(Received for publication, January 8, 1997)
From the Laboratoire des Biomembranes, URA CNRS 1116, Université Paris-Sud, Bâtiment 432, F-91405 Orsay,
France, the § Department of Biology, University of Regina,
Regina, Saskatchewan S4S 0A2, Canada, and the ¶ Department of
Biochemistry and Microbiology, University of Victoria, Box 3055,
Victoria, British Columbia V8W 3P6, Canada
Aeromonas spp. secrete the
channel-forming protein proaerolysin across their inner and outer
membranes in separate steps using the general secretion pathway. Here
we show that treating A. hydrophila or A. salmonicida with the protonophore carbonyl cyanide
m-chorophenyl hydrazone blocks the second step in
transport, secretion across the outer membrane from the periplasm,
under conditions where the ATP levels in the cell are no different than
the levels in control, secreting cells. A threshold for was
observed in the region of 120 mV, below which secretion by both species
was inhibited. Treatment of cells with arsenate, which lowered ATP
levels but did not affect
, also reduced secretion from the
periplasm, an indication that there is an ATP requirement for this step
independent of the requirement for
. Secretion across the outer
membrane was also arrested by increasing the osmotic pressure of the
medium, even though cellular ATP levels and
were not affected.
This may be due to disruption of some necessary association between the
inner and outer membranes.
Many Gram-negative bacteria are able to secrete proteins across their inner and outer membranes. Most of them use a route that has been named the general secretory pathway by Pugsley (1), who first described it while studying the secretion of pullulanase by Klebsiella oxytoca. Proteins secreted by this pathway are expressed with typical amino-terminal signal sequences and their transit across the inner membrane appears to be a Sec protein-dependent process. The second step requires a group of 12 or more genes whose products, with the exception of the outer membrane protein PulD, appear to be associated with the inner membrane. Little is yet known about the mechanism by which this apparatus enables secreted proteins to cross the outer membrane, although evidence is accumulating concerning the function of some of its components. For example, the PilD protein of Pseudomonas aeruginosa (the homolog of PulO, also called XcpA) was shown to be a type IV prepilin peptidase that is responsible for processing the prepilin-like precursors of PulG, H, I, and J proteins and their homologs in bacteria with the general secretory pathway (2). In addition, PulE and its homologues contain a consensus ATP binding site, mutations in which prevent secretion (3-5), and it was shown that the Vibrio cholerae homolog, EpsE, is an autokinase that is attached to the inner membrane via interactions with EpsL (4). Finally, PulD and its homologues are thought to form a pore in the outer membrane, based on structural properties and homology to the pIV protein involved in single-stranded DNA bacteriophage morphogenesis (6, 7).
Aeromonas spp. secrete a number of proteins, including the channel-forming toxin aerolysin, the lipase GCAT, and at least one protease. Proaerolysin secretion by A. hydrophila and by A. salmonicida containing cloned A. hydrophila aerA has been studied in the greatest detail. We have shown that the signal sequence is removed cotranslationally during transit across the inner membrane and that the protein folds and dimerizes in the periplasm before it is released from the cell (8-10). The A. hydrophila genes required for translocation across the outer membrane include exeC-N, homologues of pulC-N (11), a second operon exeAB (12), and the tapD gene, encoding the prepilin peptidase (13).
Both the protonmotive force and ATP are known to be required for
Sec-dependent secretion across the inner membrane (for a recent review, see Ref. 14), but little is known of the energetics of
the second step in secretion via the general secretory pathway. Two of
the exe gene products, ExeA and ExeE, contain sequences corresponding to the ATP-binding regions of known ABC transporters, and
direct evidence for an energy requirement for secretion across the
outer membrane has come from a study by Wong and Buckley (15). They
found that CCCP,1 which permeabilizes the inner
membrane to protons causing the collapse of pH and
,
completely prevented the release of the periplasmic pool of
proaerolysin from A. salmonicida. Secretion was also
inhibited by lowering the pH of the growth medium. These two
observations led them to propose that secretion across the outer
membrane requires a protonmotive force. However, in Escherichia coli the loss of
due to treatment with CCCP is eventually
followed by a reduction of ATP levels. In their study of A. salmonicida, Wong and Buckley (15) did not measure ATP and
therefore could not exclude the possibility that it was a diminished
supply of high energy phosphate that caused inhibition of secretion
(perhaps due to inactivation of ExeA or ExeE), rather than the change
in
. More recently, Howard et al. (16) found evidence
that ExeB, a protein with structural similarity to TonB, and ExeA form
an inner membrane complex required for secretion across the outer membrane. Mutations in the ATP binding cassette of ExeA prevented secretion, leading to the hypothesis that the process requires the
energy of phosphate bond hydrolysis provided by ExeA and that this is
transduced to the outer membrane by ExeB.
