TXE JOURNAL OF BIOLOGICAL CHEMISTRY (g 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 26, Issue of September 16, pp. 1988949896.1993 Printed in U.S.A.

Complementation of Transport-deficient Mutants of Escherichia coli a- Hemolysin by Second-site Mutations in the Transporter Hemolysin B*

(Received for publication, March 26,1993)

Fang ZhangS, Jonathan A. Shepstrj, and Victor Ling7 From the Division of Molecular and Structural Biology, The Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Ontario, M4X lK9, Canada

Hemolysin B (HlyB) is a membrane-bound transport protein composed of an amino-terminal multiple mem- brane-spanning portion followed by a conserved ATP binding sequence. Together with the inner membrane protein HlyD and the outer membrane protein TolC, HlyB is responsible for transport of the 107-kDa toxin HlyA from the cytoplasm, across both membranes of the cell envelope of Escherichia coli, directly to the medium. We have used a mutational approach to inves- tigate a postulated interaction between HlyA and HlyB. We have isolated transport-deficient mutants of HlyA altered in the C-terminal signal sequence and used one of these, a deletion of 29 amino acids, to select compen- satory mutants in the transporter protein HlyB. Fif- teen mutants located at six different sites, all mapping within the amino-terminal multiple membrane-span- ning domain of HlyB, were identified. All of the mu- tations are clustered into three groups located close to the predicted inner face of the cytoplasmic membrane. We propose that these locations are close to sites on HlyB that interact with the C-terminal signal sequence of HlyA. This interaction is likely to involve either binding of HlyA to HlyB or activation of the transport mechanism. The compensatory mutants also display different patterns of specificity in terms of their ability to transport different HlyA mutants. The fact that point mutations are able to compensate for drastic changes in the signal sequence of HlyA suggests that substrate specificity of transporters such as HlyB may shift dramatically during evolutionary history. This could account for the diversity of substrates observed for the ABC transporter superfamily in nature.

The a-hemolysin (HlyA)’ of Escherichia coli is a cytolytic toxin secreted by some uropathogenic strains, and its trans- location across the cell envelope is mediated by a unique system (1). Secretion of a-hemolysin is independent of the sec gene-mediated pathway by which proteins with amino- terminal signal peptides are routed to the periplasmic space. HlyA secretion occurs in a single step with no periplasmic intermediate and is dependent on two inner membrane pro- teins, hemolysin B (HlyB) and hemolysin D (HlyD), and an

* This study was supported by 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 be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 The first two authors contributed equally to this work. Recipient of a Natural Sciences and Engineering Research Coun-

cil of Canada studentship. II To whom reprint requests should be addressed.

The abbreviations used are: Hly, hemolysin; MNNG, N-methyl- N-nitro-N-nitrosoguanidine; LktA, leukotoxin A.

outer membrane protein TolC (1,2). The HlyB protein, which is composed of a multiple membrane-spanning domain and a cytoplasmic ATP binding domain, is a member of the ABC (ATP binding cassette) transporter superfamily. Members of this family of proteins are found in both prokaryotic and eukaryotic organisms and are involved in the translocation of a wide variety of substrates across biological membranes (3, 4).

The translocation of HlyA is dependent on a signal se- quence located in the C-terminal50 or so amino acids of the molecule. Many proteins that are not normally secreted by E. coli when engineered to contain the C-terminal signal se- quence of HlyA are then secreted in a HlyB/D dependent fashion (5, 6). Features of the signal sequence critical for recognition by the transporter have not been elucidated and may be complex. Deletions within this region cause severe reduction in HlyA export, however, detailed analyses involv- ing extensive point mutations have failed to identify a dis- crete, continuous sequence responsible for transport (7,8). In addition, a number of toxins homologous to HlyA (Pasteurella leukotoxin, Bordetella cyclolysin) and other proteins (Erwinia metalloprotease and E. coli colicin V) found in a variety of Gram-negative species have been shown to be transported by the HlyB/D system in E. coli (9-13). However, the C termini of these toxins bear little primary sequence similarity to that of HlyA. The relative transport efficiencies of these heterol- ogous proteins by the hemolysin system are generally not known. We have recently used one of these dissimilar C termini, the C-terminal 70 amino acids of leukotoxin (LktA) of Pasteurella haemolytica, to replace the signal sequence of HlyA (14). This chimeric protein could be transported by HlyB/D with efficiency equal to wild-type HlyA, indicating the LktA C-terminal70 amino acids can function as a signal recognized by HlyB/D. A common secondary structural motif, “helix-turn-helix-strand-loop-strand,” can be predicted in the C termini of both HlyA and LktA. These observations suggest that the transporter is capable of recognizing signal sequences, based on characteristics of their higher order structure, de- spite great diversity in amino acid sequence.

