FtsH-dependent Processing of RNase Colicins D and E3 Means

FtsH-dependent Processing of RNase Colicins D and E3Means That Only the Cytotoxic Domains Are Imported intothe Cytoplasm*□S

Received for publication, March 24, 2011, and in revised form, May 26, 2011 Published, JBC Papers in Press, June 23, 2011, DOI 10.1074/jbc.M111.242354

Mathieu Chauleau1, Liliana Mora1, Justyna Serba, and Miklos de Zamaroczy2

From the CNRS, UPR 9073, Institut de Biologie Physico-Chimique, 75005 Paris, France

It has long been suggested that the import of nuclease colicinsrequires protein processing; however it had never been formallydemonstrated. Here we show that two RNase colicins, E3 andD,which appropriate two different translocation machineries tocross the outer membrane (BtuB/Tol and FepA/TonB,respectively), undergo a processing step inside the cell that isessential to their killing action. We have detected the pres-ence of the C-terminal catalytic domains of these colicins inthe cytoplasm of target bacteria. The same processed formswere identified in both colicin-sensitive cells and in cellsimmune to colicin because of the expression of the cognateimmunity protein. We demonstrate that the inner membraneprotease FtsH is necessary for the processing of colicins D andE3 during their import. We also show that the signal pepti-dase LepB interacts directly with the central domain of coli-cin D in vitro and that it is a specific but not a catalyticrequirement for in vivo processing of colicin D. The interac-tion of colicin D with LepB may ensure a stable associationwith the inner membrane that in turn allows the colicin rec-ognition by FtsH. We have also shown that the outer mem-brane protease OmpT is responsible for alternative and dis-tinct endoproteolytic cleavages of colicins D and E3 in vitro,presumably reflecting its known role in the bacterial defenseagainst antimicrobial peptides. Even though the OmpT-cata-lyzed in vitro cleavage also liberates the catalytic domainfrom colicins D and E3, it is not involved in the processing ofnuclease colicins during their import into the cytoplasm.

Colicins are antibacterial toxins of Escherichia coli that arereleased into the extracellular medium in response to envi-ronmental stress conditions. Colicin D is an RNase thatcleaves the anticodon loop of all four isoaccepting tRNAArg

(1). Colicin E3 cleaves 16 S ribosomal RNA (2). Both colicinsprovoke cell death by inactivating the protein biosyntheticmachinery. Colicin producer cells are protected against bothendogenous and exogenous toxin molecules by the constitu-

tive expression of a cognate immunity (Imm)3 protein, whichforms a tight heterodimer complex with the nuclease do-main of the colicin (3, 4).Colicin E3, like most colicins, has structurally identifiable

N-terminal, central, and C-terminal domains. The first twodomains are required for translocation and receptor binding,and they “hijack” certain functions of the target cell (i.e. theBtuB receptor and the Tol system) during colicin import. TheC-terminal domain carries the cell-killing RNase function (5,6). The colicin D protein has an unusual tripartite organization.The N-terminal domain is required for both the binding of thecolicin to the high affinity, iron siderophore receptor FepA andfor its subsequent translocation across the outer membrane.The 280-residue central domain is essential for uptake (andthus for cell killing), and it is also involved in the formation ofthe colicinD-ImmDprotein complex (7). The passage of colicinD through the outer membrane is dependent on the protonmotive force of the cytoplasmic membrane, transduced by theTonB/ExbB-D system (8, 9).During the import process, when E-type nuclease colicin-

Imm complexes bind to the cell surface receptor BtuB, theirImm protein dissociates from the bound colicin and is releasedinto the external medium (10, 11). The binding of the complexper se is however not enough to liberate the Imm protein. Thedissociation of colicins E9- and E2-Immcomplexes requires theunfolding of the colicin, as it contacts the energy-transducingTol system in the periplasm (12, 13).To transfer the cytotoxic domain of the colicin molecule

across the inner membrane into the cytoplasm, nucleasecolicins need to parasitize more of the cell functions than thepore-forming colicins. The DNase domains of colicins E9 andE2 exhibit nonvoltage-gated channel forming activity in planarlipid bilayers, which involves changes in their conformation(14). Such channels, unlike those formed by pore-formingcolicins, are not directly responsible for cell killing but mayallow “self-propulsion” of the toxic domains into cytoplasm,driven by an electrostatic association of the DNase domain ofcolicin E9 with the inner membrane (15). Although such anassociation between theRNase colicin E3 and anionic phospho-lipid surfaces has been reported, no RNase colicin has beenfound to exhibit any channel forming activity (16).

* This work was supported by the Centre National de la Recherche Scienti-fique (UPR 9073) and by a studentship from the Ecole Doctorale GGC(Genes, Genomes, Cellules), Universite Paris 11 (to M. C.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: 13 rue Pierre et Marie Curie,

75005 Paris, France. Tel.: 33-1-58-41-51-54; Fax: 33-1-58-41-50-25; E-mail:[email protected].

3 The abbreviations used are: Imm, immunity; LC, large cleaved; SC, smallcleaved; PF, in vivo processed form (of RNase colicins); FS, full-size; Col,colicin; Ni-NTA, nickel-nitrilotriacetic acid; CAT, catalytic domain; R,receptor-binding.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 33, pp. 29397–29407, August 19, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Using cell extracts the in vitro cleavage of full-size colicin Dand more recently of the DNase colicin E7 was observed at theboundary of the C-terminal killing and central domains (17–19). The inner membrane signal peptidase LepB was shown tobe essential for cell killing by colicin D and also for the cleavageof colicin D in vitro in the presence of a cell extract. We andothers have proposed that proteolytic processingmay be a com-mon step in cell killing by nuclease type colicins and that itshould occur prior to or concomitant with the translocation oftheir C-terminal catalytic domain through the innermembrane(17, 19–21).OmpT, amember of the family of outermembrane endopep-

tidases (omptins), plays a role in the virulence of a variety ofpathogenic Gram-negative bacteria (22). Moreover, in agree-mentwith earlier observationsOmpThas been shown to cleaveseveral receptor-bound colicins and thus to improve the sur-vival of target bacteria exposed to colicins or other antimicro-bial peptides (23–25).In thiswork, we identified the in vivo processed forms of both

RNase colicins D and E3 as the final colicin forms present in thecytoplasm of colicin-treated bacteria. The endoproteolyticcleavage was shown to require the inner membrane proteaseFtsH, which is required for the cytotoxicity of all nucleasecolicins (26). Additionally the signal peptidase LepB is specifi-cally required for the processing and/or translocation of colicinD across the inner membrane. In vitro cleavage of colicins Dand E3 requires the outer membrane protease OmpT. How-ever, OmpT is not required for the import and toxicity ofcolicins.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids—E. coliK12 strains C600 andJM101 were used as wild-type strains. DH5� was used as thehost strain for cloning and mutagenesis. All other strains andplasmids used or constructed are listed in Table 1.Purification of Colicins—Colicin D, unlabeled or labeled in

vivo with [35S]Met (1000 Ci/mmol, Amersham Biosciences), incomplex with its immunity protein (ImmD) was purified fromanE. coli strain carrying pJF129 as described previously (17, 27).Colicin D was separated from its ImmD by gel filtration in thepresence of 9 M urea (Superose 12 HR column, GE Healthcare)and refolded by dialysis against 20 mM Tris/HCl, pH 8.0.