Here we show that reduced ATP levels are not responsible for the CCCP
effect observed by Wong and Buckley (15), thereby establishing that
there is a specific requirement for . We also show that lowering
ATP levels by treating cells with arsenate leads to a reduction in
secretion under conditions where
is not changed, evidence that
transfer across the outer membrane requires ATP as well as
.
Finally, we show that passage across the outer membrane is also
prevented by exposing cells to hyperosmotic conditions.
CB3 pNB5 (17) was grown at
27 °C to an A600 of 0.4 in Luria Bertani (LB)
medium buffered with Davis medium (18). This was supplemented with
0.2% (w/v) glucose and contained 100 µg/ml ampicillin. The cells
were then induced with isopropyl--D-thiogalactoside (1 mM) and growth was continued until they reached
A600 2. A. hydrophila Ah65 pKW206
(which is pMMB206 containing the aerA gene and its promoter)
was grown in the same way except that the growth temperature was
30 °C, isopropyl-
-D-thiogalactoside was omitted, and
chloramphenicol (2.5 µg/ml) replaced ampicillin.
To measure the potential difference () across
the bacterial inner membrane, the outer membrane must be made permeable
to the membrane potential probe
[3H]tetraphenylphosphonium bromide. The protocol designed
for E. coli (19) was used with the Aeromonas spp.
with minor modifications in the following way. One-ml aliquots of cells
grown to an A600 of 2 were centrifuged for 1 min
at room temperature in a microcentrifuge. The cells were suspended in
200-µl of 100 mM Tris, 1 mM EDTA, pH 7.8, shaken at room temperature for 10 min and centrifuged again. The
pellets were then resuspended in 400 µl of 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS), unless otherwise
stated. Where indicated, this buffer also contained 100 µg/ml
chloramphenicol (for A. salmonicida), or 20 µg/ml
tetracycline (for A. hydrophila), 0.2% glucose, and varying
concentrations of CCCP, arsenate, or sucrose.
[3H]tetraphenylphosphonium bromide (3.7 GBq/mmol) was
then added to 10 µM final concentration and the cells
were incubated for an additional 15 min (5 or 10 min in the case of the
arsenate experiments) at room temperature. Cells were recovered by
filtration on GF/F microfiber filters (Whatman) and washed twice with
the experimental buffer with or without CCCP, arsenate, or sucrose before counting. Corrections were made for nonspecific binding of the
probe as described previously by Ghazi et al. (20), by incubating cells with 40 µM CCCP for 15 min, adding
[3H]tetraphenylphosphonium bromide, and then filtering
immediately.
The cytoplasmic volumes of A. salmonicida and A. hydrophila were both estimated to be approximately 0.07 µl/2 × 109 cells by comparing the relative sizes of the two bacteria to E. coli by electron microscopy, basing the calculations on the known cytoplasmic volume of the latter (1 µl/1 × 109) cells. Aeromonas cell numbers were routinely determined from the relationship 1 × 109 cells/ml = A600 1.6.
Measurement of Cytoplasmic ATPOne-ml aliquots of cells grown to an A600 of 2 were centrifuged as described above. The pellets were resuspended in 500 µl of the buffer used for the secretion experiments and incubated for the given times. After a 1-min centrifugation, the pellets were each resuspended in 200 µl of ice-cold distilled water to which 20 µl of dimethyl sulfoxide were added to permeabilize the cell envelope (21). The suspensions were then incubated for 10 min on ice. This resulted in complete release of cytoplasmic ATP (22). Following this treatment, the samples were centrifuged for 1 min to remove debris and the supernatants were diluted 10-fold with distilled water. ATP measurements were carried out on 10-µl aliquots using an ATP photometer (SAI Technology, model 2000) and 100 µl of luciferin-luciferase assay medium (10 mg/ml; Sigma). The instrument was calibrated with ATP solutions of known concentrations.