What role HlyB plays in the transport of HlyA is not well understood. Recent biochemical evidence suggests that a fu- sion protein containing the HlyA signal sequence may bind to HlyB in E. coli membranes in a salt-resistant fashion (6). On the other hand, a construct was reported in which a drastic deletion of the HlyB was found to retain some HlyA transport activity (15). It has not been determined whether this residual transport was dependent on the C-terminal signal of HlyA. In this study, we use a genetic approach to investigate the mechanism of HlyA signal recognition by HlyB. We con- structed a series of mutants in HlyA in which the signal sequence is altered. One of these mutants, a deletion of 29 amino acids, was used to select for mutations in HlyB, which

19889

19890 HlyB Suppressor Mutants

could suppress its transport-defective phenotype. We have isolated 15 such point mutants at sites that are clustered in the amino-terminal multiple membrane-spanning domain. Our results provide evidence that the signal sequence of HlyA interacts with the amino-terminal multiple membrane span- ning domain of HlyB and that point mutations in this region of HlyB can alter substrate specificity.

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) or the pACYC-derived pLG579 (16). 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 (17). Plasmid pLG579 (containing the hlyB gene) was kindly provided by Dr. I. B. Holland (Universite Paris-Sud, Paris). The TolC gene, required for HlyA transport, is found on the chromosome of all wild-type E. coli strains. Where appropriate, cells with plasmids were grown in the presence of 50 pg/ml kanamycin, 25 pg/ml chloramphenicol, and 50 pg/ml ampicillin or 25 pg/ml tetra- cycline. Routine DNA purification and manipulations were as de- scribed (18).

Construction of HlyA Mutants-The pHApm series of amino acid substitution mutants at base pairs 2967-2989 of the hlyA gene, and pHAcr-1, a frame-shift mutation at base pair 2986 of the hlyA gene, were created by site-directed mutagenesis using the doped oligonucle- otide HlyAcharge GGTAATTTGGATGTTAAGGAGGAAAGATC- TGCCGCTTCT in which each nucleotide position contains 2% of each of the other three nucleotides. Mutations were detected by DNA sequencing. Plasmids pHAld-1 and -2 were constructed by replacing the small SalI-KpnI fragment of pLG583sk with the nucleotide se- quence 2897-CAACTTCTATCTCGTCGCCTTGA or CCTGCAGG- CATGCAAGCTTGGCGTAA, respectively, resulting in new amino acid termini of 958-YVYGHDASTtsissp and 958-YVYGHDA- STcrhasla (small letters represent amino acids that do not exist in the wild-type HlyA). Plasmid pHAadcr-1 were resolved from self- ligation of the Sal1 and BglII large fragment of pHAad. Construction of other HlyA mutants was described in detail elsewhere (14).

Hemolytic Assay-The hemolytic activity of HlyA provides a con- venient assay for testing transport efficiency. E. coli cells expressing the hly genes secrete HlyA and clear a hemolytic zone around them- selves on blood agar plates. The growth curve of cells harbouring various HlyA mutant plasmids and pTG653 and pLGCD is the same as that of wild-type HlyA. Cytoplasmic hemolytic activity was assayed in the absence of HlyB to eliminate variations due to differences in export efficiencies of the HlyA mutants. The cytoplasmic hemolytic activities of all mutants (except HlyA/lkt70) are at a level comparable with wild-type HlyA (data not shown), indicating that most mutations had no apparent effect either on the expression of HlyA or on hemolytic activity. Therefore, the size of the hemolytic zone can be used as a measure of transport efficiency.

