Vol. 173, No. 23

Functional Complementation between Bacterial MDR-LikeExport Systems: Colicin V, Alpha-Hemolysin, and

Erwinia ProteaseMICHAEL J. FATH, RACHEL C. SKVIRSKY,t AND ROBERTO KOLTER*Department of Microbiology and Molecular Genetics, Harvard Medical School,

Boston, Massachusetts 02115

Received 13 June 1991/Accepted 12 September 1991

The antibacterial protein Colicin V (ColV) is secreted from gram-negative bacteria by a signal sequence-independent pathway. The proteins that mediate the export of CoIV share sequence similarities withcomponents from other signal sequence-independent export systems such as those for oa-hemolysin (Hly) andErwinia protease (Prt). We report here that the intact HlyBD export system can export active ColV fromEscherichia coli strains lacking the ColV export proteins CvaA and CvaB. The individual Hly export genescomplement mutations in their respective ColV homologs, but do so at a lower efficiency. When CvaA or CvaBis expressed along with the intact HlyBD exporter, the Cva export protein interferes with export of ColVthrough the HlyBD system. Gene fusions and point mutations in the ColV structural gene were used to definesignals in ColV recognized by the Hly exporter. An export signal in ColV recognized by HlyBD is localized tothe amino-terminal 57 amino acids of the protein. In addition, mutations in the ColV export signal differentiallyaffect export through CvaAB and HlyBD, suggesting differences in signal specificity between the Cva and Hlysystems. The three Erwinia protease export proteins can also export active CoIV, and interference is seen whenCvaA or CvaB is expressed along with the intact Prt exporter. Functional complementation is not reciprocal;a-hemolysin is not exported through either the CoIV system or the Prt system.

Much research on protein secretion has focused on Uinder-standing the classic, signal sequence-dependent export path-way (39, 41). In gram-negative bacteria, this pathway re-quires an export complex of at least five components (theproducts of the sec genes) and specifically exports proteinsthat contain an N-terminal signal sequence. Most proteinsexported through this system are destined for the periplasmor the outer membrane.

In both prokaryotes and eukaryotes, a growing number ofproteins and peptides that are exported by signal sequence-independent pathways have been identified. In prokaryotes,these exported proteins are typically localized to the extra-cellular medium, and in each case export is mediated by a

dedicated export system (38). A rapidly expanding family ofexport proteins that mediate the extracellular secretion ofproteins, peptides, and other molbcules has been described(3). This family has been termed the MDR (multidrug resis-tance)-like family (after the first eukaryotic member de-scribed [13, 20]), the ABC transporter superfamily (whichdescribes the ATP-binding domain common to all members[25]), and the HlyB translocator family (after the first pro-karyotic member described [10]).Each member of this family is an export protein with an

N-terminal hydrophobic domain with six (27) or eight (49)putative transmembrane regions and a C-terminal domaincontaining a highly conserved ATP-binding cassette (46).Most eukaryotic members of this family contain a tandemduplication of these domains. The prototype eukaryoticmember is the MDR protein found in mammalian cells.When overexpressed, this protein exports chemotherapeuticdrugs from tumor cells (7). Other members that contain the

* Corresponding author.t Present address: Biology Department, University of Massachu-

setts-Boston, Campus, Boston, MA 02125.

tandem duplication include pfMDR, which exports antima-larial drugs from Plasmodium falciparum (12); STE6, whichexports the a-type mating factor from Saccharomyces cere-

visiae (29, 32); and CFTR, the cystic fibrosis transmembraneregulator that is defective in cystic fibrosis patients (40).Eukaryotic members that do not have a tandem duplicationinclude PMP70, a peroxisomal membrane protein (26), and aset of genes expressed in the major histocompatibility com-plex class II region of the mammalian genome whose prod-ucts are implicated in antigen presentation (6, 35, 42, 44). Inthe eukaryotic systems studied to date, only one exportcomponent has been identified for each system, although forpfMDR there is evidence of a second component (11).The prokaryotic members of the MDR-like family are

much better characterized genetically. These export systemsall include a protein with a single hydrophobic domain andan ATP-binding cassette, and they all require at least oneadditional export component. The prototype prokaryoticsystem, a-hemolysin (Hly) in Escherichia coli, requires atleast three export components (10, 45, 47). The gene for theMDR-like export component in the Hly system, hlyB, isclosely linked to the gene for the second component, hlyD.The third gene, toIC, is unlinked and encodes an outermembrane protein (36) (Fig. 1). Other proteins exportedthrough MDR-like systems in prokaryotes include the he-molysins from Proteus vulgaris and Morganella morganii(28), the adenylate cyclase from Bordetella pertussis (17),leukotoxin from Pasteurella haemolytica (43), proteases Band C from Erwinia chrysanthemi (30), hemolysin-bacterio-cin from Enterococcus faecalis (14), alkaline protease fromPseudomonas aeruginosa (21), and a factor involved ingenetic competence in Streptococcus pneumoniae (24).We recently showed that export of the antibacterial pro-

tein colicin V (CoIV) from E. coli utilizes a transport systemsimilar to that of Hly (16). Figure 1 shows a comparison

