©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hemolysin Transport in Escherichia coli
POINT MUTANTS IN HlyB COMPENSATE FOR A DELETION IN THE PREDICTED AMPHIPHILIC HELIX REGION OF THE HlyA SIGNAL (*)

Jonathan A. Sheps (§) , Ian Cheung , Victor Ling (¶)

From the (1)Division of Molecular and Structural Biology, The Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The -hemolysin transporter of Escherichia coli, a member of the ATP-binding cassette transporter superfamily, is responsible for secretion of the 107-kDa protein toxin HlyA across both membranes of the Gram-negative envelope in a single step. Secretion of HlyA is dependent on a signal sequence, which occupies the C-terminal 50-60 amino acids of HlyA. Previously, it was shown that point mutants in the transmembrane domain of the transporter HlyB could partially correct the transport defect caused by a deletion of the C-terminal 29 amino acids of HlyA. These suppressor mutations demonstrated a direct interaction between HlyA and HlyB. They also displayed suppressor effects on a broad spectrum of HlyA signal mutants. In the present study, we selected HlyB alleles that complemented an internal deletion of 29 amino acids in HlyA containing a predicted amphiphilic helix region immediately upstream from the previous deletion. This set of HlyB mutants identifies further sites in HlyB that modulate substrate specificity but display allele-specific effects on a range of HlyA signal mutants. The inability to isolate mutations with effects restricted to either half of the signal sequence suggests that the signal is not recognized in a modular fashion by the transporter but rather functions as an integrated whole. We also report the isolation of the first substrate specificity mutation, which lies within the ATP-binding domain of HlyB. This could support a model in which the region of the ATP-binding cassette between the two Walker consensus motifs involved in ATP binding interacts with either the substrate or the transmembrane domains.


INTRODUCTION

The hemolysin gene cluster (hlyCABD) is found in the chromosome or on plasmids in numerous extraintestinal isolates of Escherichia coli and is probably involved in the pathogenesis of urinary tract infections in humans(1) . Similar toxin secretion determinants (the RTX toxins) occur in a number of Gram-negative genera, such as Proteus, Morganella, Pasteurella, Bordetella, Serratia, and Erwinia(2, 3, 4, 5, 6) . The hlyA gene codes for the 107-kDa -hemolysin protein (HlyA) itself, while the inner membrane proteins HlyB and HlyD, together with the outer membrane protein TolC, are required for transporting HlyA from the cytoplasm to the growth medium(7, 8) . The HlyC protein is required for activation of HlyA to its hemolytic form by transferring a fatty acyl residue to it(9) , but this modification is not necessary for transport.

The hemolytic toxin HlyA is targeted to the extracellular medium by virtue of a C-terminal signal sequence located within the last 50-60 amino acids of the molecule(10, 11) , and this region is capable of directing the HlyB/D-dependent secretion of a reporter protein. Extensive mutagenesis has shown that the signal is tolerant of point mutations and that functional residues and motifs are dispersed throughout the length of this region(12, 13) . This C-terminal signal can be replaced with the C-terminal 70 amino acids from the related leukotoxin (LktA) of Pasteurella hemeolytica with no loss of transport function(14) . The LktA sequence has only limited amino acid identity with that of HlyA; however, on the basis of computer modeling and mutagenesis studies, both are predicted to consist of a region capable of forming amphiphilic -helices followed by a C-terminal portion with amphiphilic -sheet potential(12, 14) , suggesting a conserved higher order structure may be required for recognition of the signal. Recent studies of the HlyA and LktA signals by CD (15) and NMR spectroscopy()confirm the presence of a helical segment in the N-terminal half of the signal sequence.

To identify regions of HlyB that might interact with the HlyA signal, we selected point mutants in HlyB, which could suppress the effects of transport-defective mutants of HlyA. Our previous study (16) used a deletion of the C-terminal 29 amino acids of HlyA to select compensating point mutants in HlyB, as only large deletions in the HlyA signal produced severe transport defects. Mutations were found that clustered in the multiple transmembrane spanning domain of HlyB. These HlyB mutants caused allele-specific effects on transport of a panel of HlyA signal mutants, which provided the first genetic demonstration of a direct interaction between HlyA and HlyB. The HlyB mutants were capable of partially complementing a broad spectrum of HlyA signal mutants, preventing the identification of particular contacts.

