The Tip of the Hydrophobic Hairpin of Colicin U Is ... · Detroit, Mich.) plus 0.5% NaCl (pH 7) or on TY agar plates. When required, media were supplemented with kanamycin (50 mg/ml)

JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

Aug. 1998, p. 4111–4115 Vol. 180, No. 16

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

The Tip of the Hydrophobic Hairpin of Colicin U Is Dispensablefor Colicin U Activity but Is Important for Interaction

with the Immunity ProteinHOLGER PILSL,* DAVID SMAJS,† AND VOLKMAR BRAUN

Mikrobiologie/Membranphysiologie, Universitat Tubingen, Tubingen, Germany

Received 20 March 1998/Accepted 6 June 1998

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmicmembrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of Shigellaboydii was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunityprotein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was nolonger inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicinU hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575–582ColB), whichwas inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U.The colicin B immunity protein inactivated colicin U(575–582ColB) to the same degree. These results indicatethat the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with thecorresponding immunity proteins and is not required for colicin activity. Comparison of these results withpublished data suggests that interhelical loops and not membrane helices of pore-forming colicins mainlyinteract with the cognate immunity proteins and that the loops are located in different regions of the A-type andE1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmicmembrane, and the N and C termini face the cytoplasm.

Pore-forming colicins form voltage-dependent ion channelsin the cytoplasmic membrane of sensitive bacteria. Colicin Ubelongs to the family of channel-forming colicins (22), whichconsist of three domains responsible for translocation throughthe outer membrane (N-terminal domain), binding to the re-ceptor (central domain), and channel formation (C-terminaldomain). Crystal structures of the pore-forming domains ofcolicins A, E1, and Ia have been determined at atomic reso-lution (4, 16, 27). In the water-soluble state, the pore-formingdomains are arranged similarly and consist of a central hydro-phobic hairpin (helices 8 and 9) surrounded by eight amphi-pathic helices. The structure of the membrane pore is lessclear. Upon contact with the cytoplasmic membrane, the co-licins unfold and the hydrophobic hairpin inserts into the lipidbilayer. It is debated whether the hydrophobic hairpin is ori-ented parallel to the bilayer or whether it assumes a transmem-brane arrangement, and how its arrangement and that of theother helices change upon voltage-dependent pore formation(1, 3, 11–13, 15). In colicin Ia, at least helices 5 and 6 aretranslocated across the membrane in response to a transmem-brane voltage (21), whereas helices 8 and 9 are inserted volt-age-independent into the membrane (9). For the purpose ofthis paper, the general agreement that helices 8 and 9 areembedded in the membrane is of relevance.

Sequence similarities separate the pore-forming colicins intothe A-type (colicins A, B, N, and U) and the E1-type (colicinsE1, 5, K, 10, Ia, and Ib) colicins (Fig. 1). The correspondingimmunity proteins have been classified into the same twogroups (17, 20, 23). The colicin A immunity protein (Cai) has

four transmembrane segments, and its N and C termini arelocated in the cytoplasm (8), whereas the immunity proteins ofcolicin E1 (23) and colicin 5 (17) cross the cytoplasmic mem-brane three times, with the N terminus in the cytoplasm andthe C terminus in the periplasm.

In this study, we show that deletion of residues 575 to 583,which we propose to form the tip of the hydrophobic helicalhairpin, did not alter the cytotoxic activity of colicin U. Wefurther demonstrate that the tip sequence is a main determi-nant for the specific recognition of colicin U by the cognateimmunity protein. In addition, we determined the transmem-brane topology of the colicin U immunity protein and showthat it corresponds with the immunity protein of colicin A.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media. The bacterial strains and plasmidsused in this study are listed in Table 1. All strains were grown in a mediumcomposed of 1% Bacto Tryptone–0.5% yeast extract (TY; Difco Laboratories,Detroit, Mich.) plus 0.5% NaCl (pH 7) or on TY agar plates. When required,media were supplemented with kanamycin (50 mg/ml) or chloramphenicol (50mg/ml). The ampicillin resistance of strains carrying cui-blaM fusion genes wastested on TY agar plates supplemented with increasing concentrations of ampi-cillin (5, 25, 100, 200, and 400 mg/ml).