7549

JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 7549-75560021-9193/91/237549-08$02.00/0Copyright © 1991, American Society for Microbiology

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7550 FATH ET AL.

FIG. 1. Comparison of the CoIV, ax-hemolysin, and Erwinia protease operons (10, 16, 30). The arrows denote the direction of transcriptionfor each operon as previously described (50). The value below each gene represents the number of nucleotides in each open reading frame.The export gene cvaB and its homologs, hlyB and prtD, are lightly shaded. The second export genes, cvaA, hlyD, and prtE, are darkly shaded.The third export genes, toiC and prtF, are vertically striped. The toiC gene is located on the E. coli chromosome at min 66.4 (2) and is arequired component for both the Hly and the ColV export systems (see the text).

between the CoIV, Hly, and Erwinia protease (Prt) operons.In the ColV system, the cvaC gene encodes the bacteriocinColV, cvi confers immunity to the producer cell, and cvaAand cvaB encode the dedicated export proteins. The cvaBgene encodes the MDR-like protein, and cvaA encodes thesecond component with sequence similarity to hlyD. TolC isalso required for export of ColV (16).To determine the significance of the observed sequence

similarities between these export systems, it is important toanalyze them for functional homology. Functional comple-mentation experiments comparing some MDR-like bacterialsystems have been performed previously. Experiments com-paring a-hemolysin with the hemolysins from P. vulgaris andM. morganii showed that hlyB and hlyD could be comple-mented in trans between the E. coli, P. vulgaris, and M.morganii systems both individually and as a pair (28).Functional complementation has also been observed be-tween a-hemolysin and the P. haemolytica leukotoxin ex-porter (43), and Masure et al. (31) present evidence that theB. pertussis adenylate cyclase may also be exported by theHlyBD system. The similarity in amino acid sequence be-tween these systems is very high (between 50 and 90%).When the degree of similarity falls, the level of complemen-tation falls precipitously. Significant complementation be-tween the Erwinia protease and the Hly system was notobserved, although very small amounts of protease B wereshown to be exported through Hly (4). More recently, thecylB gene encoding the E. faecalis hemolysin-bacteriocinexporter was shown to be unable to complement an hlyBdefect (14). The level of amino acid sequence similaritybetween these systems is significantly lower (under 30%),suggesting that there is a threshold level below which highlevels of transcomplementation will not occur. The alkalineprotease of P. aeruginosa should provide a good test case forthis hypothesis. Secretion levels of the alkaline protease arevery high when the Erwinia protease exporters PrtDEF are

provided in trans but much lower when HlyBD are providedin trans (21). Once the alkaline protease export genes aresequenced, one should be able to correlate amino acidsimilarity between PrtDEF and HlyBD with levels oftranscomplementation.We analyzed functional complementation between the

CoIV system and other bacterial MDR-like export systems,specifically Hly and Prt. The levels of amino acid identitybetween all these systems are below 30%, which is below thepresumed transcomplementation threshold level. We reporthere that export of active ColV does occur in cells carryingthe Hly or Prt export genes. In particular, the two Hly exportgenes can function together to complement mutations inboth ColV export genes. At a lower efficiency, the individualHly export genes can each complement mutations in itsrespective CoIV counterpart. Using various cvaC-phoA fu-sions and point mutations in the cvaC gene, we were able todelineate signals in ColV recognized by the Hly exporter.We found that complementation is not reciprocal for a-he-molysin; neither cvaAB nor prtDEF can complement anhlyBD defect. Last, we show that the ColV export defectresulting from a toiC mutation cannot be significantly com-plemented in trans by the Erwinia prtF gene. These resultsprovide a framework in which further complementationexperiments can be carried out between other MDR-likeexport systems.

MATERIALS AND METHODS

Media and culture conditions. Except where noted, LBwas used for both liquid and solid growth media (34).Antibiotics were used at the following final concentrations:ampicillin, 150 ptg/ml; chloramphenicol, 20 jig/ml; kanamy-cin, 50 ,ug/ml; and tetracycline, 12 ,ug/ml.