Colicin E3-ImmE3 complex was purified from an E. colistrain carrying pKSJ28 and then dissociated by two chromato-graphic steps on a Mono S column (GE Healthcare) withoutand with 6 M urea, respectively, according to de Zamaroczyet al. (17).Alternatively, colicin D in complex with the ImmD protein

carrying a C-terminal His6 tag (colicin D-ImmD(Ct-His6)) wasproduced fromBL21(DE3) cells carrying the plasmid pColDI inLBmediumwith ampicillin. AtA600 � 0.5 the expression of thecolicin operon was induced by isopropyl 1-thio-�-D-galactopy-ranoside (1mM), and incubation was continued for 3 h at 37 °C.After centrifugation the cells were resuspended in 20 ml ofloading buffer (20 mM Tris, pH 8.0, 0.5 M NaCl, and 200 �g ofDNase) and then broken by one passage through a French pres-sure cell. The supernatant was loaded onto a 1-ml Ni-NTAresin column (His-select nickel affinity gel, Sigma) equilibrated

previously with the loading buffer and washed with the samebuffer, and the His-tagged colicin D-ImmD complex was theneluted with 150 mM imidazole. After the complex was denatur-ated in 9 M urea, His-tagged ImmD was retained by two pas-sages over a Ni-NTA column. Colicin D freed of ImmDpresentin the “flow-through”was concentrated (via ultrafiltration in anAmicon Ultra-4, nominal molecular weight limit 10,000; Milli-pore) after dialysis. The same protocol was used to producecolicin E3-ImmE3(Ct-His6) from plasmid pColE3I.Purification of the 12.4-kDa Colicin D Catalytic Domain in

Complexwith ImmD—The cloned catalytic domain (CAT) cor-responds to amino acids Met590–Leu697 from the C-terminalpart of colicin D. Met590 was chosen as initiating amino acidbecause it corresponds to a spontaneous break in the protein,which occurs during the crystallization of colicin D in complexwith ImmD (27). The catalytic domain in interaction with the10.9-kDa ImmD(Ct-His6) was expressed from BL21(DE3) car-rying the plasmid pCDM590 and purified as described above.The catalytic domain, used for antiserum production, althoughseparated from its ImmD partner by 9 M urea still containedsome ImmD protein. Thus, the polyclonal antiserum againstthe catalytic domain also cross-reacts with the ImmD protein.Purification of His-tagged LepB Signal Peptidase—BL21(DE3)

cells containing plasmid pLEPB, pLEPB(K145A), orpLEPB(N274K) were grown, and LepB protein expression wasinduced as described above. His-tagged LepB was purified on aNi-NTA column in the presence of 1%Triton X-100 and elutedby a gradient of 25 to 200 mM imidazole (28).N-terminal Residue Determination of the in Vitro Obtained

Small Cleaved (SC Form) Colicin Peptides—The 10.5-kDa invitro cleaved SC colicin D form was transferred from 15% SDS-PAGE to a ProBlottmembrane (Applied Biosystems) for N-ter-minal peptide sequencing with an ABI 473A automaticsequencer at the Pasteur Institute, Paris. The in vitro cleavagesite at the start of the 11-kDa SC fragment of colicin E3 waslocalized by mass spectrometric analysis performed on aMALDI-TOF-MS Voyager System 4106 at the Ecole Superi-eure de Physique et de Chimie Industrielles (ESPCI), Paris.Detection of LepB Interaction with Colicin D by Far Western

Blotting—Purified colicins or colicin D domains (2–5 �g) wereseparated on 15% SDS-PAGE, transferred to nitrocellulosemembrane, and then stained by Ponceau red. A denaturation-renaturation step according toWu et al. (29) was performed torefold the proteins. After incubation for 1 h at 37 °C with LepB(1 �g/ml) and successive washing steps, the binding of LepB tocolicins was analyzed by an anti-LepB antiserum and ECL.In Vitro Analysis of Colicin Cleavage—Extracts of E. coli

periplasmic proteinswere prepared by a classical osmotic shockprocedure adapted from the QIAexpressionist protocol (71).Alternatively, cell fractionation was performed using a sphero-plast-producing protocol based on lysozyme treatment of thecells (30). Aliquots of periplasmic extract were stored at�20 °Cin 20mM sodium phosphate, pH 6.5, 5mMMgSO4. Periplasmicprotein extracts, with orwithout the addition of purifiedOmpT(with lipopolysaccharides (LPS), 0.4 �g) (31) and/or LepB (1.5�g) or SipS, were incubated for 1 h at 37 °C in 20 mM sodiumphosphate, 5 mM MgSO4, pH 7.0, with unlabeled or [35S]Met-labeled colicin (4 and 1.5 �g, respectively). Full-size (FS) and

Endoproteolytic Processing of RNase Colicins

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cleaved colicin forms (large cleaved (LC) and small cleaved(SC)) were precipitated with acetone and then separated bySDS-PAGE, and the proteins were detected by Coomassie Bluestaining or phosphorimaging (Typhoon, GE Healthcare), re-spectively. The periplasmic extracts obtained by the two meth-ods were used in parallel for the colicin cleavage tests and gavethe same results in three repeated experiments. Polymixin B,known to form a complex with LPS, which are necessary for theactivity of OmpT (32), was added (3 �g/ml) to the reactionmixture to test the OmpT dependence of in vitro colicincleavage.Identification by “Fishing” for the in Vivo Processed Colicin

Peptides fromColicin D- or E3-treated Target Cells—50-ml cul-tures of wild-type or mutant E. coli strains carrying the plasmidpImmDwere grown in LBmedium to anA600� 0.4 at 37 °C andthen induced for 1 h with 1 mM isopropyl 1-thio-�-D-galacto-pyranoside to express the plasmid-encoded His-tagged colicinD immunity protein and with 0.05 mM 2,2�-dipyridyl. 1 mg ofcolicin D-ImmD complex was added to the cultures for 1–3 h.The cellswere harvested,washed twice, resuspended in 10ml of20 mM sodium phosphate, 150 mM NaCl, pH 7.0, and treatedwith proteinase K at 100 �g/ml for 1 h at 37 °C. After fouradditional washings, the colicin D- and proteinase K-treatedcells were resuspended in 2 ml of buffer (Tris 20 mM, and 0.5 M