Recombinant DNA techniques. Plasmid DNA was isolated with ion-exchangecolumns (Qiagen, Hilden, Germany). Standard methods were used for restrictionendonuclease analyses, ligation, and transformation with plasmid DNA (18).DNA was sequenced by the dideoxy chain-termination method (19) with an Auto-Read sequencing kit (Pharmacia Biotech, Freiburg, Germany) and an A.L.F.Automatic Sequenator (Pharmacia Biotech). Site-specific mutagenesis was per-formed by PCR (10). The nucleotide sequence of the primer used for the A-to-Treplacement at position 1857 (colicin U8 mutant) of the cua gene (22) is given asan example of the mismatch primers used for PCR (replaced nucleotide under-lined): 59-GCTAAACTAGTCGATAAAACACCCAGC-93. The sequences ofthe mutagenized fragments were verified by DNA sequencing. For the construc-tion of cui-blaM gene fusions, restriction sites that yield blunt ends were intro-duced into the cui gene. After amplification by PCR, the cui gene fragments werecloned into the pJBS636 vector, which resulted in cui-blaM gene fusions.pJBS636, a b-lactamase fusion vector, was derived from vector pJBS633 (2);pJBS636 carries the T7 promoter and the multiple cloning site of plasmid pT7-7.

Radiolabeling of proteins. cui-blaM gene fusions were under the control of thephage T7 gene 10 promoter and were transcribed by the T7 RNA polymerase,

* Corresponding author. Mailing address: Mikrobiologie/Membran-physiologie, Auf der Morgenstelle 28, 72076 Tubingen, Germany.Phone: (49) 7071 2974620. Fax: (49) 7071 294634. E-mail: [email protected].

† Present address: Department of Biology, Faculty of Medicine,Masaryk University, Brno, Czech Republic.

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which was chromosomally encoded in Escherichia coli BL21 and was under thecontrol of the lacI repressor. Cells (2 ml) in the exponential growth phase(optical density at 578 nm 5 0.4) were collected by centrifugation and thensuspended in 1 ml of a medium that contained 0.6% Na2HPO4, 0.3% KH2PO4,0.1% NH4Cl, 0.05% NaCl, 1 mM MgSO4, 0.1 mM CaCl2, 1 mM sodium citrate,0.4% glucose, 20 mg of thiamine/liter, and 0.01% methionine assay medium(Difco Laboratories). T7 RNA polymerase synthesis was induced by adding 1mM isopropyl-b-D-thiogalactopyranoside. After the culture was shaken for 60min at 37°C, 20 ml of rifamycin solution (20 mg/ml in methanol) was added toinhibit the E. coli RNA polymerase, and the culture was shaken for an additional30 min. Cells were then labeled by adding 185 kBq of [35S]methionine andincubating the culture for 15 min at room temperature. Cells were sedimented bycentrifugation, suspended in 40 ml of sample buffer, and heated for 5 min at100°C. Ten microliters was subjected to polyacrylamide gel electrophoresis(PAGE; 3% polyacrylamide stacking gel, 15% polyacrylamide running gel) in thepresence of 0.1% sodium dodecyl sulfate (SDS). The dried gel was autoradio-graphed with Kodak X-Omat S100 film.

Colicin U and the mutated colicin U proteins were labeled in vitro with[35S]methionine in a bacterial cell-free transcription-translation system (Pro-mega, Madison, Wis.) and subjected to SDS-PAGE as described above.

Colicin activity assay. Colicin activity was tested by spotting 10-fold dilutionsof colicin-containing crude cell extracts onto plates prepared with 20 ml of TYagar; the plates were then overlaid with 3 ml of low-melting-point TY agar inwhich 0.1 ml of an overnight culture of the indicator strain had been suspended(14).

