Proc. Nall. Acad. Sci. USAVol. 89, pp. 6210-6214, July 1992Microbiology

Characterization of a periplasmic thiol:disulfide interchangeprotein required for the functional maturation of secretedvirulence factors of Vibrio choleraeJOEL A. PEEK AND RONALD K. TAYLORDepartment of Microbiology and Immunology, University of Tennessee, Memphis, TN 38163

Communicated by Stanley Falkow, March 26, 1992

ABSTRACT A number of ToxR-regulated genes that en-code products required for the biogenesis or function of thetoxin-coregulated colonization pilus (TCP) of Vibrio cholraehave been identified previously by TnphoA fusions. In thisstudy we have examined the role of the product of one of thesegenes, tcpG, to which a fusion results in a piliated cell lackingall ofthe in vivo and in vitro functions associated with TCP. Ourresults show that TcpG is not an ancillary pilus adhesincomponent as suggested by the mutant phenotype but insteadis a 24-kDa periplasmic protein that shares active-site homol-ogy with several different bacterial thioredoxins and proteindisulfide isomerase, as well as overall homology with thedisulfide bond-forming DsbA periplasmic oxidoreductase pro-tein of E. coli. Corresponding activity can be demonstrated invitro for TcpG-enriched fractions from a wild-type strain but isabsent in a similarly fractionated tcpG-phoA mutant. Thephenotype conferred by a tcpG mutation was found to bepleiotropic in nature, also affecting the extracellular secretionof cholera toxin A subunit and a major protease. This suggestsa general role for TcpG in allowing a group of virulence-associated (and perhaps other) proteins that contain diulfidebonds to assume a secretion or functionally competent state.

The gram-negative bacterium Vibrio cholerae is the causativeagent of cholera, an acute diarrheal disease that can lead todeath through severe dehydration with resultant ion imbal-ance and hypoglycemia. These disease manifestations are theresult of the organism's ability to colonize the epithelialsurface of the small bowel and elaborate a potent exotoxinthat mediates the ADP-ribosylation ofthe guanine nucleotidestimulatory (G.) subunit of host adenylate cyclase (1). Ex-pression of cholera toxin and at least one of the colonizationfactors, the toxin coregulated pilus (TCP), is coordinatelyregulated within the ToxR virulence regulon (2, 3); ToxR isa transmembrane protein that activates transcription of thecholera toxin operon. TCP is a polymer composed of a20.5-kDa major pilin subunit encoded by the tcpA gene. Aseries ofTnphoA insertion mutations has defined a number ofgenes in addition to tcpA that are required for pilus assemblyand function (4, 5); TnphoA is a derivative of TnS used tocreate fusions between target genes and phoA, the Esche-richia coli gene for alkaline phosphatase. So far, all of thesegenes are coordinately regulated by ToxR, and all but one(tcpG) are closely linked and oriented in the same direction,perhaps forming an operon. A TnphoA insertion in tcpGresults in a strain that elaborates pili that appear morpholog-ically normal and yet are incapable of mediating any TCP-attributable functions. As such, 0395-derived tcpG mutantstrain KP8-% no longer autoagglutinates when grown underTCP-expressing conditions in culture, fails to mediate fucose-resistant hemagglutination, and is colonization defective to

the same extent as a tcpA mutant in infant mouse studies (4,6). This phenotype and the identification of tcpG by phoAfusion suggested that the tcpG gene product might function asa secreted pilus-associated adhesin molecule, similar to thatdescribed for other pilus systems such as type 1 fimbriae orP pili (for review, see ref. 7).The findings presented here contradict the notion that

TcpG is an adhesin molecule and instead characterize it as aperiplasmically localized protein capable of mediating thiol-disulfide interchange reactions. The tcpG mutation was alsofound to be pleiotropic, affecting the secretion of at least oneother ToxR-regulated protein, cholera toxin itself. Se-quence* homology with another oxidoreductase molecule,DsbA, which affects disulfide bond formation, suggests thatTcpG may be a member of a family of periplasmic proteinsthat assist in the conformational maturation of secretedproteins containing disulfide bonds.