1) Hemolytic zone assay was as follows. JM83 cells harbouring the desired plasmids containing hlyA, B, C, and D were plated on 5% sheep blood LB agar plates containing the antibiotics ampicillin (50 pg/ml), kanamycin (50 pg/ml), and chloramphenicol (25 pg/ml) and grown at 37 “C for 8-9 h. The transport efficiencies of the HlyA mutants were initially tested and ranked according to the size of the hemolytic zones on blood agar as determined by visual inspection (see Fig. 2, column Z, and Fig. 3). The results were confirmed by at least two independent observers and were reproducible. Colonies of the cells expressing only hlyA, C, and D without HlyB did not exhibit any hemolytic zone (see Fig. 3F), indicating that the small hemolytic zones ranked as “one” (see Fig. 3E) are dependent on the presence of hlyB.

2) Measurement of HlyA in the medium was as follows. JM83 cells harbouring the desired plasmids were grown in liquid culture with the appropriate antibiotics, and samples were taken at different growth points (Am). Cells were removed by a brief centrifugation. Supernatant (100 pl) was tested for hemolytic activity in the presence of 10 mM Tris-C1, pH 7.4, 160 mM NaCl, and 2% sheep blood (final volume, 200 pl) after 30 min incubation at 37 ‘C. Hemolysis was measured by absorbance at 420 nm after a brief centrifugation to remove unlysed blood cells. Hemolytic activity in the medium was

plotted as a function of cell density (see Fig. 1 as an example). An optical density of A6W = 0.7 (or Am = 1.0 where indicated by an asterisk) 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 a percentage of wild-type activity in each experiment. Data are presented as the mean of at least four experiments k S.D.

Results from the liquid hemolysis assay correlate well with the ranks determined by observed zone sizes (compare Fig. 2, columns Z and ZZ). A statistical test was performed to verify that the relative rankings generated by the two assays were consistent. The Spearman rank correlation coefficient R. is 0.99 between these two assays, and this value is significant at a level p = 0.01.

Mutagenesis of hlyB-In order to produce random mutants of hlyB from which to select suppressors of the transport-defective phenotype of HlyAcr-2, two different mutagenesis protocols, as described below, were 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).

1) N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) mutagenesis was as follows. A 40-ml culture of JM83 (pLG579) was grown to an optical density at 600 nm of 0.6. At this point, 40 pl of chloramphen- icol (34 mg/ml) was added. One-half h later, 40 p1 of MNNG solution (50 mg/ml in dimethyl sulfoxide) was added. Cells were incubated overnight and plasmid DNA then isolated. Mutagenized pLG579 was transformed into JM83 (pHAcr-2, pLGCD) and screened for colonies with elevated hemolytic activity.

2) Hydroxylamine mutagenesis was as follows. Plasmid pTG653 was treated with 0.8 M hydroxylamine at 70 “C for 1 h as modified from Chu et al. (19). Briefly, a stock solution of hydroxylamine (2 M hydroxylamine, 0.1 M sodium pyrophosphate, 0.002 M NaCl, pH 6.0) was prepared fresh each day. This was diluted 1.25-fold in 0.1 X SSC, and then 10 p1 was added to 10 pl of DNA (2 pg/pl) in 0.1 X SSC. (1 X SSC is 0.15 M NaC1, 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 X SSC). Precipitated DNA was redissolved in 50 pl of TE (10 mM Tris-C1 pH 8, 1 mM EDTA), and a 1/100 dilution of this was used to transform JM83 (pLGCD, pHAcr- 2).

Screening for Revertants-When plasmids containing ‘genes for

1.4 1 1.2 -

1.0-

x

0.8 - Q ( I I . (II >. ._ -

0.6- m I

0.4 -

0.2 - it

1 ++A / 0 . 0 : . , . , . , . , . , . , . , . , , I

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Cell Density (Ae00)

FIG. 1. Accumulation of secreted HlyA as a function of cell growth. E. coli cells harbouring pTG653 (hlyB), pLGCD (hlyC and -D) and the wild-type hlyA plasmid (0) or a mutant hlyA plasmid (+) (HlyAcr-1, see Fig. 2) were grown in LB medium. Cell growth was monitored by absorption at 600 nm. Samples were removed at different times for measurement of hemolytic activity in the medium (see “Materials and Methods”). Hemolysis was determined by ab- sorption at 420 nm. For each of the wild-type and mutant plasmids, two different colonies were analyzed.