RESULTS

Deletion of the tip of the hydrophobic hairpin of colicin U.The colicin U determinant consists of the genes cua, whichencodes the colicin; cui, which confers immunity to colicin U;and cul, which causes lysis of the colicin U-producing cells (22).To investigate the function of the hydrophobic hairpin of co-licin U, amino acids 575 to 583 (henceforth designated the tip)of colicin U were deleted. E. coli 5K cells transformed withplasmid pHP140 cua [colicin U(D575–583)] cui cul resultedin single colonies. A second attempt to grow the transformedcells on a nutrient agar plate failed due to cell death, whichindicates that the immunity protein could not fully inactivatethe mutated colicin. Because of the instability of E. coli 5K(pHP140), the colicin U-resistant strain HP87, which does nottake up colicin U, was transformed with plasmid pHP140. Non-immune cells devoid of uptake are resistant to pore-formingcolicins which for pore formation have to insert from the peri-plasmic side into the cytoplasmic membrane. Crude extracts ofE. coli HP87(pHP140) killed sensitive E. coli 5K cells to the same

extent as cell extracts of E. coli 5K(pDS2 cua cui cul), whichsynthesized wild-type colicin U. This result demonstrated thatthe tip of the hydrophobic hairpin is dispensable for colicin Uactivity. Colicin U(D575–583) contained in the cell extract of E.coli HP87 killed E. coli 5K(pDS4 cui) cells despite synthesis ofthe immunity protein, as demonstrated by immunity to wild-type colicin U (Table 2). This shows that colicin U(D575–583)was not recognized by the colicin U immunity protein.

Mutational analysis of the hydrophobic colicin U hairpin.Although colicins U and B display 73% sequence identity inthe pore-forming domains, they show no cross-immunity (22).Since the hydrophobic hairpins of the two colicins are only

TABLE 1. E. coli strains and plasmids used in this study

Strain orplasmid

Relevant genotypeor phenotype

Source orreference

Strains5K hsdR lacZ rpsL ser thi thr This instituteBL21 F2 hsdS gal 24HP87 5K ompA rfa This work

PlasmidspDS2 pBCSK1 carrying cua cui cul 22pDS4 pBCSK1 carrying cui cul 22pJBS636 blaM under phage T7 control T. FocaretapHP81 pBCKS1 carrying cbi This workpHP140 pDS2 cua (D575–583) This workpHP141 pDS2 cua (575 SALIAFGL 582) This workpDS101 pDS2 cua (T575S) This workpDS102 pDS2 cua (F576A) This workpDS103 pDS2 cua (A577L) This workpDS104 pDS2 cua (M578I) This workpDS105 pDS2 cua (L579A) This workpDS106 pDS2 cua (G580F) This workpDS107 pDS2 cua (V581G) This workpDS108 pDS2 cua (F582L) This workpHP131 pJBS636 cui This workpHP132 pJBS636 cui68-blaM This workpHP133 pJBS636 cui90-blaM This workpHP134 pJBS636 cui139-blaM This workpHP135 pJBS636 cui174-blaM This work

FIG. 1. Hydrophobic hairpin sequences (helices 8 and 9) of the channel-forming domains of the E1-type (colicins E1, 5, K, 10, Ia, and Ib) and A-type (colicins B,N, U, and A) colicins and of bacteriocin 28b. The hydrophobic amino acids are indicated in boldface, and the a helices of colicin E1 (4) and colicin A (16) are indicatedschematically above and below the protein sequences, respectively. The determined helical segments and the derived helices are boxed and shaded. The excised hairpintip of colicin U (solid line) and the corresponding sequence of colicin B (interrupted line) are boxed.