These results raised the question of whether the signal sequence could be divided into functional modules (perhaps along the lines of the predicted secondary structural regions), which are recognized by distinct sites on the transporter. To test this idea, we asked whether a deletion in the helical region of the signal sequence upstream from that used previously would allow selection of a different spectrum of compensating mutants.


MATERIALS AND METHODS

Bacterial Strains and Plasmids

E. coli strain JM83 was used for all manipulations. The hemolysin system was expressed in JM83 carrying three compatible plasmids. HlyA was expressed from the pSC101-based plasmid pLG583sk (14). HlyB was expressed from the pUC-derived vector pTG653(14) . Plasmid E2-2, a partial P-glycoprotein cDNA inserted into pUC19 (gift of Farida Sarangi, Ontario Cancer Institute, Toronto) was used as a vector control for the absence of HlyB. HlyC and HlyD were both expressed from the pACYC-based plasmid pLGCD(14) . HlyC is required for the addition of a fatty acyl moiety to HlyA, a modification required for hemolytic activity, which has no affect on transport(9) . HlyC was expressed in the absence of functional HlyD from the plasmid pLGC, which was constructed by partial EcoRI digestion of pLGCD and self-ligation to introduce an internal deletion of 1.3 kilobases within HlyD. Where appropriate, cells with plasmids were grown in the presence of 50 µg/ml kanamycin, 25 µg/ml chloramphenicol, and 50 µg/ml ampicillin. Routine DNA purification and manipulations were as described(17) .

Construction of HlyA Mutants

Construction of the pHApm series of amino acid substitution mutants, the pHald series of deletion mutants, and pHAdcr-1 were previously described(16) . Construction of other HlyA mutants was described in detail elsewhere(14) .

Hemolytic Assay

The hemolytic activity of HlyA provides a convenient assay for testing transport efficiency. E. coli cells expressing the hly genes secrete HlyA and clear a hemolytic zone around themselves on blood agar plates. The growth curve of cells harboring various hlyA mutant plasmids and pTG653 and pLGCD is the same as that of wild-type hlyA-bearing cells(16) . Previously, it was reported that the cytosolic hemolytic activities of the various HlyA mutants (except HlyA/lkt70) were indistinguishable from that of wild-type HlyA when assayed in the absence of HlyB(16) . This indicates that most signal mutations have no effect either on the expression of HlyA or on hemolytic activity. Therefore, the relative ranks of the hemolytic zones by size and brightness can be used as a semi-quantitative measure of transport efficiency.

Hemolytic Zone

JM83 cells harboring the desired plasmids containing hlyA, -B, -C, and -D were plated on LB agar plates containing 5% sheep blood and the antibiotics ampicillin (50 µg/ml), kanamycin (50 µg/ml), and chloramphenicol (25 µg/ml) and were grown at 37 °C for 8-9 h. The transport efficiencies of the HlyA mutants were initially tested and ranked according to a combination of size and brightness of the hemolytic zones on blood agar, as determined by visual inspection.

Measurement of HlyA in the Medium

JM83 cells harboring the desired plasmids were grown in liquid culture with the appropriate antibiotics, and samples were taken at different growth points (A). Cells were removed by a brief centrifugation. Supernatant (100 µl) was tested for hemolytic activity in the presence of 10 mM Tris-Cl, pH 7.4, 160 mM NaCl, and 2% sheep blood (final volume, 200 µl) during 30 min of incubation at 37 °C. Hemolysis was measured by absorbance at 410 nm after a brief centrifugation to remove unlysed blood cells. Hemolytic activity in the medium was plotted as function of cell density. An optical density of A = 1.0 was chosen as an arbitrary point at which to compare the relative transport efficiencies of each mutant, and hemolytic activities were interpolated to this point. Hemolytic activity was calculated as either a percentage or a multiple of wild-type activity in each experiment.

Mutagenesis of hlyB

To produce random mutants of hlyB from which to select suppressors of the transport-defective phenotype of HlyAd, the following mutagenesis procedure was used. The treatment conditions used were designed to achieve a 5-10% rate of knockout mutations (defined as a colony with no hemolytic zone when the mutant hlyB is co-expressed with hlyC, -A, and -D).