Bacterial strains and plasmids. Except where noted, thestrain used in this study was E. coli K-12 strain KS300,

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COMPLEMENTATION BETWEEN BACTERIAL EXPORT SYSTEMS 7551

supplied by K. Strauch and J. Beckwith. KS300 is MC1000recAl AphoA-pvuIl. CoIV assays were carried out on lawnsof E. coli 71-18, which is A(lac-proAB) thi supEiF' lacPlacZM15. ToIC- strains were generously provided by C.Wandersman.

Plasmids pHK11 and pHK22 were described previously(15). They were derived from pBR322 and pACYC184,respectively, and contain both ColV operons. Tn5 insertionsin pHK11 generated cvaA mutants 11-5 and 11-7 and cvaBmutants 11-2 and 11-6. Similarly, Tn5 insertions in pHK22generated cvaA mutants 22-2 and 22-8 and cvaB mutants 22-1and 22-11 (15). When plasmids pHK11-5 and pHK22-1 arecoexpressed, we see levels of export comparable to thoseseen from wild-type pHK11 or pHK22, providing evidencethat the TnS insertions in the upstream gene, cvaA, do nothave polar effects on the expression of cvaB. pLY21 is aderivative of pHK22 with an EcoRI deletion that removescvaA and most of cvaB. pLY11 is a pHK11 derivativecontaining functional cvaA and cvaB genes only (16).pHK817, provided by H. K. Mahanty, is a derivative ofpHK11 with an EcoRI-EcoRV deletion that removes all ofcvaA and all of cvaB. The CvaC57-PhoA and CvaC68-PhoAfusion-bearing plasmids have been described previously(16). The plasmids pWAM04 and pWAM554 were gener-ously provided by R. Welch. pWAM04 is a derivative ofpSF4000 containing the intact hemolysin operon cloned intothe HindIII site of pUC19 (50). pWAM554, constructed byS. Pellett, is a derivative of pSF4000 in which the uniqueApaI site within hlyD was altered, resulting in a truncatedhlyD gene. pMJF108, which was constructed for this study,is an hlyD+ derivative of pWAMO4 with an AatII deletionthat removes all of hlyCA and most of hlyB. When bothpWAM554 and pMJF108 are used to transform strainKS300, double transformants can produce and export fulllevels of a-hemolysin, showing that production of HlyB andHlyD from separate plasmids does not seem to affect theoverall levels of each protein. Plasmids pRUW4 and pHyl82were kindly provided by C. Wandersman. Plasmid pRUW4contains the Erwinia protease genes inh, prtD, prtE, andprtF cloned into the HindlIl site of pACYC184 (48). PlasmidpHyl82 contains the hlyB and hlyD genes subcloned intopACYC184 (18). Plasmid pMJF107 is an hlyC+A+ derivativeof pWAM04 that contains a HindlIl deletion of hlyD andmost of hlyB.

Manipulation of DNA. Isolation of plasmid DNA, transfor-mation of bacteria, restriction enzyme digestions, and otherroutine DNA manipulations were performed by using stan-dard procedures (1).CoIV assay. A plate assay was used to quantitate levels of

active ColV exported through the various export systems. A0.5-ml suspension of ColV-sensitive cells (E. coli 71-18) wasmixed with 3 ml of H-top agar (34) and spread on a plate ofM63-glucose (34). A single colony from the strain to beassayed was poked onto the lawn with a sterile toothpick,and the plate was incubated at 37°C overnight. A halo ofgrowth inhibition forms around the ColV-producing strain,and the area of the halo was used to quantitate the relativeamount of ColV export. The area of the zone of inhibitionwas used to calculate relative levels of ColV because CoIVdiffuses radially from the producer colony and produces acircular zone of inhibition around the producer. The mini-mum measureable area is limited by the size of the toothpickhole, which has a diameter of approximately 1.0 mm andrepresents 0.7 to 0.8% of the area of a wild-type ColV halo.This is the lower limit of the assay. For the results given

below and in Table 1, Fig. 2, and Fig. 3, all values areaverages of at least eight independent assays.Assay conditions can be manipulated to increase the sizes