NaCl, pH 7.5) with a protease inhibitor complex (CompleteEDTA-free; RocheApplied Science) and then disrupted by son-ication. The soluble fraction recovered after centrifugation wasthen loaded onto a Ni-NTA affinity column. The bound His-tagged ImmD, carrying any attached colicinmolecules that hadbeen “fished” from the cytoplasm, was eluted with 300 mM

imidazole, dialyzed, and concentrated (via ultrafiltration in anAmicon Ultracel 3K, Millipore). The processed colicin D pep-tide (ColD PF; recovered from about 10 ml of the initial cellculture)was separated from theHis-tagged ImmDon15%SDS-PAGE, identified by Western blot analysis using an antiserumto the colicin D catalytic domain, to the immunity protein or tothe full-size colicinD, and detected by ECL chemiluminescence(Immun-Star, Bio-Rad) associated with a charge-coupleddevice camera (ChemiDoc XRS� System, Bio-Rad). A similarprotocol to that used for colicin D, but without 2,2�-dipyridyl,was used to look for processing of colicin E3. Wild-type ormutant strains carrying plasmid pImmE3 encoding the His-tagged colicin E3 immunity protein were grown in LB or mini-mal medium (isopropyl 1-thio-�-D-galactopyranoside) andtreated with colicin E3-ImmE3 for 1–2 h. Proteins bound to theImmE3-His6 were purified by Ni-NTA chromatography, andWestern blot analysis was performed with an anti-colicin E3antiserum. The His-tagged ImmE3 was detected from the bot-tompart of the sameSDS-PAGEblot by an anti-His6 antiserum.Direct Immunodetection of Processed Colicin D and Colicin

E3 Peptides in the S100 Cytoplasmic Fraction of Colicin-sensi-tive Target Cells—The direct immunodetection of the in vivoprocessed product required an increased colicin D import intotarget cells and further purification to remove other cytoplas-mic proteins. A 50-ml culture of strainAD202 atA600 � 0.3 and37 °C was induced by 0.05 mM 2,2�-dipyridyl. At A600 � 0.8,0.7–2 mg of colicin D-ImmD was added to the cells for 1–2 h.After harvesting, the cells were treated with proteinase K and

washed, and the cytoplasmic fractionwas prepared as describedabove. Ribosomal proteins were eliminated from cytoplasmicextracts by micro-ultracentrifugation at 100 S (Beckman rotorTLA 100.2) to give an S100 fraction. To increase the thresholdof detection of the processed colicin D form, S100 fractionswere enriched for lower molecular weight proteins. Thus, theS100 fraction was precipitated with acetone. Proteins wereresuspended in 350 �l of 20 mM sodium phosphate buffer, pH7.0, and separated by FPLC-monitored gel filtration (Superdex75 GL 10/300). Fractions containing proteins with molecularweights lower than 35,000were pooled and precipitated by ace-tone. The concentrated and selectively enriched cytoplasmicproteins were separated on 15% SDS-PAGE and analyzed byWestern blotting as described above.In the case of colicin E3, S100 fractions, prepared from cells

grown in LB or in minimal medium (to enhance the expressionof BtuB receptor) and treatedwith 2–4mgof colicin E3-ImmE3for 0.25–2 h, were separated on 15% SDS-PAGE and analyzeddirectly by Western blotting with anti-colicin E3 antiserum.When necessary, ECL-detected proteins were quantified byImageLab software (Bio-Rad).

RESULTS

In Vitro Cleavage of Colicin D in Periplasmic Extracts—Weshowed previously that only when a crude extract of the colicinD-resistant strain A38 carrying the lepB (N274K)mutation wassupplemented with purified LepB was it capable of the endo-proteolytic cleavage of full-size colicin D molecules. However,LepB alonewas not able to cleave colicinD (17) (Fig. 1C, lane 4).We sought therefore to identify an additional component thatmight be required with LepB for colicin D cleavage. We choseto focus our investigation on periplasmic protein extracts,because the active site of the inner membrane protein LepB isoriented toward the periplasmic space (33). When LepB wasadded to the periplasmic extracts, prepared from the lepBmutant, about 50� 7%of FS 75-kDa colicinD (free of its immu-nity protein) was found to be cleaved. A 65-kDa LC form (Fig.1A, lane 2) and an about 11-kDa SC form (data not shown)werevisualized by SDS-PAGE. TheN-terminal amino acid sequenceof the SC peptide was determined to be 608KYKHAGDF-GISD619 (Fig. 2A). The in vitro cleavage site is located betweentwo lysines residues at positions 607 and 608, so that cleavageliberates a C-terminal peptide of 10.5 kDa, which correspondsin size to the previously defined minimal tRNase domain ofcolicin D (17).Outer Membrane OmpT Is the Protease Necessary for Colicin

D Cleavage in Vitro—The cleavage site, located between twobasic residues, satisfies the amino acid requirement for cleavageby the outermembraneOmpT protease (34). In the presence ofLepB, about 45�7% colicin Dwas cleaved in vitrowith purifiedOmpT-(LPS) (Fig. 1C, lane 1). However, only a marginal cleav-age of colicinD (�5%)was detectedwithOmpT alone (data notshown). We checked that colicin D cleavage was completelyinhibited when polymixin B was added to the reaction mixture(Fig. 1A, lane 3). Polymixin B is known to form a complex withthe LPS, which are necessary for the activity of OmpT (32). TheN-terminal sequence of the SC form, liberated by the purifiedLepB andOmpT together (Fig. 1B), showed that it corresponds




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to exactly the same cleavage site observed with LepB and theperiplasmic extract (Fig. 2A). We confirmed that OmpT prote-ase was responsible for the in vitro cleavage by preparingperiplasmic extracts from �ompT mutant strains (AD202 andC600ompT). These periplasmic extracts supplemented withLepB were not able to cleave colicin D. But the endoproteolyticcleavage of colicin D was efficiently restored using extractsfrom the same strains overproducing wild-type OmpT fromplasmid pML19 if supplemented with LepB (Fig. 1D, lanes 2and 3).LepB Catalytic Activity Is Not Involved in Colicin D Cleavage

by OmpT—To further analyze the function of the LepB pepti-dase, we studied in vitro the cleavage of colicin D by OmpT in

the presence of the purifiedmutant LepBprotein (K145A). Thismutation affects an essential residue in the active site of LepBwithout causing significant conformational change. Thus, itprevents, both in vitro and in vivo, the processing of the naturalsubstrates of LepB (35). Despite the loss of its normal catalyticfunction, LepB (K145A) was able to promote the in vitro cleav-age of colicin D by OmpT (Fig. 1C, lane 2). This indicates thatthe catalytic activity of LepB is not required for colicin D cleav-age in vitro. We also showed that the LepB orthologue SipS ofBacillus amyloliquefaciens could replace LepB for the cleavageof colicin D (supplemental Fig. S1), and we observed that theSipS protein fromplasmid pGDL81 efficiently restored the sen-sitivity of the strain A38 lepBmutant to colicin D.LepB Interacts withColicinD inVitro—Further evidence that

the role of LepB in colicin D processing is structural came fromthe detection of a direct interaction between purified wild-typeLepB and colicin D. We detected by Far Western blotting astrong signal indicating the formation of a complex betweenLepB and colicin D (Fig. 3B, lane 7). No interaction wasdetected with the DNase colicin E2, RNase colicin E3, or thepore-forming colicin B (Fig. 3B, lanes 1, 2, and 8), which isconsistentwith the in vivoobservation that LepB is not requiredfor the toxicity of any other colicins than colicin D. Analysis ofthe separated domains of colicinD showed that only the centraldomain (Glu314–Met590) was targeted by LepB (Fig. 3B, lanes 5and 6) but not theN-terminal domain (Met1–Glu313, 96% iden-tical to that of colicin B) (lane 3), the catalytic domain (Met590–Leu697) (lane 4), or the ImmD protein (lanes 4, 6, and 7). Thus,

