process, so that theseptation block is restricted to the cell poles. At normal levels of expression, both MinC and MinD are requiredfor the division block. We show here that, when expressed at high levels, MinC acts as a division inhibitor even

in the absence of MinD. The division inhibition that results from MinC overexpression in the absence of MinDis insensitive to the MinE topological specificity factor. The results suggest that MinC is the proximate cause

of the septation block and that MinD plays two roles in the MinCDE system-it activates the MinC-dependentdivision inhibition mechanism and is also required for the sensitivity of the division inhibition system to theMinE topological specificity factor.

In Escherichia coli, correct placement of the divisionseptum involves selection of the proper site at midcell. Thisrequires the site-specific inhibition of septation at potentialseptation sites that are present at the cell poles (9, 11). Thisprocess is controlled by the products of the minB operon,MinC, MinD, and MinE (12, 13). We have previously shownthat coexpression of minC and minD in the absence of MinEleads to the inhibition of septation at both polar and cell-internal potential division sites. Under normal conditions,the MinC/MinD division block is counteracted in a topolog-ically specific fashion by MinE, so that septation is allowedat midcell but is still prevented at the cell poles. The absenceof functional MinC or MinD protein or overexpression ofminE leads to loss of this site-specific division inhibitionprocess. This results in the frequent misplacement of divi-sion septa at cell poles, leading to the formation of smallchromosomeless minicells (13).

Further insight into the roles of the Min proteins camefrom the finding (14, 21) that MinC also plays an essentialrole in the division inhibition that results from expression ofthe dicB gene, a division inhibitor gene that is normally notexpressed (1, 2). MinC/DicB division inhibition differs fromMinC/MinD-mediated inhibition in that it is resistant toMinE (14). Because MinC is the common component of boththe MinD- and DicB-dependent division inhibition systems,it was suggested that MinC is the component that is respon-sible for the division block in both systems, with MinD andDicB functioning as activators of the MinC-dependent divi-sion inhibitor (14).A candidate for the target of the division inhibitor is the

ftsZ gene product. The ftsZ gene is an essential cell divisiongene which is part of a large cluster of genes at 2 min on theE. coli chromosome that are involved in murein metabolismand septum formation (11, 16, 22). A lack of functional FtsZleads to the formation of nonseptate filaments (10, 24, 26),and overexpression of ftsZ leads to an increase in thenumber of septa formed per unit of cell mass (27). Increasedlevels of FtsZ suppress the MinC/MinD-mediated division

* Corresponding author.

block, as well as the division block that results from coex-pression of minC and dicB (14), and certain alleles of ftsZshow an increased capacity to counteract MinC/MinD-in-duced filamentation (3). The ability of FtsZ to suppressMinC/MinD division inhibition is consistent with the obser-vation that minicells are formed when the ftsZ gene isoverexpressed in wild-type cells (27).

In this article, we show that an approximately 50-foldoverproduction of MinC blocks cell division even in theabsence of MinD and DicB. In contrast, MinD and DicB donot block division in the absence of MinC. The divisioninhibition induced by MinC overexpression is resistant tosuppression by MinE, resembling MinCIDicB-mediated di-vision inhibition and differing from the division inhibitionthat results from coexpression of minC and minD.The results of the present study define different functions

for MinC and MinD in the site-specific septation block that ismediated by the MinCDE system. They suggest that MinC isthe component of the MinCDE system that causes divisioninhibition, whereas MinD both activates the MinC divisioninhibition function and is required for the interaction of thedivision inhibition system with the MinE topological speci-ficity factor.

MATERIALS AND METHODS

Strains and growth conditions. The strains used in thisstudy were all derivatives of E. coli K-12 (Table 1). StrainsRC3F and PB129 were obtained by P1-mediated cotransduc-tion of AminCDE and aph from PB114 into UT481F andGC579, respectively, selecting for kanamycin resistance.RC8F was obtained by P1-mediated cotransduction ofAdicABC and aadA from JS279 into RC3F, selecting forspectinomycin resistance. Cultures were grown at 37°C inLB medium (23) containing the appropriate antibiotics (50,ug/ml) for plasmid maintenance unless otherwise noted.When cells were to be grown in the presence of IPTG(isopropyl-,-thiogalactoside), an overnight culture in LBcontaining 0.2% glucose was diluted 200-fold into LB; IPTGwas added, and growth was continued until harvesting, asdescribed below.

63

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64 DE BOER ET AL.

TABLE 1. Strains used

Strain Relevant genotype Reference

UT481F met thy A(lac-pro) supD r- m- TnlO/F'traD36 proAB lacIq 6lacZ36AM15(Am)

UT481 met thy A(lac-pro) supD r- m- TnlO 15RC3 UT481 AminCDE aph (Km) 15RC3F RC3/F' traD36 proAB lacIq lacZ36AM15(Am) This workRC8F RC3F AdicABC aadA (Spc') This workPB103 dadR trpE trpA tna 12PB114 PB103 AminCDE aph (Kmi) 13JS279 AIacIZYAX74 hsdR rpsL AdicABC aadA (SpcD Pmap::lacIq 4MC1000 araD,39 A(araABC-leu)7679galU galK A(Iac)X74 rpsL thi 7GC579 sfiAll thr leu pro his gal rpsL 17PB129 GC579 AminCDE aph (Kmi) This workDX1 AminCDE aph (Km') recA::TnlO 15

Phages. Bacteriophages XDB170 (Piac: :minCDE), XDB171(Piac::minC), and XDB173 (Pjac::minCD) have been de-scribed before (13).