of CoIV halos from ColV-producing strains. Under suchconditions, the ratios of halo diameter between strainsremain approximately constant. Thus CoIV halo diametershows a linear relationship with levels of exported ColVwithin the range found in these assays. Furthermore, ColVhalo sizes were found to be equivalent when the comple-menting genes were carried on plasmids with different copynumbers (pACYC184, pBR322, or pUC19 derivatives).Thus, the complementation assay does not strongly dependon gene dosage, and the absolute amount of exporter geneproduct is not rate limiting.Hemolysin assays. To assay export of a-hemolysin, the

colorimetric assay of Pellett was used (37). Briefly, strains tobe assayed were grown to an optical density (OD) of 0.7 to1.0 and pelleted, and supernatants from the strains werefilter sterilized with a 0.2-,um-pore-size acrodisc (GelmanSciences). The supernatants were used for measurement ofexternal hemolysin activity. To prepare cell lysates forinternal hemolysin assays, the cell pellets were resuspendedin 200 ml of Hly buffer (140 mM NaCl, 20 mM CaCI2), andthe cells were lysed by sonication. An assay mix of 200 RI of10% washed defibrinated sheep erythrocytes (Crane LabsInc., Syracuse, N.Y.), 600 [L of Hly buffer, and 200 ,u ofsupernatant or cell lysate was incubated in an Eppendorftube at 37°C for 30 min. Erythrocytes were pelleted, and therelease of hemoglobin was measured by spectrophotometricanalysis at a wavelength of 540 nm (OD540). The level ofhemolytic activity was quantitated as follows: -, OD540 of<0.020; +, OD540 of 0.020 to 0.300; + +, OD540 of 0.300 to0.500; + + +, OD540 of 0.500 to 1.000; and + + + +, OD540 of>1.000. For the results given above, all values are anaverage of at least four independent assays. Rapid hemolysinscreening was done by streaking the strain to be tested on aplate of tryptic soy agar with 5% sheep erythrocytes (CraneLabs Inc.).

Assay of alkaline phosphatase activity. To measure alkalinephosphatase activities of strains bearing CvaC-PhoA fu-sions, cells were grown in TB medium (10 g of tryptone and8 g of NaCl per liter) with chloramphenicol and ampicillin tothe stationary phase. Cultures were then diluted 10-fold andgrown in TB with antibiotics at 370C to the midlog phase.After growth, cultures were treated with 1 mM iodoaceta-mide to prevent spontaneous activation of cytoplasmic phos-phatase (5) and washed twice with 40 mM potassium-morpholinepropanesulfonic acid-10 mM MgCI2-1 mMiodoacetamide (pH 7.3). Washed cells were diluted fivefoldin the same buffer for OD6. determination. The rate ofp-nitrophenol phosphate hydrolysis was then measured asdescribed by Michaelis et al. (33), except that the assaybuffer was 1.0 M Tris-HCl (pH 8)-i mM ZnCl2. The exam-ples used in Fig. 2 were the results of two independent setsof assays showing full induction.

RESULTS

Hemolysin export proteins can export active ColV. Func-tional complementation was initially tested by introducing aplasmid containing the intact hemolysin operon into an E.coli strain carrying the ColV operon with TnS insertions ineither the cvaA gene or the cvaB gene. Cells with functionalexport systems export ColV into the surrounding medium.Using the plate assay described in Materials and Methods,we quantitated relative levels of ColV export. Cells harbor-

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7552 FATH ET AL.

TABLE 1. ColV assays of export through Hly and Prt

ExporteraColV activityb

Heterologous CoIV exporter (% of wild-type activity)exporter proteins proteins

None CvaAB 100HlyBD None 18-24HlyBD CvaA 10-14HlyBD CvaB 10-14HlyB CvaA 9-14HlyD CvaB 4-8PrtDEF None 16-20PrtDEF CvaA 5-9PrtDEF CvaB 5-9None None <0.8'

a All the CoIV exporter derivatives produced functional CvaC and Cviproteins. To express the following heterologous exporter proteins, the plas-mids were used as follows: HlyBD, pWAM04, which includes hlyCABD;HlyB, pWAM554, which includes hlyCAB; HlyD, pMJF108; PrtDEF,pRUW4. The CvaA and CvaB defects were due to insertion mutations inpHK11 or pHK22 described by Gilson et al. (15). To test for CoIV export ina CvaA-B- background, HlyBD was expressed with plasmid pLY21 orPrtDEF was expressed with plasmid pHK817. All the plasmids were testedindividually for CoIV production, and none was sufficient for exportingdetectable ColV. See Materials and Methods for details.

b CoIV activity was measured by calculating the area of inhibition around aproducer colony and comparing it with the area of inhibition of the wild-typeproducer, pHK11. Typical areas of inhibition for pHK11 ranged from 90 to120 mm2.