FIGURE 1. LepB-dependent in vitro cleavage of colicin D in the presence ofperiplasmic extract or OmpT. A, ColD, free of its immunity protein, wastreated with periplasmic extracts (PE) prepared from lepB (N274K) mutantstrain A38 alone (lane 1) or in the presence of purified LepB (lane 2) or LepBand polymyxin B (Poly B, lane 3). The products were separated by 8% SDS-PAGE and detected by Coomassie Blue staining: FS ColD, 75 kDa, and LC form,65 kDa. B, comparison of the sizes of the SC form of colicin D produced in vitroby purified OmpT and LepB with the cloned 12.4-kDa catalytic domain (CAT12.4) and the 10.9-kDa ImmD(6His) protein. Samples were analyzed by 15%SDS-PAGE and stained with Coomassie Blue. C, cleavage of colicin D in vitro byOmpT in the presence of purified LepB (lane 1) or LepB-K145A (lane 2). Thepositions of migration of OmpT and LepB are indicated. (Refer to supplemen-tal Fig. S1 for an analysis of colicin D cleavage by SipS of B. amyloliquefaciens,a LepB orthologue.) D, [35S]ColD was treated with periplasmic extracts of anompT-inactivated strain overproducing OmpT (PE pompT, lane 3) or not (PE�ompT, lanes 1 and 2) and supplemented with purified LepB (lanes 2 and 3). FSColD and the LC form were separated by 8% SDS-PAGE and detected byphosphorimaging.

FIGURE 2. Location of the OmpT-catalyzed in vitro cleavage sites and thein vivo processing sites in RNase colicins D and E3. A, peptide sequence ofthe junction between the central domain and the tRNase domain of colicin D.The location of the OmpT cleavage (at position Lys607–Lys608), producing invitro the 10.5-kDa SC form, and the approximate location (near Met590) of thein vivo processing site producing the 12.4-kDa ColD PF are indicated. Met590 isthe first amino acid of the cloned catalytic domain (Met590–Leu697) of colicinD. B, peptide sequence of the junction between the central R-domain andRNase domain of colicin E3. The OmpT cleavage sites, located between resi-dues Arg454 and Lys455 (within the linker) and within the peptide motifArg432–Arg440, and the corresponding LC and SC forms are indicated. (Referto supplemental Fig. S2 for a complementary analysis of the OmpT-depen-dent cleavage of colicin E3.) The approximate location of the in vivo process-ing site around position Asp420 producing the 15-kDa ColE3 PF is indicated.




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LepB recognizes the central domain, which is unique to colicinD. Moreover, we also detected a strong decrease in the interac-tion of colicin D, and especially its central domain, with thepurified LepB-mutant protein (N274K) (�10%) (Fig. 3E, lanes7, 6, and 5) as compared with that obtained with the wild-typeLepB (100%) (Fig. 3D, lanes 7, 6, and 5), whereas the interactionwith the catalytic mutant LepB protein (K145A) remainedstrong (85� 10%) (Fig. 3F, lanes 7, 6, and 5). In each blot (Fig. 3,C–F) the same amount of wild-type LepB was included (lanesC) and used as a standard to validate the quantitative compar-ison of LepB-specific bands from these blots. We checked thatthe variations in the interaction were not due to a difference inthe affinity of the anti-LepB antiserum for the LepB-mutantproteins. As shown in Fig. 3G, this antiserum has equal affinityfor the recognition of wild-type and eachmutant LepB protein.Because thewild-type andnon-catalytic LepBprotein exhibiteda similar capacity to form a complex in vitro with the centraldomain of colicin D, we verified that the plasmidpRD8(K145A), expressing the LepB mutant protein K145A,

complemented the lepB(N274K) mutant strain A38 for colicinD sensitivity, aswell as thewild-type LepB expressed by plasmidpRD8 (data not shown). These experiments indicate that coli-cin D import requires LepB but not it’s catalytic activity andpresumably necessitates a direct interaction of the toxin withLepB in the periplasm.In Vitro Demonstrated Cleavage of RNase Colicins by OmpT

Has No Role in Their in Vivo Processing—We investigatedwhether other colicins were subject to OmpT cleavage in vitroby examining the RNase colicin E3, which parasitizes the Tolsystem for its import. Using [35S]Met-labeled colicin E3, free ofits immunity protein, two LC fragments, of 47 and 45 kDa (Fig.2B), were obtained from the 58-kDa FS colicin in the presenceof periplasmic extracts prepared from either the wild-type orlepB A38 mutant strains (Fig. 4A, lanes 2 and 4). As expected,LepB is not a requirement for the in vitro cleavage of colicin E3(20). Purified OmpT restored the cleavage of colicin E3 whenthe assay was performed in an extract from anOmpT-deficientstrain (Fig. 4A, lanes 5 and 7). However, only the shorter,45-kDa LC form was detected with purified OmpT, even in theabsence of periplasmic extract (Fig. 4A, lanes 3 and 7). Thissuggests that the amount of OmpT is limiting in the periplas-mic extracts so that partial cleavage at one or the other of twosites in colicin E3 is observed (Fig. 2B).When purified OmpT isadded in excess, both sites are fully cleaved and only the shorterLC form is detected (Fig. 4A). The OmpT protease is located inthe outer membrane and oriented so that its active site is extra-cellular (36). The limited OmpT activity we observed inperiplasmic extracts may be either the consequence of itsrelease from the inner membrane into the periplasmic spaceduring its passage to the outer membrane (37) or due to somecontamination deriving from the methods used for the prepa-ration of the periplasmic extracts. Mass spectrometry analysisof the 47-kDa LC form localized anOmpT-dependent cleavagesite between residues Arg454 and Lys455 located inside the shortlinker peptide (38) (Fig. 2B). The peptide motif 432RKKKED-KKR440 carries several other consensus OmpT cleavage sites,which are presumably responsible for producing the 45-kDaLCform.Two short fragments (13- and 11-kDa SC forms) detectedby Western blotting with anti-colicin E3 antiserum carry theC-terminal nuclease domain of colicin E3 and correspondrespectively to the 45- and 47-kDa LC products, respectively(Fig. 2B and supplemental Fig. S2).