Plasmids. Plasmids pZAQ (ftsQAZ) (27), pGB2 (8),pDB184 (dicB) (14), and pCX16 (sdiA) (26) have beendescribed before.For pDB162, the BamHI-PstI minC fragment of plasmid

pDB124 (13), encoding amino acids 5 to 219 of MinC, wasligated to BamHI-PstI-digested M13mp9 DNA, giving rise tophage M13mp9.124. The BamHI-HindIII minC fragment ofthe phage DNA was next ligated to BamHI-HindIII-digestedpMLB1107, yielding pDB162A, in which the 3' end of theminC reading frame is in frame with the lacZ gene of thevector. To also bring the 5' end of the minC reading frame inframe with the translation start codon of lacZ, pDB162A wasdigested with BamHI. Sticky ends were filled in by treatmentwith Klenow enzyme, ligated, and again treated with BamHIto select molecules that had lost the BamHI site. This

1OO bp F

c24.8 Kd

H

e~ C I

t-

NN

7 ?I I ,I ItC1-

yielded pDB162 (Fig. 1), in which P,ac directs the productionof a LacZ-MinC-LacZ fusion protein that contains aminoacids 5 to 219 of MinC, accounting for 93% of the originalMinC coding sequence (13).pDB187 (Fig. 1) is a derivative of the high-copy-number

plasmid pBluescriptKS (Stratagene) in which the minD geneis under control of PIac (15).For pDB193, the 2,133-bp HindIII fragment of pZAQ,

which contains the 3' part of ftsA and the entire ftsZ gene,was inserted into the HindIII site of pGB2, thereby placingftsZ downstream of and in the same orientation as aadA.

For pDB201, the 863-bp EcoRI-BamHI fragment ofpDB171, containing the complete minC gene and the up-stream part of minD (13), was ligated to EcoRI-BamHI-treated pBluescriptKS, yielding plasmid pDB201.pDB215 (Fig. 1) is a pBluescriptKS derivative that con-

tains the minC gene downstream of the lac promoter of thevector and is completely devoid of minD sequences. The

(a)2iT1

D |- E 11

29.6 Kd 102 Kd

pDB217 (pMLB1115)pDB215 (pBluscrptlS)

pDB216 (pMLB1115)pDB208 (pBluescriptKS)

pDBI62 (pMLB107)

pDB230 (pBluesciptKS)

(b)

D 1] pDBI87 (pEluscrlptKS)

FIG. 1. Physical map of the minCDE locus and diagram of chromosomal inserts of plasmids used in this study. (a) Physical map of minCDE.Indicated are the positions of the minC, minD, and minE genes, including the locations of promotors P1 and P2 and probable transcriptionterminators Ti and T2 (13). (b) Plasmids used in this study. The insert of each plasmid is shown, and the vector in each case is indicated inparentheses. The asterisk indicates the position of a mutation in the ribosome-binding site of minC (see text). The shaded area indicates lacZ.Plasmids pMLB1107 and pMLB1115 are moderate-copy-number ColEl derivatives (13). Plasmid pBluescriptKS (Stratagene) is a high-copy-number ColEl derivative. Restriction sites: Bc, BclI; F, FokI; Hp, HpaI; N, NsiI; P, PstI; Rv, EcoRV; Sn, SnaBI; St, SlyI; X, XmnI.

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ROLES OF MinC AND MinD IN DIVISION INHIBITION 65

minC fragment extends from 29 bp upstream of the minCstart codon exactly to the minC stop codon (nucleotides 167to 891 [13]) and includes the wild-type ribosome-binding siteof minC. pDB215 was obtained by replacing the EcoRI-PstIminCSD fragment of pDB208 (see below) with that ofpDB201.pDB216 and pDB217 (Fig. 1) are derivatives of pMLB1115,

which has a lower copy number than pBluescript and carriesthe lacIq allele. pDB216 and pDB217 both carry minC down-stream of Plac. pDB216 carries the mutated (see below) andpDB217 carries the wild-type ribosome-binding site of minC.pDB216 and PDB217 were constructed by replacing theHindIII-XbaI fragment of pDB164 (PIac::minD) (13), whichincludes the entire minD gene, with the HindIII-XbaI minCfragment of pDB208 (see below) and pDB215, respectively.For pDB230 (Fig. 1), the minE gene was inserted between

PIac and the minC gene in pDB215 by ligation of theHindIII-EcoRI minE fragment of pDB151 (13) to HindIll-EcoRI-digested pDB215.