c The minimum level of detection for this assay is approximately 0.8% ofwild-type activity. See Materials and Methods.

ing these two plasmids were able to export active CoIV at 10to 14% of the level in the wild-type ColV system (Table 1).Similar levels of complementation were observed regardlessof whether the insertion mutation was in cvaA or cvaB.Hly-mediated export of ColV was then tested in a straincarrying a cvaAB deletion. This strain was able to exportactive ColV at 18 to 24% of wild-type levels, showing thatHlyB and HlyD function as a pair to export ColV in theabsence of either CoIV exporter. The level of Hly-mediatedexport of ColV was noticeably higher when CvaA and CvaBwere both absent, indicating that either ColV export proteincan interfere with Hly-mediated export.

Functional complementation by individual Hly export pro-teins. Experiments were performed to determine whethersingle components from each system could combine to formactive exporters. An Hly operon derivative with a functionalhlyB gene was cotransformed with CoIV plasmids carryingan insertion mutation in cvaB. Export of ColV from thesestrains would be seen only if CvaA and HlyB were able tointeract and form an active export complex. ColV exportfrom these strains was approximately 9 to 14% (Table 1),which was lower than export levels from strains carryinghlyBD and a cvaAB deletion. Thus CvaA and HlyB form anactive complex with lower efficiency. Similarly, an Hlyderivative carrying only a functional hlyD gene was used totransform strains with ColV plasmids carrying insertionmutations in cvaA. In this case, ColV export would beobserved only if CvaB and HlyD were able to form an activeexport complex. ColV export from these strains was approx-imately 4 to 8%, compared with 18 to 24% from strainscarrying hlyBD and a cvaAB deletion. Thus neither theCvaA-HlyB pair nor the CvaB-HlyD pair was able to exportactive ColV as efficiently as the HlyBD pair.Export of ColV-alkaline phosphatase fusions through the

hemolysin export system. A series of cvaC-phoA gene fusionshad been constructed previously to localize export signals

350

300

a

= 250

SS5 200

a.A 15 0IL0

c'a 100

50

0 -IExportel None HlyBD CvaAB None HlyBD CvaAB

Fusion CvaC57-PhoA CvaC68-PhoAFIG. 2. Alkaline phosphatase activity of CvaC-PhoA fusions in

the presence of CvaAB or HlyBD. The plasmids carrying theexporters were as follows: None, pACYC184; HlyBD, pHyl82;CvaAB, pLY21. The CvaC-PhoA fusions were described previously(16).

within ColV (16). Since alkaline phosphatase is inactivewhen localized in the cytoplasm, the level of alkaline phos-phatase activity can be used to measure the amount ofprotein translocated across the inner membrane. By usingthose fusions, an export signal was localized to the N-termi-nal 39 amino acids of the protein (16). This is in sharpcontrast to the export signal for a-hemolysin, which waslocalized to the C-terminal 52 amino acids (19).To determine whether an N-terminal export signal in ColV

can also be recognized by the Hly export machinery, plas-mids containing the cvaC-phoA fusions were used to trans-form strains expressing the Hly export proteins. Levels ofalkaline phosphatase activity were measured. The results(Fig. 2) indicate that translocation of ColV-PhoA fusionsacross the inner membrane occurred through the Hly systemat levels significantly above background levels. First, infusions CvaC57-PhoA and CvaC68-PhoA, there was a 5- to20-fold increase in alkaline phosphatase activity in the pres-ence of CvaAB. These results correlate with results reportedearlier (16). When the HlyB and HlyD proteins were presentin place of CvaAB, alkaline phosphatase levels increasedthree- to sixfold over the background level. The increase ofphosphatase activity of the CvaC-PhoA fusions in the pres-ence of HlyBD correlates well with the observed levels ofCoIV export through HlyBD. Since it has been shownpreviously that strains bearing HlyBD do not secrete PhoAlacking a signal sequence (8), we conclude that the alkalinephosphatase activity observed with the CvaC-PhoA fusionsis due to export of CvaC-PhoA through HlyBD. This indi-cates that export signals within the N-terminal 57 aminoacids of ColV are recognized by the Hly export system.

Export of CoIV export signal mutants through the hemoly-sin export system. Three export-deficient mutations withinthe amino-terminal region of ColV have been describedpreviously (16). Strains carrying these mutations produceColV, which is biologically active but exported by CvaAB ata reduced level (Fig. 3a). Export of these mutant ColV

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COMPLEMENTATION BETWEEN BACTERIAL EXPORT SYSTEMS 7553

b)

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0

CL

G14N GI 4D

30 -

25 -

20-

15

10-

5.