We examined the sensitivity of OmpT-deficient strainstoward nuclease colicins. The �ompT strains (AD202,C600ompT, and the E. coli B BL21) were all shown to be fullysensitive to colicins D and E3, as observed by the cytotoxicitytest (Fig. 4D), and toDNase colicin E2 (25). Therefore, OmpT isnot necessary for colicin import and toxicity.We also examinedthe sensitivity of the wild-type and OmpT-overexpressingstrains to colicinsD andE3. The toxicity of these RNase colicinsin complex with their cognate immunity proteins, as judged bythe growth inhibition test, diminished by 1 order of magnitudewhenOmpTwas overexpressed from a high copy number plas-mid, pML19.More importantly, colicin E3, free of its Imm pro-tein, almost completely lost its toxicity against wild-type cells.In the case of colicin D freed of its Imm protein, the wild-typecells were sensitive, whereas OmpT-overexpressing cells were

FIGURE 3. In vitro interaction of the LepB signal peptidase with colicin D.A, DNase colicin E2 (lane 1), pore-forming colicin B (lane 2), the N-terminaldomain of colicin D (lane 3), colicin D CAT with ImmD (lane 4), colicin D centraldomain (CD, lane 5), colicin D central and catalytic domains (CD�CAT) withImmD (lane 6), tRNase colicin D (FS ColD) with ImmD (lane 7), and RNase colicinE3 with ImmE3 (lane 8) were separated by 15% SDS-PAGE and detected byPonceau red staining. Molecular masses are given in kDa. B, samples are asdescribed in A. Polypeptides interacting with purified wild-type LepB weredetected by Far Western blotting with anti-LepB antiserum. C, a subset of theproteins analyzed in A was tested in the absence of any LepB (control). Thenumbers above each lane identify the proteins as related to blot A. Contami-nant signals (lane 6) are also visible in D–F. D, same blot as in C; proteinsinteracting with purified wild-type LepB were tested as indicated in B. E, sameblot as in C; proteins interacting with purified mutant LepB (N274K; resistanceto colicin D) were tested as indicated in B. The efficiency of interactionbetween mutant LepB and colicin D was quantified and expressed as a per-centage of values measured with wild-type LepB (D, 100%). To validate thequantitative comparison of LepB specific bands, the same amount of wild-type LepB was included (lanes C) in each blot (C–F). F, same blot as in C;proteins interacting with purified mutant LepB (K145A; inactive enzyme)were tested as indicated in B. G, affinity test of the anti-LepB antiserum for thewild type (wt) and each of the mutant LepB proteins (LepB K145A and LepBN274K). The same amount of protein (about 50 ng) was loaded for each sam-ple on 15% SDS-PAGE and then detected by Western blotting.




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resistant (Fig. 4D). Thus, the Imm proteins appear to efficientlyprevent the bacterial defense system mediated by OmpT fromfunctioning, as shown previously in the case of colicin E2 (25).Moreover, the Imm proteins may become essential for the kill-ing action of these colicins when the OmpT activity of targetcells increases.Identification of a Processed Form of Colicin D in the Cyto-

plasmofTargetCells by “LigandFishing”with ImmDProtein—Toverify that processing of nuclease colicins did occur and was

required for import and toxicity, we set up an in vivo fishingassay to detect a processed form of colicin D in the cytoplasmbased on its high affinity interaction with its immunity protein(27). Wild-type or OmpT-deficient bacteria expressing plas-mid-borne His-tagged ImmD protein were treated with thepurified colicin D-ImmD complex. To enhance the penetrationof colicin D into the periplasm, target cells were treated previ-ously with 2,2�-dipyridyl, which increases the expression ofFepA and TonB (39) necessary for colicin D translocationacross the outer membrane. Any colicin D fragments com-plexed with the ImmD-His6 protein and fished from the cyto-plasm were analyzed by Western blotting with anti-colicin Dcatalytic domain antiserum. The same two bandswere detectedin both the wild-type and OmpT-deficient strains (Fig. 5A,lanes 1 and 3). Because this antiserum cross-reacts with ImmD(see “Experimental Procedures”), the band with a lower masscorresponds to ImmD-His6 (10.9 kDa) as verified by a specificreaction with anti-ImmD antiserum (data not shown). Theslightly heavier band corresponds to the in vivo processed coli-cin D form (called “PF” to distinguish it from the in vitro SCform; Fig. 2A). The in vivo processed form is absent from thecytoplasm of control cells, which are not treated with colicin D(Fig. 5A, lanes 5–8 and 10), or from cells treated with colicin Dbut not induced with dipyridyl (data not shown). As expected,no processed colicin D form was found in tonB- or fepA-inacti-vated mutants, in which translocation of colicin D across theouter membrane is blocked (Fig. 5A, lanes 2 and 4). In particu-lar, there is no colicinDprocessing in the tonBmutant strainD1(R158S), which completely prevents the import of colicin D butremains competent for FepA-dependent iron uptake (Fig. 5A,lane 11) (40).Significantly, colicin D processing was abolished in the

lepB(N274K) mutant strain A38, which specifically preventscolicinD toxicity. This processing defect was complemented bythe plasmid pLEPB(wt) (Fig. 5B, lanes 1 and 2). This demon-strates that LepB does indeed have a specific functional role inthe processing of colicin D.The essential inner membrane FtsH protease was previously

observed to be required for sensitivity to nuclease colicins (26).As in the case of the lepB mutant, no processed colicin D wasdetected in an ftsH-inactivated mutant strain (in a lethality-suppressed background of strain AR3291 (Table 1)), but theprocessing was fully restored when the plasmid pFTSH wasintroduced (Fig. 5B, lanes 3 and 4). This result raised the possi-bility that FtsH may be the translocator of colicin D across theinnermembrane. By hijacking FtsH function, colicinD could beprocessed in the inner membrane, thus releasing the catalyticdomain, which is able to reach its cytoplasmic target. In thisscenario, the LepB interaction with colicin D could be essentialand thusmay facilitate the access of colicin D to the FtsH activesite.Direct Immunodetection of the Processed Form of colicin D in

the S100 Cytoplasmic Fraction of Sensitive Cells Exposed toColicin D—In the ligand-fishing experiment, the ImmD-ex-pressing cells are protected against cell killing by colicin Dtreatment, but we could not exclude the possibility that the PFmay be a consequence of proteolytic degradation, destined torid the cytoplasm of the inactive colicin D-ImmD complexes

FIGURE 4. Comparison of the in vitro and in vivo cleavage products ofcolicin E3. A, [35S]ColE3 was treated with periplasmic extracts prepared fromwild-type (lane 4, PE wt), lepB (N274K) mutant (lane 2, PE A38), ompT-inacti-vated (lanes 5 and 7, �ompT) strains, and/or purified OmpT (lanes 3 and 7). FSColE3, 58 kDa, and the LC forms, 47 and 45 kDa, were separated on 8% SDS-PAGE and detected by phosphorimaging. B, comparison of the migration ofthe in vivo ColE3 PF (lane 3) with various C-terminal peptides of colicin E3synthesized in vitro by a coupled transcription-translation Zubay-30S system(7). Peptides Lys439–Leu551 (lane 1, 12.8 kDa), Lys416–Leu551 (lane 2, 15.4 kDa),and Met429–Leu551 (lane 4, 14.1 kDa) were separated on 15% SDS-PAGE andanalyzed by Western blotting with anti-colicin E3 antiserum. C, comparison ofthe migration of the 15-kDa in vivo processed form (ColE3 PF15, lane 1) and the11- and 13-kDa SC forms of colicin E3 produced in vitro (lane 2), analyzed asdescribed in B. D, comparison of cytotoxicity of colicins D � ImmD andE3 � ImmE3 in the presence or absence of OmpT. The in vivo halo assay wasquantified by spotting aliquots of undiluted colicin (numbered 0) and serial10-fold dilutions (numbered 1 to 5) directly onto a lawn of wild-type (wt),OmpT-deficient (�ompT), or OmpT overexpressing (pOmpT) cells. Dark halosindicate a clear zone of growth inhibition (toxicity), and no clearing indicatesresistance to colicin. Turbid zones indicate marginal toxicity.