Plasmid pTG653 was treated with 0.8 M hydroxylamine at 70 °C for 1 h as previously described (16) using a procedure modified from Chu et al.(18) . Briefly, a stock solution of hydroxylamine (2 M hydroxylamine, 0.1 M sodium pyrophosphate, 2 mM NaCl, pH 6.0) was prepared fresh each day. This was added to one-quarter volume of 0.1 SSC, and then 10 µl was added to 10 µl of DNA (2 µg/µl) in 0.1 SSC (1 SSC is 0.15 M NaCl, 0.015 M Na-citrate). After incubation, the DNA was precipitated by addition of 1.05 ml of stopping buffer (70% ethanol, 0.1 M sodium acetate, 0.03 SSC). Precipitated DNA was redissolved in 50 µl of TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and a dilution of this was used to transform JM83 (pLGCD, pHAd).

Screening for Revertants

When plasmids containing genes for hlyA, -B, -C, and -D are present in E. coli JM83, the colonies generate hemolytic zones on LB agar plates supplemented with 5% sheep red blood cells and the appropriate antibiotics. When HlyAd is substituted for the wild-type HlyA, transport is reduced to about 6% of wild type (14). Hemolytic activity of HlyAd is not altered. Screening for HlyB mutants that complement this transport defect in HlyA was accomplished by transforming JM83 (pLGCD, pHAd) with hydroxylamine-treated pTG653. Colonies that produced larger hemolytic zones were restreaked three times (to ensure a consistent phenotype) before plasmid DNA was isolated.

Subcloning of Mutated hlyB Genes

To exclude mutations in sequences outside of the hlyB gene, hlyB genes from plasmids isolated from revertant strains of JM83 (pLGCD, pHAd, pTG653) were subcloned into new vectors. The EcoRI-XbaI HlyB-containing fragment of mutant strains of pTG653 was subcloned into unmutagenized vector pTZ18H. Plasmid pTZ18H is pTZ18R, in which the HindIII site has been eliminated by cutting with HindIII, blunting the ends with Klenow enzyme and religation(16) . This procedure allowed subclones (designated pTS73(n)) to be distinguished from the parental plasmid by differential sensitivity to HindIII.

pTS73(n) variants were transformed into JM83 (pHAd, pLGCD) cells and plated on blood agar to confirm that the revertant phenotype mapped to the hlyB gene.

Sequencing of Mutated hlyB Gene

Mutant clones in pTS73n were sequenced from double-stranded DNA using Sequenase (U. S. Biochemical Corp.) essentially according to the manufacturer's instructions. A set of 13 oligonucleotide primers previously prepared in our lab(19) , which span the hlyB gene and flanking sequences, was used.

Cloning to Separate a Double Mutant

In one isolate, the hlyB-containing EcoRI-XbaI fragment cloned into pTS7315 contained two mutations: a C T transition at nucleotide -81 (with respect to the start of translation of hlyB) and a missense mutation G445S in the protein sequence of HlyB. To determine which of these was responsible for the revertant phenotype of pTS7315, we subcloned each mutation into separate vectors. The wild-type hlyB vector pTG653 and pTS7315 were both cut with EcoRI and HpaI, and the homologous fragments were exchanged. This resulted in pTG65315C containing the G445S mutation and pTS7315N containing the C-81T mutation. The HindIII polymorphism between pTG653 and pTS73 was used to ensure that no vector cross-contamination had occurred.

Chloramphenicol Acetyltransferase Assay

As a control for cell leakage in the revertants, we tested one such revertant mutant (pTS733) for the release of the cytoplasmic enzyme chloramphenicol acetyltransferase into the growth medium. An overnight culture of JM83 (pTS733, pHAd, pLGCD) was spun down in a microfuge, washed twice with LB medium, and diluted 100-fold into fresh medium with appropriate antibiotics. Cells containing wild-type pTS73 instead of pTS733 were used as a control. Aliquots were taken at various time points, cells were removed by a brief centrifugation, and the conditioned medium (diluted 100- or 1000-fold in fresh medium) was assayed for chloramphenicol acetyltransferase activity essentially as described(20) .