HlyB Suppressor Mutants 19891

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 HlyAcr-2 is substi- tuted for the wild-type HlyA, transport is reduced to less than 1% of wild type. Hemolytic activity of HlyAcr-2 is not altered. Screening for HlyB mutants, which complement this transport defect in HlyA, was accomplished by transforming JM83 (pLGCD, pHAcr-2) with hydroxylamine-treated pTG653 or MNNG-treated pLG579. 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-In order to exclude mutations in sequences outside of the hlyB gene, hlyB genes from plasmids isolated from revertant strains of JM83 (pLGCD, pHAcr-2, pTG653, or pLG579) were subcloned into new vectors. the BglII-EcoRI frag- ment of mutant strains of pLG579 was ligated into pTZ18R at BamHI and EcoRI sites (designated pTG653(n), where n is the hlyB geno- type). The EcoRI-XbaI HlyB-containing fragment of mutant strains of pTG653 was subcloned into unmutagenized vector pTZ18H. Plas- mid pTZl8H is pTZ18R in which the HindIII site has been eliminated by cutting with HindIII, blunting the ends with Klenow enzyme and religation. This procedure allowed subclones (designated pTS73(n)) to be distinguished from the parental plasmid by differential sensitiv- ity to HindIII.

pTS73(n) and pTG653(n) variants were transformed into JM83 (pHAcr-2, pLGCD) cells to confirm that the revertant phenotype mapped to the HlyB gene.

Sequencing of Mutated hlyB Gene-Mutant clones in pLG579n or 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 laboratory (20), which spans the hlyB gene and flanking sequences, was used.

Construction of an HlyB Double Mutant (A146V, T251I)”Single- stranded DNA derived from plasmid pTG653(A146V), isolated as described above, was used as a template for site-directed mutagenesis (21) using the oligodeoxynucleotide CGTCGTGTTGGTGA- TATCGTTGCCAGGG. This resulted in the introduction of a Thr- 251 --.* Ile mutation into the resulting HlyB gene product along with an EcoRV site used for selection of mutagenized clones. The double mutant genotype was confirmed by nucleotide sequencing.

RESULTS

HlyA Mutants Defective in Tramport-Our overall goal was to obtain mutations in HlyB that could suppress the transport defect phenotype of a mutation in the signal sequence of HlyA. In this way, it was hoped that we would find regions of HlyB that interact with the signal sequence of HlyA. In order to simplify interpretation of such mutants, the ideal HlyA mutant against which to select a suppressor mutant in HlyB would be a point mutation that causes a large decrease in transport activity. However, the finding that the HlyB/D transporter is capable of recognizing diverse signal sequences suggests it is unlikely that single amino acid changes in HlyA would result in a dramatic reduction in function. As shown in Fig. 2, the greatest defect we have been able to achieve by point mutations is a reduction to 30% of wild-type activity. Similar observations have been made by other laboratories (7,8).

The C-terminal 27 amino acids of HlyA were proposed to interact with the transporter (7). We have obtained two hlyA mutants in which the C-terminal29 amino acids were deleted and transport activity mostly eliminated. A very small he- molytic zone (rank 1 in Fig. 3E) was observed, and less than 1% of wild-type hemolytic activity was found in the culture medium of these strains. We chose one of these, HlyAcr-2, for the next step, selection of HlyB suppressor mutants that would compensate for the low transport phenotype.

In the mutants HlyAld-1 and -2, the C-terminal 58 amino acids have been deleted. This removes essentially the entire signal sequence as defined by mutational studies. However, a small amount of residual transport activity remains; approx- imately 1% of wild-type (zone rank 2). Interestingly, this is

more activity than remains in HlyAcr-2 and -3 in which only the C-terminal29 amino acids are deleted (zone rank 1) (Fig. 2). This finding suggests that there are some sequences up- stream of the C-terminal 58 amino acids that can play a role in transport but at a very low level. That the HlyAcr-2 or -3 mutants reduce transport even further is presumably due to the random sequences added to their new C termini.