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formed during the import. Thus, we attempted to directlydetect the processed form in the cytoplasm after a shorter (0.5–1.5 h) treatment with colicin D. The C-terminal PF of colicin D,either detected directly in the S100 fraction of colicin D-treatedcells (Fig. 5C, lanes 1 and 2) or captured by using the fishingtechnique (Fig. 5C, lane 3), migrated to an identical position on15% SDS-PAGE and appeared to have exactly the same migra-tion as the 12.4-kDa cloned catalytic domain (CAT; Met590–Leu697). Comigration with the CAT implies that the in vivoprocessing site is located near position Met590 (Fig. 2A), whichis the first residue of the CAT. It should be noted that the prod-

uct (SC form) of the OmpT/LepB-dependent cleavage of coli-cin D in vitro is significantly smaller than 12.4 kDa (Fig. 1B).The difference corresponds to about 18 amino acids separatingthe start of the in vivo PF form from that of the minimal cata-lytic domain (Lys607–Leu697), defined in vitro as sufficient fortRNAArg hydrolysis (17).Identification of the Processed Form of Colicin E3 in the Cyto-

plasm of Target Cells—We wondered whether proteolyticprocessing was a characteristic of all RNase colicins and thuslooked for the processed form of colicin E3 by direct immuno-detection in the cytoplasm. The same unique peptide, with amolecularmass of about 15 kDa,was detected by an anti-colicinE3 antiserum from the S100 fractions of ompT-inactivated (Fig.6A, lanes 1 and 2) and wild-type strains (Fig. 6B, lane 8), con-firming that OmpT is not involved in the in vivo processing ofcolicin E3. The former experimentswere performed inminimalmedium, in which the low level of vitamin B12 was expected toincrease BtuB receptor expression. The same peptide, with a3-fold lower intensity, was present in colicin E3-treated cellsgrown in LB (Fig. 6A, lane 6). In both media, colicin E3 PF wasdetected efficiently after only 15 min of treatment of cells withcolicin E3-ImmE3 (Fig. 6C, lane 4). Detection of a similar levelof PF with colicin D within 15 min necessitated about 8-foldmore S100 extract (data not shown). The rapidity of the appear-ance of the PF is consistent with our observation (Fig. 5C) thatit was generated during the translocation step (allowing theentry of the catalytic domain into the cytoplasm through theinner membrane) rather than by some proteolytic degradationof longer colicin molecules that had accumulated in the cyto-plasm after their translocation. Quantification of the timecourse of the colicin E3 PF appearance (Fig. 6C) showed that itincreased 4-fold during the first hour of treatment with colicin.No processed forms were detected in the cytoplasm of tolB-

or btuB-inactivatedmutant strains (Fig. 6,A, lane 5, and B, lane3), which is in agreement with the involvement of the BtuBreceptor and the Tol system in the translocation of colicin E3across the outer membrane. In addition, we showed that FtsHdeficiency, as in the case of colicinD, prevented detection of theprocessed form of colicin E3 (Fig. 6B, lane 1). However, unlikecolicin D, colicin E3 toxicity is not eliminated by the lepB(N274K) mutation (20). We thus unexpected to observe loweramounts of the colicin E3 processed form in extracts from theA38 strain than from the wild type (Fig. 6B, lanes 7 and 8). Theamount of processed colicin E3 was restored in extracts fromthe A38 strain carrying the pLEPB(wt) plasmid (Fig. 6B, lane 6).This result suggests that even though LepB is not required forcolicin E3 toxicity, the rate of processing might be modulatedby LepB, although no interaction between colicin E3 and LepBwas detected by Far Western blotting (Fig. 3B, lane 8).The same processed fragment was detected in vivo either by

using the fishing technique, with an ImmE3-His6 proteinexpressed endogenously, or directly from the cytoplasm (Fig.6D, lane 2 compared with lane 3). The comparison confirmsthat the PF of colicin E3, which is trapped by ImmE3, corre-sponds mainly to the catalytic ribonuclease domain. This invivo detected PF has a size close to 15 kDa, as deduced from acomparison with the migration of a set of in vitro synthesizedC-terminal peptides of colicin E3 overlapping the catalytic

FIGURE 5. Analysis of the in vivo processed form of the tRNase colicin Ddetected by fishing. A, wild-type (wt) strain or strains carrying deletions (�)in tonB (lanes 2 and 6), ompT (lanes 3 and 7), fepA (lanes 4 and 8), or the R158Spoint mutation in tonB (lanes 10 and 11), all expressing the 10.9-kDa His6-tagged immunity protein (ImmD(6H)), were treated (lanes 1– 4, 9, and 11) ornot (lanes 5– 8 and 10) with ColD-ImmD. His6-tagged ImmD and bound colicinmolecules were purified from the cytoplasm as described under “Experimen-tal Procedures,” separated by 15% SDS-PAGE, analyzed by Western blottingwith anti-colicin D catalytic domain antiserum, and detected by ECL. The posi-tions of migration of the 12.4-kDa ColD PF and the 10.9-kDa His6-taggedImmD protein are indicated. B, N274K lepB mutant strain A38 alone (lane 1) orcarrying the pLEPB(wt) plasmid (lane 2) and the FtsH-deficient strain alone(�ftsH) (lane 3) or carrying the pFTSH plasmid (lane 4) were treated with ColD-ImmD and analyzed as described in A. C, comparison of the in vivo ColD PFobtained by “direct” immunodetection from the S100 cytoplasmic fraction ofa �ompT strain as described under “Experimental Procedures” (lanes 1 and 2)and that obtained by fishing with His6-tagged ImmD from a �ompT strain(lane 3). Treatment with ColD-ImmD (mg) and Western analysis are asdescribed in A. The cloned 12.4-kDa CAT is used as the molecular massreference.




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domain (Fig. 4B). This finding allowed us to estimate that thecleavage site generating PF is close to residue Asp420 of colicinE3 (Fig. 2B). TheColE3PF is about 2–4 kDaheavier than the SCforms detected after cleavage in vitro in the presence of OmpT(Fig. 4C). In fact, the in vivo processing site of colicin E3 islocated inside the C-terminal part of the central receptor-bind-ing domain.

DISCUSSION

In the present work we have demonstrated, in the case of twodifferent RNase colicins, that an endoproteolytic processingstep is essential for their import into the cell and for subsequentcell killing. Using two distinct experimental protocols, we haveshown for both colicins D and E3 that only their cytotoxicC-terminal catalytic domains (12.4 kDa for colicin D and 15kDa for colicin E3) can be detected in the cytoplasm of cellsexposed to the colicins. The same processed forms weredetected in both sensitive or immunity-protein protected cells.The PFs found in the cytoplasmare significantly longer than theminimal size of the C-terminal domains required for catalyticactivity. In the case of colicin D, the 18 additional amino acids(positions 590–607) present in the PF (Fig. 2A) were implicatedin the interactionwith the immunity protein (ImmD) and couldalso be required for tRNA target recognition (27). In the case ofcolicin E3, the 31 additional residues in the PF (positionsAsp420–Lys450) (Fig. 2B) are derived from the 100 Å long hair-pin structure of the receptor-binding (R) domain (38). This sug-gests that the R-domain should be partly unfolded to allow theRNase domain to reach the periplasmic side of the inner mem-brane. Such a conformational change of the R-domain may becompatible with its stable interaction with the receptor BtuBbecause this latter interaction involves only residues of the