Construction of minCSD. The ribosome-binding region ofthe minC gene was altered by oligonucleotide-directed mu-tagenesis, performed by the method of Kunkel et al. (20)essentially as described in the Muta-Gene in vitro mutagen-esis kit instruction manual (Bio-Rad). As a template, weused DNA from phage M13H17A21, which carries a 1,287-bpchromosomal fragment extending from 29 bp upstream of theminC start codon to the HpaI site in minD (13). Theoligonucleotide 5'-AATTGAGTAAGGaggttttATMTCAAACACG-3' (the minC start codon is underlined, and bases thatdiffer from the wild-type minC sequence are indicated inlowercase letters) was prepared on a Cyclone DNA synthe-sizer (Biosearch Inc.). DNA sequence analysis (13) of one ofthe resulting mutant derivatives (M13H17A211113) confirmedthat the 5'-CCAGG-3' (nucleotides 191 to 195 [13]) had beenreplaced by 5'-AGGTTTT-Y. This substitution places aconsensus ribosome-binding site (18) three nucleotides up-stream of the minC translation start codon and leads to lossof a FokI endonuclease restriction site located at nucleotide208 (13). The EcoRI-PstI minC fragment ofpDB201 was nextreplaced with the equivalent fragment of M13H17A211113,yielding pDB206. To remove all minD sequences from thisconstruct, we made use of a Sau3AI site that overlaps theminC translation stop codon. pDJ3206 was digested com-pletely with EcoRI and partially with Sau3AI. Fragments inthe range of 650 to 750 bp were then isolated and ligated tothe large EcoRI-BamHI fragment of pDB206. This yieldedpDB208, which carries the minC gene, including the mutatedribosome-binding site, downstream of PIac and is devoid ofany minD sequences. The mutation is referred to as minCSD.

Purification of LacZ-MinC-LacZ fusion protein. Cells ofstrain MC1000/pDB162 were grown in the presence of 0.84mM IPTG for approximately eight generations to an OD6.of 1.2. The cells were suspended in 9 ml of buffer A (20 mMTris-Cl [pH 7.5], 100 mM NaCl, 2 mM EDTA, 5 mMP-mercaptoethanol) and disrupted by four passages througha French pressure cell at 10,000 lb/in2. Phenylmethylsulfonylfluoride was added to 2 mM and DNase I was added to 1,ug/ml. After 10 min at room temperature, the homogenatewas centrifuged at 12,000 x g for 30 min. The supernatantwas removed, and the particulate fraction was resuspendedin 10 ml of buffer A containing 2% Triton X-100 and slowlyshaken at room temperature for 1 h. The suspension wascentrifuged at 12,000 x g for 30 min, and the particulatefraction, which contained virtually all of the LacZ-MinC-LacZ fusion protein in inclusion bodies, was resuspended in5 ml of buffer A. Samples of this suspension were mixed with

equal volumes of electrophoresis sample buffer (125 mMTris-Cl [pH 6.8], 4% sodium dodecyl sulfate [SDS], 20%glycerol, 1.4 M 3-mercaptoethanol, 0.001% bromophenolblue), boiled for 5 min, and electrophoresed on a preparativeSDS-polyacrylamide gel electrophoresis (PAGE) gel (7.0%T, 2.7% C). Protein bands were visualized by negativestaining with CuCl2 (19). The portion of the gel containingthe fusion protein was equilibrated in electroelution buffer(20 mM Tris-Cl [pH 8.3], 150 mM glycine, 0.01% SDS), andthe protein was then electroeluted and concentrated in anISCO concentrator device.

Preparation of antiserum. To obtain MinC-specific antise-rum, Sprague-Dawley rats were immunized with the purifiedLacZ-MinC-LacZ fusion protein, beginning with an initialintradermal injection of 100 ,ug of the protein in Freund'scomplete adjuvant and followed at intervals of approxi-mately 3 weeks by several subcutaneous injections of 50 ,ugof antigen in Freund's incomplete adjuvant.

MinE-specific antiserum was obtained in rabbits afterimmunization with a mixture of two MinE-derived syntheticoligopeptides (CRRRSDAEPHY and CVTLPEAEELK; ob-tained from Multiple Peptide Systems Inc.) conjugated tokeyhole limpet hemocyanin. The oligopeptides correspondto residues 29 to 38 and 79 to 88, respectively, of the MinEpeptide (13). Rabbits were injected intradermally with 0.5 mgof each peptide conjugate in Freund's complete adjuvant,followed by several subcutaneous injections of 0.5 mg inFreund's incomplete adjuvant at 1- to 2-month intervals.To reduce interactions with other bacterial antigens in

subsequent procedures, the MinC and MinE antisera werepreincubated for 24 h at 4°C with a whole-cell homogenate(1.6 x 109 cell equivalents per ,u of serum in 20 mM Tris-Cl[pH 7.2], 100 mM NaCl, 2 mM EDTA) of strain RC3/pMLB1107 (AminCDEPiac:::lacZ) grown in the presence of0.84 mM IPTG.

Preparation of cell extracts. Cells were grown exponen-tially in the presence of various concentrations of IPTG forapproximately six generations to an OD600 of between 0.8and 1.1. Cells were collected by centrifugation, washed oncewith 0.9% saline, suspended in 62.5 mM Tris-Cl (pH 6.8)-2%SDS-10% glycerol-1.4 M 3-mercaptoethanol-0.0005% bro-mophenol blue, and placed in a boiling-water bath for 10min. The resulting cell extracts were kept at 4°C until used.Western immunoblotting and immunolabeling. Cell ex-

tracts were subjected to SDS-PAGE (15% T, 2.7% C gels),and protein bands were electrophoretically transferred tonitrocellulose filters. Protein transfer was monitored bystaining the filters with the reversible Ponceau-S stain (19).After destaining, the filters were air dried and stored at roomtemperature.For immunolabeling of MinC, filters were incubated at

room temperature with shaking, first in TNT buffer (10 mMTris-Cl [pH 7.5], 150 mM NaCl, 0.2% Tween-20) for at least2 h and next in a 10' dilution of MinC antiserum in TNT for16 h. The filters were then rinsed several times with TNT andincubated for 2 h with affinity-purified rabbit anti-rat immu-noglobulin antibody conjugated to alkaline phosphatase (Sig-ma). After the filters were rinsed several times in TNT, thefilter-bound conjugate was detected by incubation with5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt andp-nitroblue tetrazolium chloride; (Bio-Rad) according to themanufacturer's recommendation.