G38R

CvaAB

1L TI

W.T. G14N G14D G38R

HlyBD

FIG. 3. Export of ColV point mutations through CvaAB (a) or HlyBD (b). The plasmids carrying the exporters were as follows: HlyBD,pWAM04; CvaAB, pLY11. The plasmids carrying the cvaC point mutations (G14N, pPA101; G14D, pPA107; G38R, pPA104) were describedpreviously (16).

molecules through the Hly exporter was then tested (Fig.3b). The export efficiency of mutant G38R is greatly reducedthrough both exporters. In contrast, the other two mutantColV molecules (both mutated at amino acid 14) are recog-nized differentially by the two exporters. Although they areboth greatly reduced for export through CvaAB, their exportthrough HlyBD is only slightly affected.The Erwinia protease export proteins, PrtD, PrtE, and

PrtF, can export active ColV. The extracellular secretion ofproteases B and C from the bacterium E. chrysanthemirequires the three linked genes prtD, prtE, and prtF (30).Since prtD and prtE share sequence similarity with cvaB andcvaA, respectively, we tested whether the Prt export systemwas capable of exporting active ColV. A plasmid carryingthe three Prt export genes was used to transform strainKS300 carrying the CoIV operon with an insertion in cvaA orcvaB, and levels of extracellular active ColV were assayed(Table 1). The Prt export proteins were able to export ColVfrom these cells at levels ranging from 5 to 9% of wild-typelevels. When Prt-mediated export was tested in a cvaABdeletion mutant, the level of CoIV export increased to 16 to20% of wild-type levels. Thus, a single Cva export proteinalso interferes with ColV export through the intact Prtexporter.

Hemolysin is not exported through the ColV or proteasesystem. Since the Hly and the Prt export proteins were foundto specifically recognize and export active ColV, we nexttested whether active a-hemolysin could be exportedthrough the ColV or Prt export system. For this experiment,an Hly plasmid derivative containing an hlyBD deletion wasconstructed and used to transform a strain expressing theColV or Prt export proteins. Although significant levels ofinternal a-hemolysin were observed, the ColV export sys-tem did not export detectable amounts of oa-hemolysin intothe extracellular medium (Table 2). A strain carrying an Hlyplasmid derivative with an inactive hlyD gene and a func-tional cvaA gene was also assayed for oa-hemolysin exportactivity. Hemolysin would be exported from this strain onlyif an active complex containing HlyB and CvaA could form.No detectable Hly export was seen in this system either.

Last, the Prt export proteins were not able to export activeot-hemolysin, which agrees with results reported previously(4).Complementation analysis of the third export component.

Extracellular export of both ot-hemolysin and CoIV wasrecently shown to require the product of a third, unlinkedgene, toiC (16, 47), which encodes a minor outer membraneprotein in E. coli (36). Export of proteases B and C from E.chrysanthemi also requires the presence of a third compo-nent, the linked prtF gene (30). The TolC and PrtF proteinsdisplay 23% amino acid identities. Since they were bothinvolved in similar export processes, we tested whether prtFcould functionally complement a mutation in the tolC gene inthe export of ColV. E. coli strains carrying insertion muta-tions in the toiC gene export ColV from plasmid pHK11 atgreatly reduced levels. When the prtDEF genes were ex-pressed in these strains, the level of CoIV export did notincrease detectably (data not shown). Thus, PrtF cannotcomplement the ColV export defect in strains lacking TolC.

TABLE 2. Hemolytic activities of Hly, ColV, and Prtexport components

Plasmids' Hemolytic activityb

Exporter HlyA source Internal External

None HlyCA ++++CvaAB HlyCA + + + +PrtDEF HlyCA + + + +HlyBD HlyCA ++++ ++++HlyD NoneNone HlyCAB + +HlyD HlyCAB +++ ++++CvaA HlyCAB + + + +

' All of these plasmids were harbored in E. coli KS300. The levels ofinternal and external hemolytic activity were measured as described inMaterials and Methods. The complementing plasmids used were as follows:HlyCA, pMJF107; HlyCAB, pWAM554; HlyD, pMJF108; CvaAB, pHK22;PrtDEF, pRUW4; CvaA, pHK11-2 and pHK11-6. The HlyBD-HlyCA rowwas plasmid pWAM04.

b See Materials and Methods.

a)

a0a.w

LU

Sa.)