Mutagenesis of HlyB Gene and Selection of Mutants That Suppress the HlyAcr-2 Transport Deficiency-For the random mutagenesis of hlyB, two mutagens, MNNG and hydroxyl- amine, were used. From these mutants were selected clones that could suppress the transport-deficient phenotype of HlyAcr-2. Both these mutagens are predicted to cause C-T transitions. The treatment with MNNG was undertaken in uiuo; cells harbouring the hlyB-bearing plasmid pLG579 were grown in the presence of MNNG, and plasmid DNA was isolated. In order to prevent the isolation of siblings caused by cell division and to ensure saturation of the target hlyB gene, we also used hydroxylamine to treat plasmid DNA in uitro.

To select HlyB mutants that suppressed the phenotype of the HlyA C-terminal deletion HlyAcr-2, mutagen-treated DNA was transformed into cells harbouring pHAcr-2 and pLGCD. Colonies of cells expressing wild-type HlyB, C, D, and HlyAcr-2 produce small hemolytic zones (Fig. 4A). When mutagenized HlyB plasmids were used, 90% of colonies pro- duced small hemolytic zones, and 5-10% displayed no hemo- lytic zone, presumably due to mutations that destroyed hlyB expression or function. Colonies exhibiting larger hemolytic zones were rare These latter colonies were restreaked several times to confirm that the phenotype was consistent. Approximately 600,000 colonies were screened (total for both mutagenesis protocols). Six revertant colonies were isolated from MNNG-treated DNA and 10 from hydroxylamine- treated DNA. The HlyB-containing plasmid DNA was iso- lated from each clone. The hlyB gene was subcloned from the mutated plasmids to fresh vectors and retransformed into JM83 (pHAcr-2, pLGCD) cells to ensure that the phenotype mapped to the gene itself. One clone isolated from hydroxyl- amine-treated DNA lost its suppressor phenotype on subclon- ing and was not examined further. Sequencing of the rest revealed that all mutant clones had single point mutations changing an amino acid in HlyB (Table I). Some mutants additionally contained a silent mutation (not shown). From MNNG-treated DNA, three clones were isolated in which alanine 146 was changed to valine, one clone of Leu-158 --., Phe and two clones of Thr-251 + Ile. From hydroxylamine- treated DNA were isolated four independent clones of Asp- 259 + Asn, four of Asp433 + Asn, and a single clone with the mutation Ala-269 + Val. The appearance of multiple clones at the same sites suggests that the screening was extensive enough to obtain all possible mutants (within the limits of the mutagens) that are able to complement the transport deficiency of HlyAcr-2. Although both mutagens cause C-T transitions, the two gave rise to mutations at different sites, which may be due to some sequence preferences on the part of each mutagen.

All six point mutants isolated in HlyB are located in the predicted cytoplasmic regions of the molecule and all are located close to predicted membrane-spanning segments (Fig. 5). The fifteen mutant clones isolated fell into three clusters. These “hot spots” may be regions of the HlyB molecule important for its interaction with the HlyA C-terminal signal sequence. As an initial test to determine whether these mutant sites exhibit any synergistic effect, we constructed a HlyB mutant that combines two of these mutations, Ala-146 --., Val

19892 HlyB Suppressor Mutants

958 988 998 1024 I I I I

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HlyA - - - + - +--+ - + YVYGHDASTY GSQDNLNPLI NEISKIISAA GNFDVKEERS AAsLLQLSGN ASDFSYGRNS ITLTASA#

000000 00000000 A A A A A A A A A A A A A A A A A

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36t19 88tlO

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HlyAcr-2 - - - + - +-" -+ YVYGHDASTY GSQDNLNPLI NEISKIISAA GNFDVKEFDL DRCP#

A A A A A A A A A

FIG. 2. C-terminal sequences of wild-type and mutant HlyA. A, wild-type HlyA from E. coli LE2001; B, amino acid substitution mutants; C, HlyA mutants in which the C-terminal 29 amino acids are replaced; D, HlyA mutants in which the C-terminal 58 amino acids are removed, and E, internal deletion mutants. The altered amino acids in B and the replaced amino acid sequences in C and D are indicated. Dashed lines indicate identity with wild-type sequence. In E, the [ ] indicates the position where the amino acids are deleted. # indicates the end of the molecule. 4 indicates HlyA mutants described in Ref. 14. Columns Z and ZZ, transport efficiency of HlyA mutants. E. coli cells harbouring pTG653 (wild-type HlyB), pLGCD (HlyC and -D), and the indicated HlyA plasmids were grown either on blood agar plates or in liquid culture and tested by the size of hemolytic zones (column I ) of colonies (see Fig. 3) and the secretion of HlyA in the medium as a percentage of wild type (column 11). Samples in cell culture were taken for hemolytic assay at different growth points (A,) (see Fig. 1). The data presented in this figure represent hemolytic activity interpolated to & = 0.7 (or & = 1.0 where marked with an asterisk), a point arbitrarily chosen at which to compare the relative transport efficiency of each mutant. Data are presented as means of at least four experiments & S.D. F, HlyA and HlyAcr-2 C-terminal sequences with charges and predicted structural features indicated (14); solid line, a- helix; 0, @-strand; A, @-turn.