Extracts from a AminCDE strain (75 ,ug of protein) and awild-type strain (38, 75, and 150 ,ug of protein) were run inparallel to the experimental samples on the same gel toprovide standards for quantitation. The experimental sam-

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66 DE BOER ET AL.

ples were diluted with cell extract from a AminCDE strain toa point at which the intensity of the MinC band fell within therange of the wild-type standards. The stained bands werequantitated by reflective densitometry of the immunoblots.To control for possible variations in protein transfer, thevalues for the 25-kDa MinC band were normalized to theintensity of a 34-kDa protein species in the cell extracts thatcross-reacted with the secondary antibody and which did notappear to vary in concentration among samples. The amountof MinC in the experimental samples was then determinedby comparison with the wild-type samples.The procedure for detection of MinE was essentially as

described above except that MinE-specific rabbit antiserumwas used as the primary antibody and goat anti-rabbitimmunoglobulin alkaline phosphatase-conjugated antibody(Sigma) was used as the secondary antibody.Other procedures. Phase microscopy was performed on

glutaraldehyde-fixed cells as described previously (13). Cellswere defined as Min- (minicell phenotype) when the culturecontained many cells with polar septa, large numbers ofspherical minicells, and substantial numbers of short fila-ments (Fig. 2a, b, g, and h). Cells were defined as Sep-(filamentation phenotype) when essentially all of the cells ina culture consisted of nonseptate filaments (Fig. 2c, e, and f).Protein concentration was determined by the Bradfordmethod with bovine immunoglobulin G as a standard (5).

RESULTS

MinC-mediated division inhibition. Evidence that MinCwas capable of blocking cell division in the absence of MinDcame from studies of the effects of high levels of minCexpression in AminCDE strains. To vary the expression ofminC over a wide range, a DNA fragment containing thecomplete minC open reading frame was placed downstreamof the lac promoter in the moderate-copy-number plasmidpMLB1115 and the high-copy-number plasmid pBlue-scriptKS. Cells containing the Piac::minC plasmids werethen grown in the presence of IPTG to induce minC expres-sion. The cellular concentration of MinC was determined byquantitative immunoassay, and the division phenotype wasdetermined microscopically.As shown in Table 2 and Fig. 2e, IPTG induction of minC

fromn the high-copy-number pBluescript derivative pDB215in the AminCDE host RC3F caused a general inhibition ofcell division, leading to the accumulation of large numbers oflong filamentous cells. The cellular concentration of MinCwas increased 55-fold over wild-type levels under theseconditions (Fig. 3). In contrast, division was not affectedwhen minC was induced from the moderate-copy-numberplasmid pDB217, leading to a fourfold increase in cellularMinC (Table 2), or when RC3F/pDB215 was grown in theabsence of IPTG.To further define the effects of MinC concentration on the

division pattern in the AminCDE host, we constructedPlac::minC plasmids in which the translational efficiency ofMinC was increased. This was accomplished by creating amore efficient ribosome-binding site by site-directed muta-genesis of minC upstream sequences. When the mutatedPiac::minC construct (Piac::minCSD) was present in the mod-erate-copy-number vector pMLB1115 (in pDB216), the cel-lular MinC concentration was increased 25-fold over wild-type levels, compared with a 4-fold increase from thecorresponding Piac::minC' plasmid (pDB217) grown at thesame concentration of IPTG (Table 2). This led to a moder-ate increase in cell length (Fig. 2d). When Piac::minCSD was

FIG. 2. Effects of different levels of minC expression on divisionphenotype in the presence and absence of minD, minE, ftsZ, andsdiA. Cells were grown in the presence of 0.05 mM IPTG (a, b, andc) or 0.5 mM IPTG (d through h) prior to preparation for phasemicroscopy. (a) RC3 (AminCDE). (b) RC3(XDB171) [AminCDE(Pjac::minC)I. (c) RC3(XDB173) [AminCDE(P1a,::minCD)]. (d) RC3/pDB216 (AminCDE/Piac::minCSD). (e) RC3F/pDB215 (AminCDEIPiac::minC). (f) RC3F/pDB230 (AminCDE/Piac::minEC). (g) RC3F/pDB215/pDB193 (AminCDE/Piac::minC/ftsZ). (h) RC3F/pDB215/pCX16 (AminCDE/Piac::minC/sdiA). The diagonal length of eachpanel represents 100 ,um.

present in the high-copy-number pBluescriptKS vector (inpDB208), approximately 15% of the cells grew as longfilaments even in the absence of IPTG (data not shown). Thedivision block that occurred in the absence of IPTG presum-ably reflected the basal level of minC expression from PIac,since it could be suppressed by addition of glucose to themedium. The finding that the Plac::minCSD constructs weremore effective in inducing a division block than the

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ROLES OF MinC AND MinD IN DIVISION INHIBITION 67

TABLE 2. Effect of increased MinC concentration on division phenotype'

