From the
The
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
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
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.
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 the pHApm series of amino acid substitution
mutants, the pHald series of deletion mutants, and pHA
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.
To produce random mutants of hlyB from which to
select suppressors of the transport-defective phenotype of HlyA
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
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 HlyA
To exclude mutations in sequences outside of the hlyB gene, hlyB genes from plasmids isolated from
revertant strains of JM83 (pLGCD, pHA
pTS73(n) variants were transformed into JM83 (pHA
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.
In one isolate, the hlyB-containing EcoRI-XbaI fragment cloned into pTS73
As a control for cell leakage in the revertants, we tested
one such revertant mutant (pTS73
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
JM83 (pTS73
(A269V), pHA
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 HlyA
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) (). HlyA
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 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
A model has been proposed
in which the C-terminal-predicted
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.
The -fold increase of
efficiency in suppressors as compared with wild-type HlyB is determined
by the liquid hemolytic assay of secreted HlyA
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.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-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.
-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.
Bacterial Strains and Plasmids
Construction of HlyA Mutants
dcr-1 were
previously described(16) . Construction of other HlyA mutants
was described in detail elsewhere(14) .
Hemolytic Assay
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
d,
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).
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, pHA
d).
Screening for Revertants
d is substituted
for the wild-type HlyA, transport is reduced to about 6% of wild type
(14). Hemolytic activity of HlyA
d is not altered. Screening for
HlyB mutants that complement this transport defect in HlyA was
accomplished by transforming JM83 (pLGCD, pHA
d) 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
d, 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.
d, pLGCD)
cells and plated on blood agar to confirm that the revertant phenotype
mapped to the hlyB gene.
Sequencing of Mutated hlyB Gene
Cloning to Separate a Double Mutant
15
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
pTS73
15, we subcloned each mutation into separate vectors. The
wild-type hlyB vector pTG653 and pTS73
15 were both cut
with EcoRI and HpaI, and the homologous fragments
were exchanged. This resulted in pTG653
15C containing the G445S
mutation and pTS73
15N 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
3) for the release of the
cytoplasmic enzyme chloramphenicol acetyltransferase into the growth
medium. An overnight culture of JM83 (pTS73
3, pHA
d, 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 pTS73
3 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) .
Selection of HlyB Mutants That Suppress the
HlyA
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 bxTransport Deficiency
d 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 hlyA
d
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 hlyA
d, 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.
15). All confirmed revertant
strains containing a mutation within the sequenced region produced
larger hemolytic zones than wild-type HlyB when transporting HlyA
d (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 HlyA
d. 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, pTS73
3 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 (pTS73
3, pHA
d, pLGCD) (HlyB A269V), filledcircles, and two cultures of JM83(pTS73, pHA
d,
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).
d, 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.
d 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.
d 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 HlyA
d 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 HlyA
d
complementing HlyB mutants. Therefore, we cannot say that we have
exhaustively screened the library for potential HlyA
d suppressors.
dcr-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.
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.
-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.
d (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
, 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.