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35% identical, they may determine the specificity for the im-munity proteins (Fig. 1). To investigate whether the tip of thecolicin B hydrophobic hairpin specifies interaction with thecolicin B immunity protein, the tip of the colicin U hairpin wasreplaced by the tip of the colicin B hairpin (SALIAFGL), whichresulted in colicin U(575–582ColB). Transformation of E. coli5K with plasmid pHP141 Col U(575–582ColB) yielded unsta-ble cells, similar to transformants carrying pHP140. Therefore,strain HP87 was transformed with pHP141, and the crude co-licin extract was tested on sensitive E. coli 5K cells; colicinU(575–582ColB) was as active as wild-type colicin U (Table2). The colicin B immunity protein reduced colicin U(575–582ColB) activity 10-fold (Table 2). The SALIAFGL sequencewas also recognized by the colicin U immunity protein, whichreduced the activity of colicin U(575–582ColB) 10-fold (Table2). Since the colicin B immunity protein did not fully inactivatecolicin U(575–582ColB) and the colicin U immunity proteindid not inactivate colicin B, either the SALIAFGL sequence isnot the only recognition site for the colicin B immunity proteinor SALIAFGL assumes somewhat different conformations inthe two colicins.

To investigate which of the eight inserted amino acids ofcolicin U(575–582ColB) are responsible for the interactionwith the immunity proteins, single point mutations were intro-duced in the tip of the hydrophobic hairpin of colicin U (Table1). The eight mutants isolated were fully active on E. coli 5Kcells (Table 2). Colicins U1, U3, U4, U5, and U7 were inacti-vated by the colicin U immunity protein to the same extent aswild-type colicin U (Table 2). Colicin U6 was less inactivatedby the colicin U immunity protein (Table 2) and renderedE. coli 5K[pDS106 cua (G580F) cui cul] unstable. Colicin U2cross-reacted with the colicin B immunity protein, which indi-cates that the F576A replacement altered the immunity spec-ificity. Although E. coli 5K(pDS4 cui cul) was also not fullyimmune to colicin U2 (Table 2), no instability was observedafter transformation with DS102 cua (F576A). All mutant co-licin U proteins were synthesized in similar amounts andshowed the expected size, as determined by SDS-PAGE (Fig.2). The unknown band below the colicin U proteins wasformed in the in vitro protein synthesis system used and wasalso observed previously (22).

Topology of the colicin U immunity protein (Cui) in the cy-toplasmic membrane. The hydropathy profile of the Cui im-munity protein shows four hydrophobic segments that are pre-dicted to form four a helices across the cytoplasmic membrane(Fig. 3). To support this model, hybrid proteins between the

colicin U immunity protein and b-lactamase (BlaM) were con-structed. Only hybrid proteins with fusion sites located in theperiplasm should allow cells to grow as single colonies on nu-trient agar plates supplemented with at least 5 mg of ampicil-lin/ml (2). The Cui68-BlaM hybrid in loop 1 conferred resis-tance to 200 mg of ampicillin/ml, whereas cells expressing theCui-BlaM hybrids with fusion sites at positions 90 and 174formed no colonies on plates containing 5 mg of ampicillin/ml.Cells that synthesized the Cui139-BlaM hybrid were resistantto 100 mg of ampicillin/ml, indicating a localization of residue139 of Cui in the periplasm. The transmembrane topology ofCui corresponds to that of the colicin A immunity protein(Cai) (8), with which it shares 45% sequence identity (22).When examined by SDS-PAGE (Fig. 4), the hybrid proteinseach displayed the molecular mass as calculated consideringthe fusion sites in the Cui immunity protein and the matureform of the BlaM b-lactamase. There were also proteolytic deg-radation products of the hybrid proteins (bands below the hy-brid proteins) and bands corresponding to the resistance pro-teins.

The colicin U immunity protein confers cross-immunity tocolicin A but not to colicin B (22). The immunity of Cui shouldtherefore be specified by amino acids that are common to Caibut not to Cbi. There are only 30 such residues, and they aredistributed along the entire Cui sequence (data not shown),

TABLE 2. Sensitivity of E. coli strains to colicin U and colicin U mutants

Colicin Amino acid sequenceaColicin activityb

5K 5K(pDS4 cui) 5K(pHP81 cbi)