MATERIALS AND METHODSCloing the tcpG-phoA Fusion and Intact Gene. Chromoso-

mal DNA isolated from strain KP8-96 was digested withBamHI, ligated into similarly digested pBR322, and trans-formed into E. coli strain MC1061 (8-10). Transformants wereselected on agar containing kanamycin (45 ,g/ml), ampicillin(100 ,.g/ml), and the alkaline phosphatase chromogenic sub-strate X-P (5-bromo-4-chloro-3-indolyl phosphate; 40 Itg/ml).Two antibiotic-resistant blue colonies resulted. Both recom-binant plasmids were shown by restriction analysis to containa 6.7-kilobase (kb) BamHI fusion fragment, of which 1.7 kbwas Vibrio DNA and the remainder was from TnphoA. Theplasmid used throughout this study is referred to as p8-96.1.The intact gene was cloned by inserting an antibiotic cartridgeadjacent to the tcpG coding region on the Vibrio chromosome.The antibiotic cartridge was utilized as a cloning markerresulting in the tcpG clone pATG1 (unpublished data).DNA Sequence Determination. The BamHI fragment of

p8-96.1 and the BamHI/Pst I fragment of pATG1 weresubcloned into the appropriate restriction sites of M13mpl8and transformed into JM103 (11) derivative strain JF626 (J.Felton). Additional subclones generated in both M13mpl8and M13mp19 were used to determine the DNA sequencefrom both strands by the dideoxynucleotide chain-termination method with the universal lac, phoA, and addi-tional 20-base-pair (bp) synthetically generated primers. Se-quence analyses were performed utilizing Wisconsin Genet-ics Computer Group Algorithms (12).

Antibodies Dircted Apinst TcpG. Kyte and Doolittleanalysis (13) indicated a strong hydrophilic peak correspond-ing to residues 108-130 of the predicted amino acid sequenceofthe tcpG open reading frame (see Fig. 4B). A 24-amino acidpeptide, peptide 1, corresponding to this region plus a C-ter-

Abbreviations: TCP, toxin-coregulated pilus; X-P, 5-bromo-4-chloro-3-indolyl phosphate; PDI, protein disulfide isomerase.*The sequence reported in this paper has been deposited in theGenBank data base (accession no. M93713).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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minal cysteine residue to facilitate keyhole limpet hemocy-anin (KLH) coupling, was synthesized on an Applied Bio-systems peptide synthesizer. The KLH-coupled TcpG pep-tide was resuspended in phosphate-buffered saline (PBS;0.154 M NaCl/0.67 M KCl/1.9 mM Na2HPO4/0.1 mMKH2PO4) and emulsified at a 1:1 ratio with Freund's completeadjuvant or with Ribi's adjuvant according to reconstitutioninstructions provided by Ribi Immunochem. Rabbits werebled for preimmune sera and then immunized with 150-200,Ag of the antigen. After a routine immunization protocol, therabbits were bled and then administered a booster with75-150 ,ug of antigen solubilized in PBS (for Freund's rabbit)or Ribi's adjuvant (for Ribi's rabbit) (14). Either antiserumwas used to detect TcpG by Western immunoblot (15), andboth are collectively referred to as "anti-TcpG antiserum."

Purification of TcpG. Cultures of 0395 were grown in LBmedium (pH 6.5) at 30'C (TCP-expressing conditions) to anoptical density of 1.7-1.9 at 600 nm as described (2). Thesecells were chilled on ice and pelleted at 10,000 x g for 10 min.Cells were then resuspended in cold PBS at a 20-fold concen-tration. A stock solution of polymyxin B sulfate at 10 mg/mlin PBS was added to the cells to a final concentration of 2mg/ml. This mixture was gently stirred in an ice bath for 10-12min. Polymyxin B-treated cells were removed by centrifuga-tion at 10,000 x g for 10 min. The supernatant was thendialyzed against 10 mM Tris.HCl/1 mM EDTA, pH 6.8,overnight at 4°C and concentrated about 4-fold by ultrafiltra-tion using an Amicon PM10 membrane at 50 psi (1 psi = 6.89kPa) of N2 at 4°C. The retentate was applied to a column ofDEAE (DE 52) cellulose (2.6 x 15 cm) that had been equili-brated with 10 mM Tris-HCl/1 mM EDTA, pH 6.8 at 4°C.These ionic conditions were such that TcpG was eluted withthe flow-through of the DEAE column. Flow-through frac-tions were analyzed by Western blot, and TcpG-containingfractions were pooled and concentrated as above. The con-centrated TcpG fractions were applied to a column of G-100Sephadex (1.6 x 96cm) that had been equilibrated with 50mMTris HCl/1 mM EDTA, pH 6.8. Fractions of 1 ml each werecollected at a flow rate of 12 ml/hr. TcpG-containing fractionswere identified by Western blot and by insulin assay.