R-domain that are near the tip of the coiled coil structure (41).The partial unwinding of the termini of the coiled coil R-do-main (41, 42) facilitates both the insertion of the N-terminalpart of the translocation-domain through OmpF and then thesubsequent unfolding of the C-terminal part of colicin E3, thusallowing the catalytic domain to reach the cytoplasmicmembraneso that it can enter the cytoplasm (Fig. 7) (42). In agreementwith aspecific cascade of events during their import (depicted for bothcolicins in Fig. 7), we show that the processing of colicinsDandE3does not occur if their translocation across the outermembrane ispreventedbymutationsaffecting their receptors (FepAorBtuB)orthe energy transducer Ton or Tol system (Figs. 5 and 6).Our work has shown that the inner membrane, ATP-depen-

dent, and membrane-anchored protease FtsH (43) is essentialfor the processing of both colicins D and E3 and/or the trans-location of the PF into the cytoplasm (Fig. 7). In particular, weshow that there is a strict correlation between the loss of sensi-tivity of ftsH mutants to nuclease colicins, as reported previ-ously (26), and the absence of PF in the cytoplasm of colicin-treated, FtsH-deficient cells. It was shown previously thatsensitivity to nuclease colicins requires both the protease andATPase activities of FtsH (26). Our tentative conclusion is thatthe FtsH endopeptidase is the catalytic enzyme required forcolicin processing.The usual biological function of FtsH consists of the disloca-

tion and degradation of misfolded or damaged membraneproteins (43) by “pulling” them into cytoplasm (44, 45). FtsHrather than unfolding the substrates itself, preferentially acts onalready unfolded proteins (46, 47). The presence of an unstruc-tured terminal tail of membrane substrates that penetrate intothe cytoplasm appears to be important in initiating FtsH-de-

TABLE 1E. coli strains and plasmids

Strain or plasmid Genetic description Source

StrainA38a C600 lepB (N274K), colBS/colDR Ref. 17AD202 ompT1000::kan Ref. 59C600ompT C600 ompT1000::kan (by P1 transduction from AD202) This workBL21(DE3) �ompT Ref. 60UT5600 �(ompT-fepA-C)266 Ref. 61C600tonB C600 tonB::TN5 Ref. 62D1a D10 tonB (R158S), colBS/colDR Ref. 40JCL8789 188 nadA::Tn10, �tolB-pal, TetR Ref. 63JCL11650 169 btuB::Tn10, TetR Ref. 63AR3291 sfhC zad::Tn10, ftsH3::kan Ref. 64

PlasmidpLEPB(wt) pACYC184, lepAB (at SphI/BamHI), CmR This workpRD8 pING-1 (araBC), lepAB, AmpR Ref. 65pRD8(K145A) pING-1 (araBC), lepAB(K145A), AmpR This workpLEPB pET23, (tag-His6)lepB, AmpR Ref. 35pLEPB(K145A) pET23, (tag-His6)lepB(K145A), AmpR Ref. 35pLEPB(N274K) pET23, (tag-His6)lepB(N274K), AmpR This workpML19 pUC19, ompT, AmpR Ref. 66pSTD113 ftsH, AmpR Ref. 67pFTSH pACYC184, ftsH (at SalI/HinDIII), CmR This workpJF129b pColD-CA23, colicin D operon cda-cdi-cdl Ref. 68pColDI pET11a, cda-cdi(tag-His6) (at NdeI/BamHI), AmpR This workpCDM590 pET11a, the 3�-domain of cda (starting at Met590)-cdi(tag-His6) (at NdeI/BamHI), AmpR This workpIMMD pTRAC, cdi(tag-His6) (at NdeI/BamHI), AmpR This workpIMME3 pTRAC, cei(tag-His6) (at NdeI/BamHI), AmpR This workpKSJ28b pColE3-CA38, colicin E3 operon cea-cei-cel Ref. 69pColE3I pET11a, cea-cei(tag-His6) (at NdeI/BamHI), AmpR This workpGDL81 sipS (lepB orthologue from B. amyloliquefaciens) Ref. 70

a Strain sensitive to colicin B but resistant to colicin D.b cda, cdi, and cdl are genes for colicin D its immunity protein and bacteriocin release protein; cea, cei, and cel are the corresponding genes for the colicin E3 operon.




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pendent proteolysis (48). It is conceivable that DNase colicinshave a similar access to FtsH, because their nuclease domainexhibits an endogenous channel-forming activity, possiblyallowing its self-propulsion into the cytoplasm (14, 15).An electrostatically mediated interaction of the nuclease

domain of E type colicins with the inner membrane has beenreported (16). This could lead to (or maintain) a partial unfold-ing of the catalytic domain so that the unstructured C-terminalend may be recognized by FtsH as a substrate (Fig. 7). FtsH-de-pendent ATP hydrolysis could contribute to the translocationof the RNase domain toward the proteolytic active site residingin the central pore of FtsH (49, 50).

The normalmode of action of FtsH is a processive proteolyticdegradation, which liberates 10–26-residue oligopeptides intothe cytoplasm (43). However the in vivo cleavage of RNasecolicins D and E3 generates exceptionally long C-terminal pep-tides of 108 and 132 residues, respectively. This major differ-ence compared with its usual substrates may be correlated witha particular degradation pathway shown in vitro for FtsH. Aftertranslocating an internal loop of a model substrate to the pro-tease chamber, the proteolysis is initiated from an internal site.This liberates some discrete fragments of about 30 kDa, and theproteolysis proceeds processively in theC- toN-terminal direc-tion (50, 51).Previously we isolated a mutation in the gene for the signal

peptidase lepB (N274K) (strain A38), which renders the cellresistant to colicin D killing. We have now demonstrated thatLepB plays a specific role in colicin D processing and translo-cation (Fig. 5). The analysis of twomutations in lepBproves thatthe function of LepB in colicin import is not enzymatic. K145ALepB has no catalytic activity (35), but it still restores sensitivityto colicin D of the N274K lepB mutant strain and is able tointeract in vitrowith colicin D almost as well as wild-type LepB(Fig. 3). In contrast, strain A38 with the N274K lepBmutationshows no apparent deficiency with respect to the processing ofnormal LepB substrates (17). In addition to the absence of anyPFin the N274K lepBmutant strain, we also showed in vitro a largedecrease in the interaction between the N274K LepB protein andthe central domain of colicin D (Fig. 3). Together these results areconsistentwitha structural roleofLepB,whichcould interactwiththe central domain of colicin D and modify the structure of thetRNase domain so as to allow proteolysis by FtsH. Specific “adap-tator proteins” have been shown to be required for recognition ofother soluble protein substrates by FtsH (43).An interesting question is: why does colicin D processing

require a specific interaction with LepB, but colicin E3 doesnot? Cytotoxicity of DNase colicins has been shown to dependupon the net positive charge of their DNase domain, essentialfor their interaction with the inner membrane (26). Similarly, aproductive interaction of the RNase domain of colicin E3with theinner membrane could be ascribed to its high net positive charge(�11). In contrast, theweak positive charge of the tRNase domainof colicinD(�2)maybe insufficient to allowanelectrostatic inter-actionwith the innermembrane.Thus, the interactionof colicinDwith the membrane may be ensured or stabilized by a direct con-tact between its central domain and the LepB protein at theperiplasmic side of the inner membrane (Fig. 7).It is striking that there are four parallel requirements for bac-

terial toxin import into mammalian cells and nuclease colicinimport into E. coli (52, 53). Import into both the eukaryotic andprokaryotic systems necessitates: (i) hijacking of a quality con-trol system of protein folding; (ii) endoproteolytic cleavage ofthe toxin during translocation; (iii) a chaperone protein toaccess the membrane translocator; and (iv) ATP hydrolysis.In addition to colicin D, other nuclease colicins, e.g. rRNase

colicin E3 (Fig. 4) and DNase colicins E7 (18, 19) and E24 (25),have been shown to be cleaved in vitro by periplasmic extracts,