1-

f-0C

01

IL.

11 0

100

90

80

70

60

50.

40

30

20.

1 0

0.W.T.

Exporter:

v - . .. ..

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7554 FATH ET AL.

DISCUSSION

The export systems for CoIV, a-hemolysin, and Erwiniaprotease share significant sequence similarities, and eachincludes a component that has sequence similarity to themultidrug resistance protein found in human tumor cells. Inthis study, we tested functional homologies among the threebacterial export systems. We found that the Hly and Prtexport systems were able to complement CvaA and CvaBdefects and export active ColV from E. coli cells. Exportthrough the intact HlyBD system in a cvaAB deletion strainwas compared with export through CvaA-HlyB and CvaB-HlyD complexes. The level of ColV export observed whenHlyBD was present in a CvaAB deletion strain was higherthan that observed when individual ColV and Hly exportproteins were expressed together. Therefore, neither theCvaA-HlyB complex nor the CvaB-HlyD complex was asactive as HlyBD. The results obtained do not allow us todistinguish whether diminished export results from ineffi-cient interaction of the exporters or from the formation of a

less active complex.Export of ColV through HlyBD and through PrtDEF was

greater when both CvaA and CvaB were absent, demonstrat-ing that the presence of a single ColV export protein inter-fered with heterologous export. There are at least twopossible explanations for this effect. The single Cva exportprotein might interact with the ColV substrate and sequesterit away from the heterologous export machinery. Alterna-tively, the single Cva export protein might form a less activecomplex with its heterologous export component, resultingin decreased levels of active exporter. It is worth noting thatCvaA and CvaB produce approximately equal levels ofinterference. This suggests that both CvaA and CvaB can

individually interact with either CoIV or the complementaryexport component.

In order for HlyBD and CvaAB to export a proteinsubstrate such as a-hemolysin or ColV, several types ofinteractions must occur. The export proteins must be prop-

erly localized, they must recognize each other and interact toform a functional complex, and they must recognize thesubstrate protein and mediate its translocation. It is possiblethat domains within each of the export proteins have sepa-

rable functions and that only a subset of these functions isconserved between systems. Our results suggest this to bethe case. HlyB and HlyD act together to recognize andexport ColV. Although some signals involved in optimizingColV export are clearly not present in the HlyBD exporter,the observed levels of export demonstrate that the domain inHlyBD sufficient for substrate recognition and export is stilllargely conserved between the ColV and Hly systems. Incontrast, the formation of active CvaA-HlyB and CvaB-HlyD complexes occurs at a lower level, suggesting that thedomain involved in protein-protein interaction between theexporters is less well conserved.The complementation observed between Cva and Hly

contrasts with that observed with the hemolysins from P.vulgaris, M. morganii, and E. coli. Koronakis et al. (28)showed that individual export proteins can complement eachother fully in these systems. This suggests that all of thefunctional domains for Hly export are conserved among

these exporters. In other studies, the ability of the hemolysincomponents to complement other exporters was analyzed byusing only the intact HlyBD pair (4, 21, 31, 43). Analysis ofprotein export in strains with single export proteins ex-

changed can provide additional information about functionaldomains conserved among these systems.

Using cvaC-phoA fusions and point mutations in the CoIVstructural gene, we have begun to define the structuralsignals in ColV that are recognized by the Hly system. Thenormal CoIV export signal was localized previously to theN-terminal 39 amino acids (16), while the normal exportsignal in oa-hemolysin was localized previously to the C-ter-minal 52 amino acids (19). There is only marginal similarityin primary and secondary structure between these twoexport signals. However, structural recognition must occurfor HlyBD to export active ColV. Our studies show that thephoA fusion to codon 57 of cvaC has significantly morealkaline phosphatase activity when either Cva or Hly exportproteins are present. These results localize the export signalin ColV recognized by HlyBD to the N-terminal 57 aminoacids.