Insulin Assay. The catalyzed reduction of insulin in thepresence of dithiothreitol was measured turbidimetrically at600 nm as described (16). Reaction mixtures contained 500 ,ulofinsulin at 1 mg/ml in 0.1M potassium phosphate buffer, pH7.0/2 mM EDTA and 3-20 ug of sample. Water was addedto a final volume of 1 ml. The reaction was started with theaddition of 1-5 ,ul of 100 mM dithiothreitol. Measurementswere taken at 60-sec intervals for 60-80 min. Measurementswere taken again at -24 hr to assure that equivalent reducingpotential was contained in all of the samples. Slopes of thedithiothreitol control and the reaction samples were deter-mined, and activities were calculated as described (16). Theassay parameters were first worked out by utilizing lyophi-lized E. coli thioredoxin (Sigma) that had been resuspendedin 0.1 M potassium phosphate buffer, pH 7.0/2 mM EDTA.Once optimized, the assay indicated a thioredoxin activity of2.9 OD/min per mg of protein slightly below the activityreported by Sigma of 3.0-5.0 OD/min per mg.

RESULTSIdentification of TcpG. Antiserum prepared against syn-

thetic TcpG peptide 1 was used to identify TcpG in total-cellprotein extracts. Samples prepared from strains RT110.21(tcpA-phoA), iB1 (toxR-), 0395 (wild type), and KP8-%(tcpG-phoA) were separated by SDS/PAGE and analyzed byWestern immunoblot (Fig. 1). A 24-kDa protein recognizedby the TcpG antibodies was detected in RT110.21 and 0395protein extracts and to a lesser extent in iB1. This apparentregulation by ToxR is consistent with the manner in which thetcpG gene was originally identified (4). The KP8-% lane

A B C D EW -92.5

TcpG-PhoA -*-Fusion

Fusion toDegradation

TcpG 4-N

Migration -* -

Front

-46

iA -30

-21.5

_ -14.3

FIG. 1. Immunoblot identification of TcpG. Whole-cell extractswere resolved by SDS/PAGE and probed with anti-peptide 1 TcpGantiserum. Extracts in lanes are from the following strains: A,RT110.21 (tcpA); B, iB1 (toxR); C, KP8-96 (tcpG-phoA); and D,0395 (wild type): Lane E shows molecular mass markers in kDa.

lacked the 24-kDa protein and instead expressed a 64-kDaprotein that cross-reacted with both anti-TcpG and anti-alkaline phosphatase antisera (data not shown).

Subcellular Locliation of TcpG. Since the anti-peptideantibody appeared to be specific for TcpG, it was utilized tolocalize the TcpG protein. If TcpG were to function as anadhesin molecule as was initially expected, it should beexposed on the cell surface. Repeated attempts utilizingwhole-cell ELISA, immunofluorescence, and immunoelec-tron microscopy of intact bacteria failed to detect TcpG onthe exterior of the bacteria (data not shown). This suggestedthat TcpG is not surface-exposed or that the native epitopesare inaccessible or unrecognizable by the peptide-generatedantibodies. Since phoA fusion data indicated that the TcpGmolecule was exported beyond the cytoplasm, a fractionationtechnique with polymyxin B sulfate was used to localizeTcpG to either the periplasm or the membrane fraction (4,17). Cells were washed and then treated with polymyxin B,allowing the periplasmic contents to be solubilized. Poly-myxin B-treated cells were pelleted by centrifugation, leavingthe periplasmic contents in the supernatant. The pellet andsupernatant were then examined for the presence ofTcpG. AWestern blot of polymyxin B-fractionated 0395 probed withanti-TcpG antibodies showed that all of the detectable TcpGwas released from the periplasm and contained in the super-natant fraction (Fig. 2). Fig. 2 also shows a duplicate blotprobed with anti-TcpA antibodies that shows TcpA in thepellet fraction. Thus, TcpA and TcpG are separated indifferent fractions, further suggesting that TcpG does notfunction as an adhesin molecule but rather works by modu-lating TCP function, perhaps at a step during pilus assembly.The periplasmic location of TcpG was also demonstrated byAnti-TcpA

mspAnti-TcpGm

IFIG. 2. Immunoblot of polymyxin B-frac-

tionated 0395. Washed cells were treated withpolymyxin B and then centrifuged at 16,000 x gfor 10 min to separate the periplasmic contents(supernatant) from the treated cells (pellet).Proteins from corresponding fractions (lanes Sand P) were resolved by SDS/PAGE and sep-arately probed with anti-TcpA (Left) or anti-TcpG (Right) antisera. Immunoblots were over-

developed to enhance detection of traceamounts of antigen.