Colicin U-WT 575 T F A M L G V F S583 2 i 2Colicin U(D575–583) 575 - - - - - - - - - 583 2 2 2Colicin U(575–582ColB) 575 S A L I A F G L S 583 2 1 1Colicin U1 575 S F A M L G V F S583 2 i 2Colicin U2 575 T A A M L G V F S583 2 (1) 1Colicin U3 575 T F L M L G V F S583 2 i 2Colicin U4 575 T F A I L G V F S 583 2 i 2Colicin U5 575 T F A M A G V F S 583 2 i 2Colicin U6 575 T F A M L F V F S 583 2 0 2Colicin U7 575 T F A M L GG F S 583 2 i 2Colicin U8 575 T F A M L G VL S 583 2 i 2

a Identities are indicated in boldface.b A 102-fold-diluted colicin sample yielded a clear zone of growth inhibition. 0, undiluted colicin solution; i, immune. (1), both an undiluted and a 10-fold-diluted

colicin sample yielded a turbid zone of growth inhibition. The experiments were repeated at least three times with the same results.

FIG. 2. SDS-PAGE of radiolabeled colicin U proteins. The genes of colicinU (lane 1), colicin U(D575–583) (lane 2), colicin U(575–582 ColB) (lane 3), andthe colicin U mutant proteins U1 to U8 (lanes 4 to 11) were synthesized in vitro.The 25-kDa bands (marked by a star) represent the chloramphenicol transacety-lase encoded on pBCSK1. The arrow indicates the position of the colicinproteins. Numbers on the right indicate positions of molecular mass standards inkilodaltons.

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indicating that different parts of the Cui protein may partici-pate in the specific recognition of the colicins.

DISCUSSION

In this study, we showed that residues 575 to 583 (designatedthe tip) of the hydrophobic hairpin are not required for colicinU activity. Deletion of nine residues which included five resi-dues of the predicted helix 8 and the loop between helix 8 andhelix 9 (Fig. 1) resulted in a fully active colicin. Full activity ofcolicin U(D575–583) demonstrates that deletion of the tip doesnot alter the relative arrangement of helices 8 and 9 and of theother membrane helices in a way that prevents pore formation,which includes binding to the cytoplasmic membrane, unfold-ing, insertion into the membrane, and assembly of the helicesto a pore. The length of the truncated helix 8 is similar to thelength of helix 8 of the E1-type colicins (Fig. 1). Helix 9 couldalso be shortened when helix 9 residues are included in theconnecting loop of the deletion derivative. Either full-lengthhelices of 17 and 18 residues required to span a lipid bilayerare not necessary for pore formation of the A-type colicins andof the E1-type colicins or the helices lengthen upon binding tothe membrane (3, 4). Replacement of the eight colicin Uresidues by eight colicin B residues did not alter colicin Uactivity, which supports the conclusion that there is no strictrequirement regarding the length and the sequence of the tip.

Deletion of residues 575 to 583 abolished inactivation ofcolicin U by the colicin U immunity protein. Lack of immunityto colicin U(D575–583) probably indicates recognition of thetip sequence, or a portion of it, by the immunity protein.Replacement of the 575 to 582 region by the correspondingregion of colicin B expanded immunity specificity in that theresulting colicin U(575–582ColB) was inhibited by the colicin

B immunity protein and by the colicin U immunity protein.This result indicates that the tip represents an important im-munity-conferring region of colicin U, and correspondingly ofcolicin B, but it is not the only specificity-determining regionbecause of the cross-immunity. Analysis of single point muta-tions in the tip of the hairpin of colicin U revealed that phe-nylalanine 576 contributes to immunity specificity since its re-placement by alanine in colicin U2 caused cross-immunity withthe colicin B immunity protein and a decrease in inhibitionby the colicin U immunity protein. The G580F substitutionin colicin U6 somewhat lowers immunity conferred by thecolicin U immunity protein but does not alter the immunityspecificity. Reduction of specificity and reactivity may becaused by a distortion of the colicin B tip conformation incolicin U, and/or colicin regions outside the tip contribute tothe interaction with the immunity protein. Furthermore, dele-tion of the tip may affect the conformation of a binding regionof the immunity protein outside the tip which results in areduced reactivity and specificity with the immunity protein.Apart from the unsolved molecular details, our data lead us topropose that the tip of the hydrophobic hairpin in colicin Uand in colicin B represents a major determinant for the inter-action with the corresponding immunity proteins. We considerthese results as representative for the interaction of the A-typecolicins with their immunity proteins.