Interestingly, HlyBD is able to secrete a PhoA-HlyAhybrid into the extracellular medium (22), whereas CvaAB isonly able to translocate CvaC57-PhoA across the innermembrane (16). CvaC57-PhoA exported by HlyBD does notappear to reach the extracellular medium; bacterial coloniescontaining CvaC57-PhoA and HlyBD and grown on LBmedium plus 5-bromo-4-chloro-3-indolyl phosphate are bluebut do not form blue halos. The reason for the differences infusion protein localization between these systems is notknown.The export-deficient point mutations within the N-termi-

nal region of CvaC provide additional information aboutspecific amino acids in ColV recognized by the two systems.In particular, each of the two mutations in amino acid 14profoundly reduces export of ColV through CvaAB whilereducing export through HlyBD (relative to that of wild-typeCoIV) only slightly. Thus, although amino acid 14 has animportant role in Cva-mediated export, it has a minor role inHly-mediated export. In contrast, the G38R mutation re-duces export through CvaAB to approximately 10% ofwild-type levels and completely abolishes export throughHlyBD. Therefore this residue is an important component ofthe export signal for both systems.These results show that, although HlyBD and CvaAB

recognize a common domain at the N terminus of ColV,specific amino acids within this domain are differentiallyrecognized by each export system. This type of analysis canbe extended further; as more export mutants are isolated andanalyzed, additional specific residues recognized by eachsystem can be identified and compared. Furthermore, sup-pressor mutations that restore export function can be iso-lated within cvaAB and hlyBD. Analysis of such mutationswill enable us to compare specific amino acids in CvaAB thatinteract with ColV with those in HlyBD that interact withColV. This system therefore provides an excellent means foridentifying, at one level, protein regions that interact as wellas for identifying, at a finer level, the precise amino acidsresponsible for determining specificity in protein-proteinrecognition.

It is interesting to note that export of a-hemolysin by theColV or protease export system was not detected. There areseveral possible explanations for the finding that functionalcomplementation was not reciprocal. Our results with thepoint mutations support the hypothesis that the HlyBDexporter requires fewer export signals in its substrate thandoes the CvaAB exporter. Specifically, Cva-mediated ColVexport is perturbed by substrate mutations at residues 14 and38, whereas Hly-mediated export of ColV does not requirethe wild-type residue at amino acid 14. The evidence that thesignal requirements for CvaAB are more stringent could

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VOL. 173, 1991 COMPLEMENTATION BETWEEN BACTERIAL EXPORT SYSTEMS 7555

explain why hemolysin is not exported by the CvaABsystem.Another possible factor controlling specificity is the size of

the protein normally translocated by each export system.The substrate normally exported by HlyBD is a-hemolysin,a large 107-kDa protein (9, 23). In contrast, the Erwiniaproteases are 50 to 55 kDa in size (48) and ColV is only 6 kDain size (15). The levels of protein export through the variousexport systems correlate well with the size of the normalsubstrate of the system. HlyBD can export ColV rather welland protease B at a very low but detectable level, PrtDEFcan export ColV rather well but does not measurably exporta-hemolysin, and CvaAB cannot detectably export a-hemo-lysin. By the criterion of substrate size, HlyBD is the leaststringent export system, PrtDEF has an intermediate level ofstringency, and CvaAB is the most stringent. On the otherhand, export of proteins through the least stringent system,HlyBD, is known to be highly specific (4, 19). The fact thatColV is exported by HlyBD points to a conserved structuraldomain in ColV that is recognized by the HlyBD exporter.We have performed complementation experiments be-

tween several prokaryotic signal sequence-independent ex-port systems, each of which includes an MDR-like compo-nent. The finding that prokaryotic MDR-like componentscan be exchanged intact to produce partly functional heter-ologous systems indicates a high degree of functional homol-ogy among these members of the MDR family. Furthermore,these findings lay a foundation for future analyses in whichspecific domains of CvaB can be replaced with analogousdomains from the more distant eukaryotic MDR-like rela-tives. The resulting chimeric proteins can then be tested aspart of the CvaAB system for the ability to export CoIV.Such analyses can distinguish between regions of theseMDR-like transport proteins that control substrate speci-ficity and regions that are essential for their general exportfunction. More generally, this line of experimentation canhelp elucidate steps in the export pathway that are commonto the diverse substrates of the MDR-like export family.

ACKNOWLEDGMENTS

We thank Rodney Welch (University of Wisconsin MedicalSchool) for providing us with the plasmids pWAM04 and pWAM554and Cecile Wandersman (Institut Pasteur) for providing toiC strainsand the Erwinia plasmid. We also thank Shahaireen Pellett forproviding us with hemolysin assay protocols and Alan Derman forhelpful comments on alkaline phosphatase assays.The work was supported by Cystic Fibrosis Foundation grant

Z138, National Science Foundation grant DMB-8813612, and PublicHealth Service grant AI25944 from the National Institutes of Health.M.J.F. was the recipient of a National Science Foundation Predoc-toral Fellowship, R.C.S. was a Science Scholar at the Mary In-graham Bunting Institute, and R.K. was the recipient of an Ameri-can Cancer Society Faculty Research Award.

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