and Thr-251- Ile. This mutant exhibits the same phenotype (assayed by hemolytic zone on blood agar) as the Ala-146 - Val single mutant. In this case, the double mutation does not have an additive effect beyond that seen with a single muta- tion. However, it does not rule out possible additive effects of other double mutants in untried combinations.

Characterization of the HlyB Mutations-The increased ability of the HlyB mutants to transport HlyAcr-2 is shown by their increased hemolytic zones (Fig. 4) and quantified by liquid hemolytic assay (Table I).

To assess whether the increased transport caused by the hlyB mutations was specific to the interaction between the C- terminal signal of HlyAcr-2 and HlyB, we tested the ability of the HlyB mutants to transport another HlyA mutant, HlyAcr-3. HlyAcr-3 is similar to HlyAcr-2 in that the last 29 amino acids of the natural signal sequence are removed but differs in the random sequence appended to the C terminal (Fig. 2). Both mutants have comparably low transport effi- ciencies. None of the suppressor mutations in HlyB comple- mented the transport defect in HlyAcr-3. In addition, the transport deficiency of HlyAcr-2 could not be corrected simply by increasing the expression level of wild-type HlyB by 10- fold.' These results suggest a specific interaction between the mutated sites on HlyB and the C terminal of HlyAcr-2.

Other HlyA C-terminal mutants were tested for restored transport competence in the presence of the HlyB suppressor

J. Sheps and Sarah Childs, unpublished result.

mutants with variable results (Table 11). Table I1 shows changes in transport efficiency associated with the hlyB mu- tants relative to wild-type hlyB for each hlyA mutant. Our intention was to observe changes in substrate specificity that result from the HlyB mutations, and no attempt has been made to quantify the magnitudes of these effects. Each column of this table provides a profile of specificity for each mutant hlyB. All of the HlyB mutants are unimpaired in their ability to transport wild-type HlyA. The HlyB mutants can comple- ment certain HlyA mutants to some extent. It appears that the effect of the mutations in HlyB has been to widen the specificity of transport (at least with respect to a few HlyA variants) while leaving the normal transport function intact.

One noteworthy result is that HlyB, D433N, and D259N are impaired in their ability to transport HlyA/lkt7O (Table I1 and Fig. 6). Wild-type HlyB transports this construct as efficiently as it does wild-type HlyA (14). Thus, HlyB, D433N, and D259N represent the first HlyB mutants in which trans- port specificity has been shifted by mutation and not simply widened.

DISCUSSION

We have adopted the strategy of isolating suppressor mu- tations as an approach for identifying interactions between a ligand and transporter as a basis for investigating substrate specificity. In this study, we have sought for mutations in HlyB (transporter) that complement a transport-defective

HlyB Suppressor Mutants 19893

mutant of HlyA (ligand) in which the C-terminal signal sequence has been drastically truncated. Fifteen mutant clones were found having single amino acid changes that fell into three clusters mapping to the multiple membrane-span- ning domain in the amino-terminal half of the protein. We have attempted to isolate suppressor mutants in HlyB using another, nonoverlapping, deletion in the C terminal of HlyA (HlyAad, Fig. 3E) . Preliminary results indicate the isolation

.-- I I

efficiency of HlyA

I

i mutants ranked by FIG. 3. Transport

s ize of hemolytic zone. E. coli cells harbouring pTG65.7, pLGCD, and various HlyA plasmids were plated on blood agar. The ranks of hemolytic zone sizes are as follows (given in parentheses). A, pLG583sk (wild-type) (rank 7); B, pHApmLK (rank 5); C, pHAcr-1 (rank 4); D, pHAld-1 (rank 2); E, pHAcr-2 (rank 1). The E. coli cells in F harbour pLG583sk and pLGCD. Because the HlyB protein is not expressed, HlyA cannot be secreted from these cells; therefore, no hemolytic zone is observed.

of point mutants in similar locations in HlyB (data not shown). These findings constitute the first genetic evidence for the interaction of HlyA and HlyB. This interaction ap- pears to be localized to the amino-terminal multiple mem- brane-spanning domain of HlyB on cytoplasmic loops close to predicted transmembrane segments (Fig. 5).