Line Strain Relevant genotype Phenotypeb( MinC concn(fold wild-type concn)

1 RC3 AminCDE Min- 02 UT481 minCDE+ wt 13 RC3/pDB217 AminCDE/PIac: :minC Min- 44 RC3/pDB216 AminCDEIPIac::minCSD a/ 255 RC3F/pDB215 AminCDE/Pjac::minC Sep- 556 RC8F/pDB215 AminCDE AdicABCIPIac::minC Sep- 447 RC3F/pDB230 AminCDE/Pjac::minEC Sep- 50' Cells were grown in the presence of 0.5 mM IPTG. MinC concentration was determined by quantitative immunoblotting and is expressed as multiples of the

concentration in wild-type cells. Cell phenotype was determined by light microscopy, as defined in Materials and Methods. For lines 3 through 7, when grownin the absence of IPTG, the cells showed the Min- phenotype.

b wt, wild-type phenotype; Min-, minicell phenotype; Sep-, filamentation phenotype; a/, phenotype similar to the Min- phenotype of the RC3 host except thatthe cells were approximately two to four times longer (see Fig. 2d). Anti-MinC immunoblots of cell extracts from the experiments shown on lines 1, 2, 4, and 5are illustrated in Fig. 3.

PIac::minC' constructs supports the view that the divisioninhibition resulted from the increased concentration ofMinCrather than from increases in minC transcription or minCcopy number.

Overexpression ofminC thus can induce a general block inseptation even in the absence of MinD. The reverse is nottrue, since division was not blocked when the cellularconcentration of MinD was increased to approximately 5%of total cell protein (approximately 200-fold over the wild-type level [15]) in the absence of MinC by IPTG induction ofAminCDE cells containing pDB187 (Plac::minD) (Fig. 4).MinC-induced division inhibition is independent of DicB

and SfiA. Expression of the division inhibitor gene dicB inthe presence of MinC leads to a MinD-independent divisionblock (14, 21). We therefore considered the possibility thatDicB might be substituting for MinD in the MinC-mediateddivision block that occurred in the absence of MinD. Thisseemed unlikely because under normal conditions, dicBexpression is strongly repressed (1). However, it remainedpossible that a very small amount of DicB protein might besufficient to cause division inhibition at high concentrationsof MinC or that high levels of MinC might increase dicBexpression to a level sufficient to activate the MinC/DicBinhibition reaction.We therefore asked whether the MinC-induced division

block required the dicB gene product by introducing pDB215(Pjac::minC) into strain RC8F (AdicB AminCDE). When minCoverexpression was induced by growth of RC8F/pDB215 inthe presence of IPTG, there was a general block in celldivision that was indistinguishable from that seen in the dicB+host RC3F (AminCDE) (Table 2). We conclude that theMinD-independent division inhibition that occurs in the pres-ence of high levels of MinC is not dependent on DicB.The MinC-mediated division block that occurred in the

l 2 3 4

FIG. 3. Detection of MinC in immunoblots. Lane 1, RC3(AminCDE). Lane 2, UT481 (minCDE+). Lane 3, RC3/pDB216(AminCDEfPiac::minCSD). Lane 4, RC3F/pDB215 (AminCDEIPiac::minC). For the corresponding cell phenotypes, see Fig. 2 and Table2.

absence of MinD was also independent of the SfiA divisioninhibitor that is induced as part of the SOS response to DNAdamage (25). Thus, filamentation occurred when high levelsof MinC were induced from pDB215 in cells that lacked afunctional sfiA gene (PB129 [AminCDE sfiA]). Similar resultswere obtained with cells that were unable to induce sfiAexpression via the RecA-mediated inactivation of the LexArepressor (strain DX1 [AminCDE recA::TnlO]) (data notshown).MinC-mediated division inhibition is not suppressed by

MinE. Under normal conditions, MinC/MinD-mediated divi-sion inhibition is suppressed by MinE, thereby permittingdivision to proceed normally (13, 14). We therefore asked

a b66-36-29-24-

MinD- n/_0\(MinC)

14-

I z I

FIG. 4. Effect of MinD overproduction in the absence of MinC.(a) Strain RC3F/pDB187 (AminCDE/Piac::minD) was grown in thepresence of 1 mM IPTG prior to preparation for phase microscopy.The diagonal of the panel represents 100 ,um. (b) Coomassieblue-stained SDS-PAGE gel (35 ,ug of protein each). Lanes 1 and 2,strains UT481 (minCDE+) and RC3F/pDB215 (AminCDE/Piac::minC), respectively, grown in the presence of 0.5 mM IPTG.The Sep- division phenotype of the sample used for lane 2 is shownin Fig. 2e. Lane 3, strain RC3F/pDB187 (AminCDEfPiac::minD),grown in the presence of 1 mM IPTG; the cells were obtained fromthe culture shown in panel a. The difference in the intensities of theMinC band in lane 2 (not visible as a separate band) and the MinDband in lane 3 (the major protein species) illustrates the fact that, inthe absence of MinC, MinD fails to inhibit division even whenpresent at a level that is considerably higher than the concentrationof MinC that is capable of inducing a division block in the absenceof MinD. The positions of molecular mass markers (sizes in kilodal-tons) are indicated. Arrows indicate the positions of MinD (themajor protein species in lane 3) and MinC (as determined byimmunoblot analysis).