RESULTS

Selection of HlyB Mutants That Suppress the HlyAd bxTransport Deficiency

Our goal has been to isolate mutations in HlyB at residues that may interact with the signal sequence of HlyA. To this end, we have used HlyA signal sequence mutants with severe transport defects(16) . To generate seriously defective alleles of hlyA, it was necessary to use deletion mutations that removed substantial portions of the signal sequence. The previously characterized HlyAd allele (Fig. 1) retains only approximately 6% of wild-type transport competence and lacks the N-terminal half of the signal sequence (14) (residues 968-985 of HlyA, the wild-type signal continues to residue 1024). This level of background hemolysis is approximately 10-fold greater than that produced by HlyAcr-2 (Fig. 1), against which we had previously selected HlyB suppressor mutations. Nevertheless, we were able to isolate hlyB alleles with hlyAd suppressor activity with fair efficiency.


Figure 1: Hemolysin A C-terminal signal sequence mutants. Sequence at top shows the C-terminal 67 amino acids of HlyA, with changes in mutant strains indicated below. Amino acids are numberedbackwards from the C terminus. The box of lowercaseletters in HlyAcr-2 indicates a mutant amino acid sequence resulting from fusion of the hlyA gene with downstream vector sequences. A stop codon is present after the last printed amino acid. Construction of the pHApm series of point mutants was described in Ref. 16 and that of the deletion mutants in Ref. 14. The effects of these mutations on transport by HlyB/D are indicated in Table II. Solidrectanglesabove the wild-type HlyA sequence indicate the positions of the putative -helices observed by NMR spectroscopy.



Selection of HlyB mutants was carried out by screening a library of chemically mutagenized pTG653, containing hlyB. Mutagenized plasmids were transformed into E. coli JM83 cells containing plasmids pHAd and pLGCD, containing hlyAd, and hlyC and hlyD, respectively, and plated on blood agar plates with appropriate antibiotics. Colonies of transformants that generated larger hemolytic zones (compared with wild-type pTG653 transformants) were picked and restreaked several times to ensure a stable phenotype. Plasmid DNA was isolated and, after retransfection to fresh host cells as a final check on the revertant phenotype, was purified again and sequenced.

Approximately 30,000 colonies were visually inspected, and the hlyB inserts from nine apparent revertants were subcloned to fresh vector pTZ18H. Seven of these apparent revertant hlyB bred true, and all seven contained missense mutations in the coding region (). Two apparent revertant isolates of hlyB did not breed true when subcloned, and when sequenced they were confirmed to be wild type. One confirmed revertant strain was found that contained one sense mutation and one mutation in the upstream non-coding region (strain 15). All confirmed revertant strains containing a mutation within the sequenced region produced larger hemolytic zones than wild-type HlyB when transporting HlyAd (Fig. 2A), secreted more protein as monitored by gel electrophoresis (Fig. 2B), and secreted more hemolytic activity when grown in liquid culture ( Fig. 3and ). The two mutations found in strain 15 were subcloned separately into unmutagenized vectors as described under ``Materials and Methods.'' The revertant phenotype followed the sense mutation in 15 (G445S). A vector containing the non-coding mutation (a C to T transition at -81 from the HlyA translation start) produced hemolytic zones indistinguishable from wild-type hlyB.


Figure 2: Secretion of mutant HlyA mediated by wild-type or a suppressor HlyB. A, hemolytic zone assay. E. coli carrying the appropriate plasmids were grown on blood agar as described under ``Materials and Methods.'' Effects on transport of HlyA mutant HlyAd was evaluated by the size and brightness of the ``halo'' around each colony. Upperpanel, expressing wild-type HlyB; lowerpanel, expressing suppressor HlyB A269V. B, SDS-polyacrylamide gel analysis of secreted HlyAd. Cell-free culture medium was trichloroacetic acid precipitated, and the precipitate dissolved and was subjected to SDS-polyacrylamide gel electrophoresis analysis on an 11% gel as previously described (14). Leftlane, pTS73 expressing wild-type HlyB; rightlane, pTS733 expressing HlyB A269V. Arrowhead, HlyA (107 kDa). Molecular mass is shown in kDa.