Among ABC transport proteins, it has been noted that sequence conservation is highest within the nucleotide bind- ing domains and declines within the amino-terminal trans- membrane domain. This has led to the suggestion that the differences in substrate specificity between ABC transporters are a function of their divergent transmembrane domains. Mutations in the transmembrane domains of ABC transport- ers have been shown to alter substrate specificity. In the cystic fibrosis-associated CFTR protein (a chloride channel), mutations in positively charged amino acids within trans- membrane helices alter the halide selectivity of the protein (22). In the multidrug transporter, P-glycoprotein mutations in the cytoplasmic loop before the third transmembrane seg- ment (23) and in the sixth (24) and eleventh transmembrane segments (25) differentially affect the transport of various drugs. All of our HlyB mutations were found to lie within the domain containing the predicted transmembrane segments. Our findings contribute to the general picture that determi- nants of substrate specificity in ABC transporters are located in the multiple membrane-spanning domains. A greater un- derstanding of the determinants of substrate specificity in the hemolysin transporter may have implications for understand- ing the mechanism of action of the recently identified major histocompatibility complex-linked transporters of antigenic peptides, which must be able to transport a broad range of peptides into the lumen of the endoplasmic reticulum (26). It may be that the ability of the hemolysin transporter to rec- ognize multiple signal sequences is related to the ability of P- glycoprotein to transport a wide variety of lipophilic drugs.

We have created a set of HlyB mutants in which substrate specificity has been altered. When tested against different HlyA mutants, the HlyB mutants show different specificity profiles (Table 11). The substrate specificity of these HlyB mutants has been widened. These HlyB mutants have an

FIG. 4. Secretion of HlyAcr-2 by HlyB suppressors. E. coli cells harbouring pHAcr-2, pLGCD, and wild-type or reverted HlyB plasmids were plated on blood agar. A, wild-type HlyR in pTG653; B, A146V; C, L158F; D, T251I.

19894 HlyB Suppressor Mutants

TABLE I HlyB suppressors

Codon change A:kn;!id Mutagen No, of Increase

effciencv of

-fold”

GCC + GTC A146V MNNG 3 CTT + TTT L158F

4 MNNG

XCT + KTT T251I 1 12

MNNG 2 2.5 - GAC +&C D259N Hydroxylamine 4 18 GCA + GTA A269V Hydroxylamine 1 10

~~

- G A T - g T D433N Hydroxylamine 4 4.6 The -fold increase of efficiency in suppressors as compared with

wild-type HlyB is determined by the hemolytic assay of secreted HlyA (see “Materials and Methods”).

A X

B

C 136-AKFDFTWFIPAIIKYRRIFIETLWSVFLQLF-167

V F

I N V 246RRVGDTVARVRELDQIRNFLTGQALTSVLDLL-277

N 423-PVIRLAQIWQDFQQVGIS440

FIG. 5. Location of suppressor mutations in HlyB. A , muta- tions in HlyB plotted on a linear representation of the protein. The number of Xs at a location corresponds to the number of isolates of each genotype. Shaded boxes indicate transmembrane spans; black boxes represent the nucleotide binding consensus. B, the topology of HlyB in the inner membrane (indicated by the shaded area) of E. coli is based on topological studies by Gentschev and Goebel (6). The sites of amino acid changes in HlyB suppressors are marked. The ATP binding site is indicated with a stippled circle. C, the amino acid sequences surrounding the suppressor mutations are shown. Under- lined regions are predicted to lie within the membrane.

enhanced ability to transport certain HlyA mutants without loss of the ability to transport wild-type HlyA. Two of the HlyB mutations (D259N and D433N) are of particular inter- est, since they cause a defect in transport of HlyA/lkt70. We have shown previously that the HlyA/lkt7O construct, in which the signal sequence of HlyA is replaced with that of the homologous toxin leukotoxin A, is transported by wild- type HlyB with equal efficiency (14). The secondary structural