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immunoelectron microscopy of Lowicryl thin sections ofstrain 0395 and Western analysis after EDTA-lysozymefractionation (data not shown).

Purification of TcpG. To investigate potential activities ofTcpG by in vitro assays, the protein was purified from theperiplasmic space as described in Materials and Methods.Absorbance profiles from the G-100 elution of KP8-% and0395 samples were similar, with peak material consistentlybeing eluted at characteristic molecular weights. Only theamplitude of these peaks varied from run to run, possiblybecause of slightly differing culture conditions. While theSDS/PAGE protein profiles ofcorresponding peaks from bothstrains appeared to be identical (Fig. 3 Left), a duplicate gel runsimultaneously and analyzed by Western blot with anti-TcpGantibodies showed a 24-kDa band for 0395 (lanes M-Q in Fig.3 Right) but no 24-kDa cross-reactive band in the correspond-ing lanes for KP8-96. Interestingly, while TcpG was thusapparently absent in KP8-96, other fractions from this straincontained either a 20-kDa or 29-kDa cross-reactive protein(Fig. 3 Right, bands a and b). These bands have been notedpreviously in whole-cell samples of KP8-96, where the pre-dominate cross-reactive band is always the 64-kDa hybridprotein, with bands of lesser intensity from 20-35 kDa, sug-gesting that these smaller bands are degradation products ofthe hybrid protein since they are only seen in KP8-% andnever in 0395.TcpG Thiol-Disulfide Interchange Activity. Comparison of

the predicted amino acid sequence of TcpG to entries in theSwiss protein data base using the TFASTA algorithm (18)revealed homology to the reactive redox site of protein disul-fide isomerase (PDI) (19) and several different bacterial thiore-doxins (20) (Fig. 4A), suggesting that a similar activity mightbe attributable to TcpG. The most widely used method formonitoring the activity of E. coli thioredoxin during isolationis an insulin assay developed by Holmgren (16) that spectro-photometrically records the precipitation of the insoluble Bchain that is produced when the interchain disulfide bonds ofinsulin are reduced. For strains KP8-% and 0395, G-100elution profiles were used to choose samples from each of thecharacteristic elution peaks to be assayed for redox activity.Due to time-dependent loss of activity of the samples, theywere assayed as they were eluted from the G-100 column orshortly thereafter. Subsequent to the assay, the elution peaksfrom both strains appeared to contain identical protein profileswith the exception ofTcpG being absent in the KP8-% profile(Fig. 3). Based on the difference in the slope of the reactionsample to that ofthe dithiothreitol control, samples taken fromthe area ofthe 0395 elution profile that contained protein in the24-kDa range (corresponding to lane P in Fig. 3) showed anactivity of 2.7 OD/min per mg of protein (Fig. 5). Multiple

A B C D E F If1 I J K L M N 0 P Q R S

APDI: E K K N F Vf YAP WUGXQL.AThioredoxin: Ela D W iA E :1g3: P E M I A AATcpG: IIENShI D NKMIT*ADsbA: A G AuQ LIEHEE F ..U Y Qua@ E V