Figure 3: Secretion of mutant HlyA during growth of E. coli carrying wild-type or a suppressor HlyB. Secretion of mutant HlyA (HlyAd) was measured using a liquid hemolysis assay as described under ``Materials and Methods.'' Three cultures of JM83 (pTS733, pHAd, pLGCD) (HlyB A269V), filledcircles, and two cultures of JM83(pTS73, pHAd, pLGCD) (HlyB WT), opencircles, were grown in parallel, and aliquots were taken at various time points. Each data point represents one time point from one culture.



Six of the point mutations were found within, or close to, the membrane-spanning domains of HlyB, as was the case for all the mutations found in our previous study (Fig. 4). All mutations that mapped to soluble domains of HlyB were predicted to lie within the cytoplasm. One mutation (V599I) was found within the predicted ATP-binding domain between the two Walker consensus sequences for ATP binding (Fig. 4). This region in HlyB may therefore be involved in linking substrate binding and/or transport to ATP hydrolysis either through direct contact with the substrate or via close contact with the transmembrane helices with which it interacts. A similar situation may exist in the histidine permease of Salmonella typhimurium, where there is evidence that the ATP-binding subunit (HisP) interacts with both the transmembrane subunits and the substrate(21, 22) .


Figure 4: Location of suppressor mutations in HlyB. A, mutations in HlyB plotted on a linear representation of the protein. The number of circles at a location corresponds to the number of isolates of each genotype. Filledcircles represent mutations selected for their ability to revert the transport defect of HlyAd. Opencircles are those sites previously identified (16), which complement the transport defect of HlyAcr-2. There is some overlap in these phenotypes, however, as indicated in Table II and Ref. 16. Shadedboxes indicate transmembrane spans (23), and blackboxes represent the nucleotide-binding consensus (33). B, the topology of HlyB in the inner membrane (indicated by the shadedarea) of E. coli is based on topological studies by Gentschev and Goebel (23). The sites of amino acid changes in HlyB suppressors are marked. The ATP-binding site is highlighted with a stippledcircle. Filled and opencircles are as described in A.



To confirm that the set of HlyB mutants discussed here do not result simply from leakage of cells associated with expression of mutant HlyB alleles, a number of control experiments were performed. All the HlyB alleles originally selected for their ability to secrete HlyAcr-2 (opencircles in Fig. 4) were transfected into JM83 (pLGC, pLG583SK) cells and plated on blood agar. In the absence of intact HlyD, none of these HlyB mutants produced a hemolytic zone larger than that seen with the same cells expressing the control plasmid E2-2. The zone caused by leaked HlyA under these conditions was less than that associated with over-expression of -galactosidase from a wild-type pTZ18R vector in the absence of hlyB (data not shown).

JM83 (pTS73 (A269V), pHAd, pLGCD) cells were also assayed for their leakage of chloramphenicol acetyltransferase into the growth medium, compared with cells bearing wild-type pTS73. While A269V was associated with an approximately 3.5-fold increase in the amount of HlyA secreted to the medium (Fig. 3), the amount of chloramphenicol acetyltransferase detected in the medium at A = 1.0 was about half that seen in wild-type cultures. The reason for this difference is not clear, as the growth rates were the same for both genotypes (data not shown), but this effect is in the opposite direction from what one would expect from a HlyB mutation, which results in nonspecific leakage or export of cytoplasmic proteins.

The substrate specificity profile of HlyB A269V shows the least specificity of all the HlyB alleles (). However, it produces levels of secreted HlyA approximately four times higher when transporting HlyAd than with HlyAcr-2 (approximately 21 and 5% of wild-type hemolysis, respectively) ( and Ref. 16). Were the revertant phenotype solely the result of nonspecific leakage of HlyA caused by the mutated HlyB and not the product of a specific interaction between HlyA and HlyB, one would expect the secretion level to be similar regardless of the HlyA allele studied. It is unlikely that other alleles of HlyB, whose secretion-enhancing effects are restricted to certain HlyA alleles, are the result of nonspecific effects.