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3inE

1 2 3 4 5 6

FIG. 5. Overproduction of MinE from plasmid pDB230(Plac::minEC). Cells were grown in the presence of 0.5 mM IPTG.MinE-specific antiserum was used to stain extracts of the followingstrains: lane 1, RC3 (AminCDE), 50 ,ug; lane 2, UT481 (minCDE+),50 ,ug; lanes 3 to 6, 0.2, 0.5, 1.0, and 10.0 ,ug of protein, respectively,from extracts of strain RC3F/pDB230 (AminCDEIP,ac::minEC).

whether the division inhibition that resulted from high levelsof MinC in the absence of MinD was also sensitive to MinE.To test this possibility, minE was expressed together with

minC from the high-copy-number plasmid pDB230 (Piac::minEC). When cells containing this plasmid were grown inthe presence of IPTG (in strain RC3F/pDB230[AminCDEIP1?ac::minEC]), MinC was increased 50-fold to alevel that was sufficient to block cell division in the absenceof MinE (RC3F/pDB215 [AminCDE/Pjac::minC]) (Table 2).Under these conditions, the cellular concentration of MinEwas increased more than 100-fold over the wild-type level, asshown by immunoblot analysis (Fig. 5). As shown in Fig. 2f,IPTG induction of RC3F cells containing the Plac::minECplasmid led to a filamentation phenotype that was indistin-guishable from that of RC3F/pDB215 (Piac::minC). We con-clude that the MinD-independent division inhibition thatoccurs with high levels of MinC is resistant to MinE.MinC-mediated division inhibition is suppressed by FtsZ.

The MinC/MinD- and MinC/DicB-mediated division inhibi-tion reactions can be suppressed by overexpression of theessential cell division gene ftsZ (14). To determine whetherthe filamentation phenotype that results from high levels ofMinC in the absence ofMinD and DicB is also suppressed byincreased concentrations of FtsZ, we introduced plasmidpDB193 (ftsZ) into cells containing pDB215 (Plac::minC). Asshown in Fig. 2g and Table 3, the presence of the ftsZplasmid resulted in suppression of the IPTG-dependentfilamentation phenotype of RC3F/pDB215 (AminCDEIPiac: :minC).

Similar results were obtained when the cellular FtsZ levelwas increased by introduction ofplasmid pCX16 (sdiA) (Fig. 2hand Table 3). The sdiA gene in pCX16 is a positive regulator offtsQAZ transcription, leading to a 1.5- to 2-fold increase incellular FtsZ concentration (26). This is sufficient to preventthe MinC/MinD-, MinC/DicB-, and SfiA-mediated divisionblocks (26) and, as shown here, is also sufficient to suppress theMinC-dependent division inhibition. Thus, a modest increasein cellular FtsZ concentration results in suppression of theMin-mediated division inhibition phenotype.The ability of increased levels of FtsZ to suppress the

Min-mediated division inhibition did not reflect an effect ofFtsZ on the cellular concentration of MinC. This was shownby the finding that the high level of MinC protein in RC3F/pDB215 cells was unaffected by the presence of pDB193(ftsZ) or pCX16 (sdiA) (Table 3). FtsZ also failed to affectMinC levels when minC was expressed from its normalchromosomal copy in wild-type cells. This was shown by thefinding that pZAQ (ftsQAZ), previously shown to increasethe FtsZ level sixfold (26, 27), did not significantly affect thecellular concentration of MinC in wild-type cells (Table 3).Thus, FtsZ does not suppress MinC function by decreasingits concentration.MinD and DicB do not act by increasing the concentration of

MinC. As described above, in the absence of MinD and DicB,a 55-fold increase in MinC concentration over the wild-typelevel is needed to cause complete division inhibition and a25-fold increase is needed to cause even modest inhibition, asshown by the accumulation of moderately elongated cells. InminCDE+ cells, however, the wild-type level of MinC issufficient to cause a division block at the cell poles. Thisimplies that the role of MinD in MinC/MinD-mediated divi-sion inhibition is to stimulate MinC activity rather than toincrease the cellular concentration of MinC. To confirm this,we measured the levels of MinC in AminCDE cells in whichminC was expressed either alone or together with minD. Asshown in Table 4, the cellular concentration of MinC in strainRC3(XDB173) [AminCDE (Piac::minCD)], in which septationwas blocked by coexpression of minC and minD, was notsignificantly increased over the MinC level in strainRC3(ADB171) [AminCDE (Piac::minC)], in which MinD wasabsent. The MinC concentration was also unaffected whenseptation was blocked by coexpression of minC and dicB inRC3(XDB171)/pDB184 [AminCDE (Pjac: :minO1/P.d :dicB](Table 4). Therefore, the Min-dependent division blocksinduced by MinD and DicB are not due to secondary changesin the cellular level of MinC, indicating that MinD and DicBact by stimulating the ability ofMinC to block septation ratherthan by increasing its concentration.The MinC concentration was also unchanged when the

filamentation phenotype induced by expression of minCDwas suppressed by the coexpression of minCD and minE instrain RC3(XDB170) [AminCDE (Pjac::minCDE)] (Table 4).Thus, the ability of MinE to suppress the MinC/MinDdivision block was also not due to a secondary change in thecellular concentration of MinC.