4 M. de Zamaroczy, unpublished data.

FIGURE 6. Detection and analysis of the in vivo processed form of theRNase colicin E3. A, cells grown in minimal (Min) or LB medium were treatedwith the quantities indicated (mg) of ColE3-ImmE3 complex. The proteins ofthe S100 cytoplasmic fractions (S100 �ompT (lanes 1–3, 6, and 7) or S100 �tolB(lanes 4 and 5)) were separated directly on 15% SDS-PAGE and analyzed byWestern blotting with anti-colicin E3 antiserum. ColE3 PF was revealed byECL, quantified, and expressed as a percentage of the value measured in LB(lane 6, 100%). The molecular mass of reference proteins is given in kDa. FSColE3 is shown (58 kDa). B, cells of FtsH-deficient (lanes 1 and 2, S100 �ftsH),btuB-inactivated (lanes 3 and 4, S100 �btuB), lepB mutant alone (lanes 5 and 7,S100 lepB-N274K) and carrying the pLEPB(wt) plasmid (lane 6, S100lepB�pLEPB), and wild-type (lanes 8 and 9, S100 wt) strains grown in LBmedium were treated with ColE3-ImmE3 and analyzed as described in A.ColE3 PF was quantified as in A and expressed as a percentage of the valuemeasured in S100 wt (lane 8, 100%). C, time course of the processing of colicinE3 performed in ompT-inactivated cells grown in minimal medium andtreated with ColE3-ImmE3 for the times given (min). The amount of ColE3 PFin S100 �ompT extracts was quantified and expressed as a percentage of theamount detected after 15 min (lane 4, 100%). D, comparison of the size ofColE3 PFs detected after treatment with ColE3-ImmE3 either by fishing (lane2) in the �ompT-ImmE3(6H) strain grown in LB or by direct immunodetection(lane 3) from an S100 �ompT cytoplasmic fraction. The ImmE3 protein (lanes 1and 2) was revealed by immunodetection with an anti-His6 tag antiserum,performed separately with the bottom (boxed) part of the same blot.




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and in all cases the in vitro cleavagewas strictly dependent uponOmpT. In this workwe have shown that the cleavage of colicinsD and E3 by purified OmpT (or by periplasmic extracts) occursat a different site from the authentic in vivo processing site wehave mapped (Fig. 2). The OmpT-dependent colicin cleavagesobserved in vitro (Figs. 1 and 4) appear to reflect the role ofOmpT at the cell surface as a part of the bacterial defense sys-tem to rid itself of extraneous and nefarious proteins (54). Thedegradation of colicin D by OmpT at the cell surface presum-ably requires the attachment of colicin-Imm complex to theFepA receptor (Fig. 7) and then is accelerated by the release ofthe immunity protein (Fig. 4). In contrast, we have shown thatOmpT cleavages clearly are not involved in the import ofcolicins into target cells. Contrary to a previous hypothesis (19),there is no evidence for periplasm-dependent processing dur-ing the import of nuclease colicins into the cytoplasm.It is interesting to note, however, that the productive in vivo

processing of colicin D by FtsH and the in vitro observed cleav-age by OmpT both require LepB. It is conceivable that theseendoproteolytic cleavages of colicin D freed of its Imm proteinrequire a similar folding of the C-terminal part of the colicin D

molecule to allow access of FtsH or OmpT to their distinct butrather closely located cleavage sites preceding the catalyticdomain. The appropriate conformation of the C-terminal partof colicin D for these cleavages seems to be mediated by thespecific structural interaction of the colicin D central domainwith LepB.

Acknowledgments—We are grateful to Jackie Plumbridge for helpfuladvice throughout this work and in-depth discussion of the manu-script.We also thank A. Kramer and J. Jongbloed for the generous giftsof purified OmpT and SipS proteases, respectively; R. E. Dalbey forplasmid pRD8 and pLEPB (wt and K145A); P. Bouloc for strain 1734(transduced from AR3291); and J. C. Lazzaroni and A. Vianney formutant strains btuB::Tn10 and �tolB-pal.

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FIGURE 7. Model of the import mechanisms for RNase colicins E3 and D. Path 1a, the BtuB receptor with bound colicin E3 and the recruited OmpF porinconstitute the outer membrane translocon, which requires the Tol system to drive colicin import toward the periplasm. The assembly of the translocon maylead to the unfolding of the CAT prior to its translocation across the outer membrane (Kurisu et al. (41) and reviewed by Kleanthous (55)). Path 1b, the bindingof colicin D to the FepA receptor followed by the interaction of TonB with the FepA cork domain and the N-terminal part of colicin D triggers the translocationof the CAT across the outer membrane (reviewed by Braun et al. (6) and de Zamaroczy and Chauleau (9)). The translocation mechanism of the CATs is unknown,although for both colicins the Imm protein should be released first from the receptor bound colicin. Recent evidence suggests that the Imm protein release isenergy-dependent (56). The crystal structure of the conserved N-terminal domain of colicin D shown bound to FepA is adapted from that of colicin B (57).Colicin E3 and possibly colicin D remain attached to their receptors, whereas the CATs traverse the periplasm and contact the inner membrane (indicated bydotted lines and a question mark). Path 2a, electrostatic association of the RNase domain of colicin E3 (CAT) with the anionic phospholipid surface of the innermembrane (16) (indicated by a pink X) may monitor the contact with FtsH. Path 2b, interaction of LepB with the central domain (CD) of colicin D is presumablyan essential requirement for colicin D to enter the cytoplasm. Residues Lys145 and Asn274 are localized in the LepB crystal structure by red and blue spheres,respectively (adapted from Paetzel et al. (58)). Path 3, the processing and translocation mechanisms of both RNase colicins are unknown but require an activeFtsH (ATPase/protease) to allow the PFs to reach the cytoplasm. Path 4, the nucleases imported by PFs cleave either 16 S rRNA (colicin E3) or tRNAArg (colicin D),preventing protein synthesis (1, 2) and thus leading to target cell death.




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Mathieu Chauleau, Liliana Mora, Justyna Serba and Miklos de ZamaroczyCytotoxic Domains Are Imported into the Cytoplasm

FtsH-dependent Processing of RNase Colicins D and E3 Means That Only the

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FtsH-dependent Processing of RNase Colicins D and E3 Means