Characterization of the HlyB Mutations

Mutant forms of hlyB in pTS73 were transformed into E. coli JM83 strains carrying various HlyA signal sequence mutants (). The results were complex as all the HlyB mutants can revert different sets of HlyA mutations to some degree. In no case is there a loss of the ability to transport wild-type HlyA. The HlyB mutants showed differential sensitivity to the effects of mutations within the signal sequence of HlyA, suggesting the C terminus of HlyA interacts directly with HlyB, though not necessarily with the mutated residues of HlyB themselves.

Certain combinations of HlyA and HlyB mutants had their activity quantitatively assayed by liquid hemolysis. In general, the increase in HlyA secretion found in the set of HlyB mutants reported here is less than that seen with HlyB alleles selected for complementation of HlyAcr-2 in our earlier study (16) (). HlyAd is much more transport competent than HlyAcr-2, and the smaller relative effects seen here still result in higher levels of hemolysis. In general, the mutants in HlyB, which were selected for their ability to revert HlyAcr-2(16) , were less effective at complementing HlyAd than the HlyB mutants reported here (data not shown), which may explain why they were not re-isolated in this screen even though the same hydroxylamine-mutagenized hlyB plasmid library was used. The only exception was A269V, which alone was isolated independently from both screens. The present screen did, however, isolate a new mutation, A269T, at this same site. Unlike our previous screen for revertants (16), we did not find multiple independent isolates of HlyAd complementing HlyB mutants. Therefore, we cannot say that we have exhaustively screened the library for potential HlyAd suppressors.


DISCUSSION

In this paper, together with our previous one(16) , we have identified a distribution of amino acid positions where changes can affect substrate specificity in HlyB. The pattern of specificity changes demonstrates a direct contact between the HlyA and HlyB proteins. HlyB mutants selected for their ability to compensate for a given deletion in the C-terminal of HlyA may also complement a non-overlapping deletion (). For example, all the HlyB mutants reported in this study can partially correct the transport defect of HlyAdcr-1, a HlyA mutant in which none of the original signal sequence remains, as well as HlyAld-1, in which the entire signal sequence was deleted (16) (). This indicates that sites within HlyB are able to interact with positions in HlyA that either lie outside the C-terminal 58 amino acids or with sequences within this region that would not normally be recognized. The latter possibility could imply that novel sequences introduced into the C terminus of HlyA are recognized by positions in HlyB that would normally interact with the signal or that positions which may not function in recognition of the normal substrate are recruited to allow recognition of the new substrate. The observation that single point mutants in HlyB can correct defects associated with deletions in both halves of the signal sequence, while retaining differential sensitivity to certain signal mutants, suggests that these positions in HlyB are involved in forming a single binding pocket for the entire C terminus signal of HlyA.

Of interest is the observation that one of the HlyB mutants is defective in transport of HlyAed (a deletion of a charged cluster in the HlyA signal, Fig. 1). This result raises the possibility that in this case there is a direct interaction between a mutated residue in HlyB and HlyA. This HlyB mutation is in a charged residue and causes a defect in the absence of the ``charged cluster'' in HlyA. It is possible that this residue in HlyB (Arg-212) normally interacts with one of the charged residues in this cluster or at a site close to this in the HlyA molecule. Because of this result, we investigated the ability of this HlyB mutant to transport a set of point mutants in this region of HlyA ( Fig. 1and ). No difference was observed between mutant and wild-type HlyB with respect to any of these HlyA mutants except for HlyApmLKT, in which a small effect on zone size was noticed. The E K substitution in HlyApmLKT (Fig. 1) may introduce a positive charge into HlyA, which could be repelled by the abnormal lysine in HlyB R212K. The HlyB mutations we obtained previously, which, like R212K, produced a specific defect in secretion of a particular HlyA allele, were both alterations in charged residues (D259N and D433N, see Fig. 4and Ref. 16). This may point to a role for electrostatic interactions in the mechanism of substrate discrimination by HlyB. Experiments to test this possibility are being undertaken. The other mutant sites we have isolated represent positions that are either involved in recognition of the signal sequence, or of other regions of HlyA such that they can compensate for the loss of signal function, or are changes that lessen the specificity of HlyB and allow mutant substrates to be transported more readily.