DISCUSSION

We have previously shown that MinC is the commoncomponent of the MinC/MinD and MinC/DicB divisioninhibition systems (13, 14). This suggested that MinC was

likely to be the effector of the division block in both systems,

TABLE 3. Effect offtsZ overexpression on MinC levels and division phenotypea

Line Strain Relevant genotype Phenotypeb MinC concn (fold wild-type concn)

1 RC3F/pDB215/pGB2 AminCDE/Pjac::minC/vector Sep- 43.02 RC3F/pDB215/pDB193 AminCDE/PIac::minC/ftsZ Min- 47.03 RC3F/pDB215/pCX16 AminCDE/PIac::minClsdiA Min- 43.04 PB103 minCDE+ wt 1.05 PB103/pZAQ minCDE+lftsQAZ Min- 0.9a Cells were grown either in the presence of 0.5 mM IPTG (lines 1, 2, and 3) or in the absence of IPTG (lines 4 and 5) and examined as described in Table 2,

footnote a. When the strains indicated in lines 1 through 3 were grown in the absence of IPTG, the cells showed the Min- phenotype. pGB2 is the vector usedto construct pDB193 and pCX16.

b See Table 2, footnote b.

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ROLES OF MinC AND MinD IN DIVISION INHIBITION 69

TABLE 4. Effects of MinD, DicB, and MinE on MinC concentration and division phenotypea

Line Strain Relevant genotype Phenotypeb MinC concn(fold wild-type concn)

1 RC3 AminCDE Min- 0.02 UT481 minCDE+ wt 1.03 RC3(ADB170) AminCDE(P1ac::minCDE) wt 1.54 RC3(ADB173) AminCDE(P1ac::minCD) Sep- 1.35 RC3(ADB171)/pGB2 AminCDE(P1ac::minC)Ivector Min- 0.96 RC3(ADB171)/pDB184 AminCDE(P1ac::minC)IdicB Sep- 0.6

a Cells were grown in the presence of 0.05 mM IPTG and examined as described in Table 2, footnote a. For lines 3 through 6, when grown in the absence ofIPTG, the cells showed the Min- phenotype. In pDB184, dicB is constitutively expressed under control of the aadA promoter of the vector (pGB2) (14).

b See Table 2, footnote b.

with MinD and DicB acting as independent activators of theMinC-mediated division inhibition process (14).The present studies support the view that MinC is the

proximate cause of the division inhibition in these systemsby showing that a 40- to 50-fold increase in the cellularconcentration of MinC leads to a complete division block inthe absence of MinD and DicB. In contrast, overproductionof MinD or DicB (14, 21) in the absence of MinC has noapparent effect on the division pattern. The inhibitory mol-ecule that ultimately interacts with the septation machinerycould be MinC itself or another molecule whose synthesis oractivity depends on MinC.There are several possible reasons why elevated levels of

MinC could lead to inhibition of septation in AminD AdicBcells. It is possible that a small fraction of MinC is normallypresent in an activated form even in the absence of MinD orDicB. In this case, division inhibition would result if theMinC concentration were raised to a high enough level,without the need for further activation. Alternatively, othermolecules that either are present at low concentrations orhave a low affinity for MinC may be able to assume the roleof MinD or DicB in activating MinC. This would also lead todivision inhibition if the MinC concentration were increasedsufficiently.The mechanism by which MinD and DicB activate the

MinC-dependent division inhibition process, leading to divi-sion inhibition at low concentrations of MinC, is not known.Direct measurement of MinC protein excluded the possibil-ity that MinD or DicB activates the division inhibitionreaction by increasing the concentration of MinC. Otherpossibilities remain to be investigated. For example, MinDand/or DicB could directly modify the MinC protein, acti-vating it as an inhibitor of septation. It is also possible thatMinD and/or DicB acts indirectly, for example, by promot-ing the interaction between MinC and its molecular target.

It has previously been shown that the MinC/MinD divisioninhibition activity can be modified by MinE (13). When theminC, minD, and minE gene products are expressed atnormal levels, MinE prevents the division inhibitor fromblocking division at midcell without affecting the septationblock at the cell poles. In addition, at high levels of MinE,the MinC/MinD-associated division block is suppressed atthe cell poles as well as at midcell, leading to minicellformation (13). It is not known how MinE suppressesMinC/MinD-mediated division inhibition.The present study showed that the MinC-mediated divi-

sion inhibition that occurs in the absence of MinD and DicBis resistant to MinE. In this regard, it differs from theMinC/MinD-mediated division block and resembles the di-vision inhibition that is mediated by MinC/DicB (14). Thisindicates that MinD rather than MinC is responsible for the

sensitivity of the MinC/MinD division inhibition system toMinE.Thus, MinC and MinD play different roles in the site-

specific septation block that is mediated by the MinCDEsystem. The results are most easily explained by a model inwhich MinC is the component that causes division inhibi-tion, whereas MinD plays two roles-it activates the MinCdivision inhibition function and is the component of thesystem that is responsible for interaction with the MinEtopological specificity factor. The simplest way in whichMinE could suppress MinC/MinD division inhibition withoutsuppressing division inhibition mediated by MinC/DicB orby high levels of MinC alone would be by blocking theactivation of MinC by MinD. Further work will be needed totest this and other possibilities.

ACKNOWLEDGMENTS

This work was supported by grants from the National ScienceFoundation and the National Institutes of Health.