B1 MKKLFALVATLMLSVSAYAAQFKEGEHYQVLKTPASSSPWSEFFSFYCP 50

1 MEKIWLALAGLVLAFSASAAQYEDGKQYTTLEKPVAGAPQVLEFFSFFCP 50

51 HCNTFEPII... AQLKQQLPEGAKFQKNHVSFMGGNMGQAMSKAYATMIA 97

51 HCYQFEEVLHISDNVKKKLPEGVEOTKYHVNFMGGDLGKDLTQAWAVAMA 100

98 LEVEDKMVPVMFNRIHTLRKPPKDEQELRQIFLDEGIDAAKFDAAYNGFA 147

101 LGVEDKVTVPLFEGVQK. TQTIRSASDIRDVTINAGIKGEEYDAAWNSFV 149

148 VDSMVRRFDKQFQDSGLTGVPAVVVNNRYLVQGQSVKS....,.LDEYFD 191

150 VKSLVAQQEKAADVQLRGVPAMFVNGKYQLNPQGMDTSN)VFVQQYAD 199

192 LVNYLLTLK 200

200 TVKYLSEKK 208

FIG. 4. (A) Active site homologies. TcpG residues 36-58 arealigned with the region surrounding the catalytic site (boxed) of ratliver PDI (19), thioredoxin from Corynebacterium nephridii (20), andthe highly related E. coli DsbA protein (21). (B) Overall homologybetween TcpG (top line) and DsbA was determined by the algorithmof Needleman and Wunsch (22). Vertical lines show identity;comparison value 2 0.50; ., comparison value 2 0.10.

0395 purification analyses yielded thio-disulfide interchangeactivities from 2.7 to 4.2 OD/min per mg. This activityremained constant with a doubling ofthe sample concentration(data not shown). Thus, the "thioredoxin-like" reactive site ofTcpG can function as a thiol-disulfide interchange site. Sam-ples taken from analogous areas of the KP8-96 protein profilehad no activity (Fig. 5).

It was noted that one KP8-96 sample, corresponding tolane C in Fig. 3, had increased performance (0.64 OD/min permg) over similar nonactive 0395 fractions. Subsequent West-ern analysis revealed that the assayed sample contained theprobable degradation product of the fusion protein describedabove (Fig. 3 Right, band a). Since the fusion protein containsthe redox site from TcpG, the activity of this sample may beattributable to this degradation product or to a compensatoryoverexpression of another redox molecule or redox reactionintermediary.Plelotpic Nature of the tcpG-phoA Fusion Mutation. The

thiol-disulfide interchange activity of TcpG led us to investi-gate whether another ToxR-regulated disulfide-containingmolecule, cholera toxin itself (23), was affected by a tcpG

A B C D F F G HI I J K L IM N o P Q R S

a o

-TcPG TcpGx * -b *-21.5 v

FIG. 3. 0395 and KP8-96 G-100 gel filtration protein profiles. Samples were resolved by SDS/PAGE and stained with Coomassie blue (Left)or were transferred to nitrocellulose and probed with anti-TcpG (Right). Lanes: A, a polymyxin B supernatant from 0395; B-J, KP8-96 elutionfractions; K-S, corresponding 0395 elution fractions.

Proc. Natl. Acad. Sci. USA 89 (1992)

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0

0.2-0

0.1

0 10 20 30 40 50 60

Time, min

FIG. 5. Oxidoreductase activity assay of 0395 and KP8-% gelfiltration samples. Each reaction mixture contains 500 /4I of insulinat 1 mg/ml to which -3 itg of protein sample was added, and thevolume was then adjusted to 1 ml. The reactions were started withthe addition of 1 A.l of 100 mM dithiothreitol to the reaction cuvette,and the OD was recorded at 600 nm for 60 min. o, DTT control; *,0395 sample corresponding to lane P of Fig. 3; and *, correspondingKP8-96 sample.

mutation. To assess the effects of TcpG on toxin, cultures ofKP8-96 and 0395 were grown to an equivalent OD at 600 nmunder toxin-expressing conditions. Both whole-cell and su-

pernatant samples were resolved by SDS/PAGE and analyzedby Western blot (Fig. 6) with polyclonal anti-holotoxin anti-serum (provided by J. Mekalanos). Most notable was thefinding that the toxin A subunit profiles were markedly dif-ferent between the two strains. The A subunit of 0395 wasfound in the unnicked A form in the whole-cell extracts, andboth the unnicked A and Al forms in the supernatant. KP8-96,on the other hand, showed elevated levels ofunnicked A in thecellular extracts, but reduced levels of secreted unnicked Aand virtually no Al form in the supernatant. In contrast thesecreted B subunit appeared to be at wild-type levels or abovefor strain KP8-96. Toxin secretion remains defective in a tcpGnull mutant as well (unpublished data), indicating that the toxinA subunit secretion defect seen in the tcpG-phoA mutant is not

a b c d

FIG. 6. Comparison of KP8-96 and 0395 toxin profiles. KP8-%and 0395 cultures were centrifuged at 10,000 x g for 10 min to pelletcells. The culture supernatants were concentrated -20-fold by usingan Amicon PM10 membrane at 50 psi at 40C. Proteins from thesupernatant and from whole-cell samples were resolved by SDS/PAGE and immunoblotted with anti-whole toxin antibodies. Lanes:a, 0395 whole-cell sample; b, 0395 culture medium; c, KP8-%whole-cell sample; d, KP8-96 culture medium.