A model has been proposed in which the C-terminal-predicted -sheet half of the signal sequence interacts directly with HlyB to activate transport, while the upstream, helical half of the signal causes the association of HlyA with the membrane and allows presentation of the extreme C-terminal to the transporter(12) . The HlyA signal sequence is sufficient to target a fusion protein to the membrane(23) . Recent results (15) indicate that in aqueous solution the HlyA signal is unstructured but that a membrane-mimetic environment induces the formation or stabilization of -helical structures in both halves of the signal sequence (see Fig. 1). A role for the predicted helical region in interacting with the HlyB/D transporter is suggested by recent mutagenesis studies, which have shown specific amino acids within the N-terminal half of the signal sequence (24) to have large influence on transport. A previous revertant study (16) showed that in HlyA mutants with the C-terminal 29 amino acids (the predicted -sheet domain) deleted, there was still a specific interaction with HlyB. The evidence presented here suggests that both N- and C-terminal halves of the C-terminal signal are involved directly in interacting with a single binding pocket formed by the transmembrane domain and attendant intracellular loops of HlyB. Our results indicate that the role of the N-terminal half of the signal sequence cannot be regarded as solely a membrane-targeting domain but rather that both halves of the signal sequence collaborate in recognition by the transporter.

The HlyB protein is a member of the ABC transporter superfamily, a group of membrane bound, ATP-dependent proteins responsible for the secretion of a wide array of substrates in both bacteria and eukaryotes(25) . Substrate specificity can be modulated by sequences within the membrane-spanning domains in several ABC transporters. Mutations at charged residues in the cystic fibrosis-associated chloride channel CFTR alter halide selectivity(26) , and substrate preference in the mammalian multidrug transporter P-glycoprotein can be modified by mutations within the transmembrane domain(27, 28, 29, 30, 31, 32) . We propose that substrate recognition in ATP-binding cassette transporters occurs through a common mechanism, involving homologous regions of the proteins (the transmembrane domain), even though substrate itself is not well conserved. In line with this, we note that even in transporters with a single natural substrate (such as HlyA), recognition occurs through multivalent interactions with substrates (or signal peptides) that contain multiple, overlapping recognition features. This characteristic of ATP-binding cassette transporters may underlie the ability of individual transporters to recognize multiple substrates as well as the diversity of substrates known for the ATP-binding cassette transporter superfamily.

  
Table: HlyB suppressors

The -fold increase of efficiency in suppressors as compared with wild-type HlyB is determined by the liquid hemolytic assay of secreted HlyAd (see ``Materials and Methods'' and Fig. 3). All experiments performed using duplicate cultures of mutant HlyB were grown in parallel with duplicate cultures of wild-type HlyB. To normalize for day-to-day variation in the hemolysis assay -fold increase is by comparison to wild type in the same experiment except for T220I and G445S, which are by comparison with the mean wild-type values for the entire set of experiments.


  
Table: The substrate specificity of HlyB suppressors

The hemolytic phenotypes of various combinations of HlyA and HlyB mutants were tested. The size of hemolytic halos of colonies from each suppressor was visually compared with that of the same HlyA allele expressed with wild-type HlyB on the same day. , increase; , decrease, NC, no change in the size of zones by comparison to wild-type hlyB. All data are from cells plated in duplicate. Transport efficiency of different HlyA mutants by wild-type HlyB is listed in the second column from left (from Refs. 14 and 16). All experiments were done with duplicate plates. All HlyA mutants are described in Fig. 1 and/or Ref. 16.



FOOTNOTES

*
This work was supported by a grant from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Natural Sciences and Engineering Research Council of Canada Studentship.

To whom reprint requests should be addressed. Tel.: 416-924-0671 (ext. 4988); Fax: 416-926-6529.

Yin, Y., Zhang, F., Ling, V., and Arrowsmith, C. (1995) FEBS Lett., in press.


ACKNOWLEDGEMENTS

We thank our colleagues at the Ontario Cancer Institute for help during the course of these experiments. In particular, we are indebted to Fang Zhang, whose work first showed the isolation of complementing mutations in the HlyA/B system to be practical. We thank Sarah Childs for critical comments on the manuscript and for the patience to stare at numerous blood agar plates. We also thank Peter ebo (Institut Pasteur) for suggesting the chloramphenicol acetyltransferase assay control and helpful discussions.


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