The killing activity of a series of colicin A-colicin B chimericproteins on immune indicator strains has revealed the hydro-phobic region between amino acids 530 and 577 as the mainimmunity-specifying determinant of the colicin A sequence (7).Our data confine the region that specifies interaction with theimmunity protein to the tip of the hairpin of colicins U and B.A nontoxic derivative of colicin A bound to the cytoplasmicmembrane does not form an open channel but reacts with theimmunity protein, which shows that the immunity protein caninactivate the colicins without opening the channel (5, 6). Incolicin E1, which represents another type of pore-forming co-licins, amino acid substitutions of residues 440, 443, 444, 474,and 477, located in the segments between helices 5 and 6 andhelices 7 and 8, respectively (4), reduced protection by theimmunity protein (28). For colicin 5 (E1-type colicin), we haveshown that residues 405 to 424 of helix 6, corresponding toresidues 437 to 456 of colicin E1, are important for the inac-tivation by the immunity protein (17). Since the site of inter-action on the immunity protein is located in the cytoplasmic

FIG. 4. SDS-PAGE of [35S]methionine-labeled wild-type Cui (lane 3),Cui68-BlaM (lane 4), Cui90-BlaM (lane 5), Cui139-BlaM (lane 6), and Cui174-BlaM (lane 7) (indicated by arrows) expressed in E. coli BL21 transformed withthe corresponding plasmids. Lane 1 shows the neomycin phosphotransferase(indicated by a dot) expressed by the vector plasmid pJBS636; lane 2 shows theprecursor and the processed form of b-lactamase (BlaM) (indicated by stars).Numbers on the right indicate positions of molecular mass standards in kilodal-tons.

FIG. 3. Predicted arrangement of the colicin U immunity protein in the cy-toplasmic membrane of E. coli. H1, H2, H3, and H4 denote the transmembranea helices; L1, L2, and L3 denote loops. PP, periplasmic space; CM, cytoplasmicmembrane; CP, cytoplasm. Arrows and numbers indicate the locations of thefusion sites of the constructed Cui-BlaM hybrid proteins.

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loop and the inner leaflet of the cytoplasmic membrane (17),residues 405 to 424 of colicin 5 have to be deeply inserted inthe cytoplasmic membrane. Apparently, E1-type and A-typecolicins differ with regard to the membrane locations of theinteraction sites with the cognate immunity proteins, and thereacting interhelical loops are located in different regions ofthe E1-type colicins and the A-type colicins.

The amino acid sequence of bacteriocin 28b (probably iden-tical to colicin L) of Serratia marcescens displays the highestsequence similarity to colicin A (26). However, it lacks fiveresidues in the tip of the hairpin (Fig. 1) which, according tothe results presented in this paper, it would not need becauseproducer cells are not protected by an immunity protein (25).

With the identification of the hydrophobic hairpin tip as asite of interaction with the immunity protein, the location ofthe tip within the membrane gains interest. During the multi-step membrane insertion process, the location of the tip mostlikely differs after the voltage-independent primary insertion ofthe hydrophobic hairpin and after the subsequent response tothe transmembrane voltage. The known transmembrane ar-rangement of the immunity protein and the identification ofthe site of interaction on the immunity protein should allowdetermination of whether A-type colicins are inactivated priorto or after pore formation. b-Lactamase hybrid proteins re-vealed four transmembrane segments of the colicin U immu-nity protein and that the N and C termini of the immunityprotein face the cytoplasm. This arrangement agrees with thetransmembrane topology of the colicin A immunity protein (8)and suggests that all immunity proteins of A-type colicins dis-play similar structures.

ACKNOWLEDGMENTS

We thank K. Hantke for fruitful discussions and K. A. Brune forcritical reading of the manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft(SFB 323, project B1, foreign guest grant to D.S.; Graduiertenkolleg“Mikrobiologie,” fellowship to H.P.).

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