due to a general secretion defect caused by the improperlocalization of the TcpG-PhoA hybrid protein.The tcpG mutant was also found to be defective in protease

secretion. This was evident from growth of the wild-type andmutant strains on casein agar, where the parent caused asubstantial clearing around the colonies, that was barelyevident around equally sized colonies of KP8-96 or a tcpGdeletion/insertion mutant strain (data not shown).Homology Between TcpG and DsbA. Recently, a periplasmic

protein named DsbA has been identified in E. coli that alsoshares primary structural homology with the active site ofdisulfide oxidoreductases. Furthermore, this protein has beenshown to be required for the efficient disulfide bond formationof several periplasmic and outer membrane proteins in vivo.Like TcpG, DsbA can also catalyze the reduction of insulin invitro (21). Comparison of the amino acid sequences of TcpGand DsbA using the Needleman and Wunsch program (22)revealed 63% similarity, with a striking 40%o identity, betweenthe two proteins (Fig. 4B). Neither of these proteins sharessignificant homology with the other oxidoreductases beyondthe region surrounding the active site, suggesting that DsbAand TcpG form a distinct class of enzymes involved in theformation of disulfide bonds within the periplasm.

DISCUSSIONThe tcpG mutant strain KP8-96 elaborates TCP colonizationpili that appear morphologically normal by transmissionelectron microscopy of negatively stained samples, yet fail tomediate colonization in vivo, and are functionally deficient byseveral in vitro assays (4, 6). This phenotype suggested thattcpG would encode a surface or pilus-associated adhesinmolecule, or possibly a product that mediated pilus functionin some other manner. The findings presented here areconsistent with the latter interpretation. By using a variety oftechniques, no detectable TcpG was found on the exterior ofthe cell or associated with assembled TCP pili. Instead, TcpGappears to be localized exclusively to the periplasm. Thepredicted amino acid sequence of TcpG initially revealedhomology to both thioredoxin and PDI, centering around thethiol-disulfide interchange sites of these molecules. We wereable to show that the homologous redox site in TcpG canfunction in vitro with a level of activity comparable to that ofE. coli thioredoxin. Thus, TcpG might interact with targetproteins that contain disulfide bonds. Consistent with thishypothesis, the major TcpA pilin subunit contains an intra-chain disulfide bond (unpublished data; ref. 24). The aminoacid sequence encompassed by this disulfide bond is hydro-phobic in nature, and it is likely that this domain is exter-nalized, leading to the very hydrophobic characteristics ofthe pilus (2). This domain has been implicated in the functionof TCP-mediated colonization based on its recognition bymonoclonal antibodies that provide passive immunity toexperimental cholera (24). A similar carboxyl cystine loop inthe type 4 pilin of Pseudomonas aeruginosa PAK has alsobeen proposed to be an exposed adhesive domain (25, 26).Preliminary findings have indicated that KP8-96 pili areseveralfold less hydrophobic than wild-type pili, yet the pilinin these defective structures retains its disulfide bond (un-published data). The presence of the disulfide-bonded pilin inthe tcpG mutant may not be surprising, since atmosphericoxidation would still be expected to occur. Thus, the TCPdefect in the tcpG mutant may be attributable to the domainbetween the disulfide bond misfolding, resulting in the hy-drophobic area in this region being internalized.The properties of TcpG, coupled with the ToxR regulation

of its expression, prompted us to examine the effect of thetcpG-phoA fusion on other disulfide-containing, ToxR-regulated molecules such as cholera exotoxin, where both thetoxin A and B subunits contain disulfide bonds (21, 27).Western analysis of whole cholera toxin revealed a defect in