REFERENCES1. Bejar, S., F. Bouche, and J.-P. Bouche. 1988. Cell division

inhibition gene dicB is regulated by a locus similar to lambdoidbacteriophage immunity loci. Mol. Gen. Genet. 212:11-19.

2. Bejar, S., and J.-P. Bouche. 1985. A new dispensable geneticlocus of the terminus region involved in control of cell divisionin Escherichia coli. Mol. Gen. Genet. 201:146-150.

3. Bi, E., and J. Lutkenhaus. 1990. Interaction between the minlocus and ftsZ. J. Bacteriol. 172:5610-5616.

4. Bouche, F., and J.-P. Bouche. 1989. Genetic evidence that DicF,a second division inhibitor encoded by the Escherichia coli dicBoperon, is probably RNA. Mol. Microbiol. 3:991-994.

5. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

6. Cahill, K. B., and G. G. Carmichael. 1989. Deletion analysis ofthe polyomavirus late promoter: evidence for both positive andnegative elements in the absence of early proteins. J. Virol.63:3634-3642.

7. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of genecontrol signals by DNA fusion and cloning in Escherichia coli.J. Mol. Biol. 138:179-207.

8. Churchward, G., D. Belin, and Y. Nagamine. 1984. A pSC101-derived plasmid which shows no sequence homology to othercommonly used cloning vectors. Gene 31:165-171.

9. Cook, W. R., P. A. J. de Boer, and L. I. Rothfield. 1989.Differentiation of the bacterial cell division site. Int. Rev. Cytol.118:1-31.

10. Dai, K., and J. Lutkenhaus. 1991. ftsZ is an essential celldivision gene in Escherichia coli. J. Bacteriol. 173:3500-3506.

11. de Boer, P. A. J., W. R. Cook, and L. I. Rothfield. 1990.Bacterial cell division. Annu. Rev. Genet. 24:249-274.

12. de Boer, P. A. J., R. E. Crossley, and L. I. Rothfield. 1988.Isolation and properties of minB, a complex genetic locus

VOL. 174, 1992

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nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

b on

30

Dec

embe

r 20

21 b

y 27

.75.

160.

240.

70 DE BOER ET AL.

involved in correct placement of the division site in Escherichiacoli. J. Bacteriol. 170:2106-2112.

13. de Boer, P. A. J., R. E. Crossley, and L. I. Rothfield. 1989. Adivision inhibitor and a topological specificity factor coded forby the minicell locus determine proper placement of the divisionseptum in E. coli. Cell 56:641-649.

14. de Boer, P. A. J., R. E. Crossley, and L. I. Rothfield. 1990.Central role for the Escherichia coli minC gene product in twodifferent cell division-inhibition systems. Proc. Natl. Acad. Sci.USA 87:1129-1133.

15. de Boer, P. A. J., R. E. Crossley, and L. I. Rothfield. The MinDprotein is a membrane ATPase required for the correct place-ment of the E. coli division site. EMBO J., in press.

16. Donachie, W. D., and A. C. Robinson. 1987. Cell division:parameter values and the process, p. 1578-1593. In F. C.Neidhardt, J. E. Ingraham, K. B. Low, B. Magasanik, M.Schaechter, and H. E. Umbarger (ed.), Escherichia coli andSalmonella typhimurium: cellular and molecular biology, vol. 2.American Society for Microbiology, Washington, D.C.

17. George, J., M. Castellazzi, and G. Buttin. 1975. Prophageinduction and cell division in E. coli. III. Mutations sfiA and sfiBrestore division in tifand lon strains and permit the expressionof mutator properties of tif. Mol. Gen. Genet. 140:309-332.

18. Gold, L., and G. Stormo. 1987. Translational initiation, p.1302-1307. In F. C. Neidhardt, J. E. Ingraham, K. B. Low, B.Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Esche-richia coli and Salmonella typhimurium: cellular and molecularbiology, vol. 2. American Society for Microbiology, Washing-ton, D.C.

19. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory man-ual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

20. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid andefficient site-specific mutagenesis without phenotypic selection.Methods Enzymol. 154:367-382.

21. Labie, C., F. Bouche, and J.-P. Bouche. 1990. Minicell-formingmutants of Escherichia coli: suppression of both DicB- andMinD-dependent division inhibition by inactivation of the minCgene product. J. Bacteriol. 172:5852-5855.

22. Lutkenhaus, J. 1990. Regulation of cell division in E. coli.Trends Genet. 6:22-25.

23. Mifler, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

24. Pla, J., M. Sanchez, P. Palados, M. Vicente, and M. Aldea. 1991.Preferential cytoplasmic location of FtsZ, a protein essential forEscherichia coli septation. Mol. Microbiol. 5:1681-1686.

25. Walker, G. C. 1987. The SOS response of Escherichia coli, p.1346-1357. In F. C. Neidhardt, J. E. Ingraham, K. B. Low, B.Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Esche-richia coli and Salmonella typhimurium: cellular and molecularbiology, vol. 2. American Society for Microbiology, Washing-ton, D.C.

26. Wang, X., P. A. J. de Boer, and L. I. Rothfield. 1991. A factorthat positively regulates cell division by activating transcriptionof the major cluster of essential cell division genes in Esche-richia coli. EMBO J. 10:3363-3372.

27. Ward, J. E., and J. Lutkenhaus. 1985. Overproduction of FtsZinduces minicell formation in E. coli. Cell 42:941-949.

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/jour

nal/j

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