TABLE I1 The substrate specificity of HlyB suppressors

In each row of the table, the E. coli cells harboring pLGCD and an hlyA (as indicated in the leftmost column) containing plasmid and an hZyB suppressor (as indicated in the top of each column) were plated on blood agar plates in triplicate. The size of hemolytic halos of colonies from each suppressor is visually compared with that of the same HlyA expressed with wild-type HlyB in the same experi- ment. t, increase; J, decrease; NC, no change in the size of zones by comparison with wild-type hlyB. Transport efficiency of different HlyA mutants by wild-type HlyB is listed in the second column from the left (Fig. 2 and Ref. 14).

HlyA HlyB

Wild-type A146V L158F T251I D259N A269V D433N %

A, HlyAcr-2“ C0.5 T t t t T t Bb

Wild-type 100 NC NC NC NC NC NC HlyAed 70 NC NC NC NC NC NC HlyAcr-3 <0.5 NC NC NC NC NC NC

HlyAtd 1 5 t T t t t t HlyAcvd 6 t t t t t N C

c‘

HlyAld-1 1 t t t t t t HlyAld-2 1 N C T t t t T HlyAcr-1 19 t t t t T N C

D, HlyA/lkt70d 100 NC NC NC J NC 1 “A, HlyAcr-2, the HlyA mutant with which the HlyB mutants

were selected. All HlyB mutants increase transport efficiency. B, HlyA and mutants thereof whose transport activity was unal-

tered by any of the HlyB mutants. e C, HlyA mutants whose transport efficiencies were improved by

some or all of the HlyB mutants. dD, An HlyA construct in which the C-terminal 58 amino acids

were replaced with the C-terminal 70 amino acids of LktA of P. haernolytica. Transport activity was unaffected or reduced by HlyB mutation (see Fig. 6).

features already mentioned in connection with HlyA are also predicted to be present in the C terminal of LktA (7, 14). However, the LktA C-terminal signal bears little primary sequence similarity to that of HlyA. Therefore, the molecular surfaces that HlyA and LktA present to their transporter must be quite different. This suggests a model in which the transporter recognizes the C termini of HlyA and LktA by using different contacts although overall transport is equally efficient. The isolation of HlyB mutants with a defect in HlyA/lkt70 transport but that are unimpaired with respect to the wild type HlyA suggests that the affected residues are involved in the recognition or transport of HlyA/Lkt70 but are dispensable for HlyA transport. The isolation of HlyB mutants that can distinguish between the two signal se- quences suggests that recognition of the HlyA and LktA signals by HlyB proceeds by distinct, though probably over- lapping, mechanisms. It is expected that further investiga- tions of HlyB mutants such as D259N and D433N will reveal insights into the mechanism of pleiotropic recognition by an ABC transporter.

The diversity of substrates transported by the various mem- bers of the ABC transporter family presents an evolutionary puzzle. The demonstration that it is possible, in HlyB, to widen substrate specificity while retaining wild-type function through simple point mutations suggest that it would be easy, evolutionarily, for ABC transporters to rapidly shift their substrate spectra to include novel substances. The observation that many ABC transporters possess the ability to transport a range of substrates (often substrates associated with other ABC transporters) such as STEG-mediated valinomycin re- sistance (27), P-glycoprotein associated transport of yeast a

HlyB Suppressor Mutants 19895

FIG. 6. Secretion of HlyA/lkt70 by HlyB suppressors. E. coli cells harbouring pHA/lkt70, pLGCD, and wild-type or reverted HlyB plasmids were plated on blood agar. A, wild-type HlyR in pTS73; B, A269V; C, D259N; D, D433N.

factor and C1- channel activity (28, 29), and transport of numerous bacterial toxins by HlyB/D (9-13) suggests that the evolutionary plasticity of ABC transporters may be an extension (as well as a source of) of their shared functional diversity.

Acknowledgments-We thank Dr. Peter Juranka for pioneering work in establishing the hemolysin system in our laboratory. We thank Sarah Childs for inspiration and our colleagues at the Ontario Cancer Institute for critical reading of the manuscript.

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