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the secretion of A subunit, while the B subunit was unaf-fected or present in greater than wild-type levels. Since theA subunit must associate with the B pentamer to be exported(28), it seems likely that the conformations required for thisassociation are not efficiently achieved in the tcpG mutant.The pleiotropic nature of a tcpG mutation was also seen toinclude a significant decrease in the amount ofactive secretedprotease by such mutants. A likely protease to be affected isthe soluble hemagglutinin/protease that contains four cys-teine residues and shares extensive homology with elastase ofP. aeruginosa (29) where these residues have been shown tobe disulfide-bonded (30).While the exact roles and mechanisms of TcpG function

are not yet defined, insight may be provided from thefunctions attributable to related thiol-disulfide interchangemolecules. For example, a protein with a homologous redoxsite, PDI, is a soluble multifunctional homodimer (2 x 57,000)found on the luminal side of the endoplasmic reticulummembrane (19). PDI has been shown to catalyze thiol-disulfide interchange reactions in protein substrates and hasalso been closely correlated to the process of secretoryprotein synthesis and secretion (31, 32). Thus, proteinsinvolved with disulfide bond formation may be important inmediating protein conformation during the export process.The idea that protein conformation is dictated solely by

amino acid sequence has long been considered the funda-mental principle behind protein folding (33). This model wasinitially reinforced by in vitro refolding studies of smallproteins following denaturation and may be analogous towhat usually occurs in the cytoplasm of cells. However,spontaneous protein folding based on primary structure failsto take into account the multiple physiochemical environ-ments encountered by translocated polypeptides or the ca-talysis of disulfide bonds at a physiological rate. In vitrofolding assays with proteins that contain disulfide bonds oftengive low yields (34). Thus, while bacterial disulfide-bondedproteins may spontaneously oxidize once they are secretedbeyond the cytoplasm, it may not occur at a physiologicalrate without catalysis by an oxido-reductase protein such asTcpG. Given the properties of the TcpA pilin in the tcpGmutant, TcpG may have two roles in polypeptide maturation,one as a thiol oxidant and possibly also as an isomerase/chaperone similar to BiP (binding protein) (35), where TcpGmay act to locate and guide portions of polypeptide chainsinto a state whereby complex surfaces can form. Thesesurfaces, such as the externalized hydrophobic domain ofTcpA, might otherwise be energetically unfavorable andwould rarely form under physiological conditions.This analysis suggests that the successful maturation of

many translocated polypeptides would require the assistanceof oxido-reductase enzymes on the distal side ofmembranes.The presence of a global class of periplasmic thiol-disulfideinterchange proteins is supported by the independent findingsof Bardwell et al., who have isolated the protein DsbA fromthe periplasm of E. coli that participates in disulfide-bondformation of several periplasmic and outer membrane pro-teins (21). As shown in Fig. 4, comparison ofthe E. coli dsbAgene product to TcpG reveals 40%o identity throughout theentire sequence, extending beyond the active site. Thisconservation of primary structure and function suggests thepresence of a unique class of bacterial periplasmic proteinsnecessary for the functional maturation of some secretedproteins and illuminates another step in the secretion path-way of bacterial proteins. Consistent with this function thetcpG gene product has a higher basal level than most ToxR-regulated gene products and is further induced under ToxR-expressing conditions (4). This response may reflect a cellularmechanism for maintaining the functional conformation ofexported proteins before and during times of high expression

of translocated polypeptides, which in this case represent acoregulated set of secreted virulence factors.

The authors thank L. Hatmaker, P. Hoffman, J. Katze, M.Kaufman, J. Mekalanos, and K. Peterson for contributing strains,reagents, equipment, and technical advice and for participating ininsightful discussions. We also thank J. Bardwell and J. Beckwith fordiscussing results prior to publication. This work was supported byU.S. Public Health Service Grant AI-25096 to R.K.T.; J.A.P. is therecipient of National Institutes of Health Predoctoral Training GrantAI-07238.

1. Mekalanos, J. J., Collier, R. J. & Romig, W. R. (1979) J. Biol.Chem. 254, 5849-5854.

2. Taylor, R. K., Miller, V. L., Furlong, D. B. & Mekalanos, J. J.(1987) Proc. Natl. Acad. Sci. USA 84, 2833-2837.

3. Miller, V. L., Taylor, R. K. & Mekalanos, J. J. (1987) Cell 48,271-279.

4. Peterson, K. M. & Mekalanos, J. J. (1988) Infect. Immun. 56,2822-2829.

5. Taylor, R. K., Shaw, C. E., Peterson, K. M., Spears, P. & Mekal-anos, J. J. (1988) Vaccine 6, 151-154.

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