Cell Fractionation and Measurement of Proaerolysin ActivityOne-ml aliquots of the cells grown to A600 2 were centrifuged and resuspended in the specified medium containing chloramphenicol (100 µg/ml) or tetracycline (20 µg/ml) and incubated at 25 °C for the given times. Cells were then centrifuged (1 min, room temperature), and aliquots of the supernatants were taken for measurement of proaerolysin activity. The cells were resuspended in 1 ml of 33 mM Tris-HCl, pH 7.5, 20% sucrose, 1 mM EDTA and incubated for 5 min. After a 2-min centrifugation, the pellets were osmotically shocked by rapid resuspension in 200 µl or 1 ml of ice-cold distilled water. After a further 5-min incubation, the suspensions were centrifuged and aliquots of the supernatants were taken for measurement of proaerolysin concentration.
Proaerolysin activity in A. salmonicida was measured essentially as described by Wong and Buckley (15). Proaerolysin activity in A. hydrophila was measured in the same way except that rat erythrocytes, which are more sensitive to aerolysin, were substituted for human erythrocytes (23).
We have previously shown that
A. salmonicida pNB5 contains a sizeable pool of
cell-associated proaerolysin (approximately 1.5 µg in 1 ml of culture
with an A600 of 2.0), virtually all of which is
located in the shockable fraction. The second step in secretion,
transport across the outer membrane, can be measured by following the
decline in this pool with time or by following the appearance of
proaerolysin in the medium. Washed cells resuspended in fresh medium
containing chloramphenicol to prevent synthesis of new protoxin are
used (15, 17). Changes in periplasmic proaerolysin levels in the
presence and absence of CCCP are compared in Fig. 1. In
the absence of CCCP, the amount of proaerolysin associated with the
cells decreased with time, so that after 20 min, only 10% remained.
There was a corresponding increase in the amount of protoxin outside
the cells as we found before (not shown). In contrast, when cells were
treated with 40 µM CCCP, most of the proaerolysin
remained in the periplasm. This is consistent with the results of our
previous experiments in which we used a higher concentration of CCCP
(15). We also examined the effect of CCCP on the release of periplasmic
proaerolysin from A. hydrophila cells containing the plasmid
pKW206. These cells contain a smaller periplasmic pool of proaerolysin
(50-100 ng of proaerolysin in 1 ml of cells at an
A600 of 2.5). When suspended in fresh medium containing tetracycline, these cells secrete the entire pool of accumulated proaerolysin within 5 min (data not shown). Again the
addition of CCCP inhibited secretion, but in this case, even in the
presence of 40 µM CCCP, 40-50% of the protein could be recovered in the supernatant (compare Fig. 2).
Relationship between the Transmembrane Potential and Secretion of Proaerolysin from the Periplasm
When the transmembrane potential
of A. salmonicida was determined by measuring the
accumulation of the radiolabeled lipophilic cation
tetraphenylphosphonium bromide, the value obtained was 170 ± 10 mV (negative inside; mean ± S.D. of three independent measurements), in good agreement with the membrane potentials that have
been reported for other Gram-negative bacteria (24, 25). As with
E. coli (26), declined when A. salmonicida was incubated with increasing concentrations of CCCP. The values obtained were 165, 123, 112, 107, 88, and 0 mV for 0, 2, 5, 10, 20, and
40 µM CCCP, respectively.
In a parallel experiment, secretion of proaerolysin was measured using
cells treated with the same concentrations of CCCP for 5 min. Treatment
with Tris-EDTA was omitted to minimize reduction of the periplasmic
pool, which would otherwise have occurred as a result of secretion
during the incubation (A. salmonicida cells would release
75% of the periplasmic pool during the 15 min needed for the treatment
and subsequent centrifugation steps and A. hydrophila would
release all of it). The results in Fig. 2, A and
B, illustrate the dependence of the release of proaerolysin
from the periplasm on the value of . It may be seen that when
reached 120 mV, secretion started to decrease (more proaerolysin
remained in the periplasm), and when it fell below approximately
90-100 mV, periplasmic levels no longer changed. Thus a threshold
membrane potential of approximately 120 mV is required for efficient
secretion of proaerolysin from both bacteria.
Experiments with E. coli have shown
that depolarization of the cells using a protonophore can affect the
level of cytoplasmic ATP (27). It was thus necessary to consider that
the reduction in the concentration of high energy phosphate was the
actual cause of the block in secretion we observed, rather than the
change in . To determine whether this was the case, cytoplasmic
ATP was measured at various times in control A. salmonicida
pNB5 cells and in cells treated with 40 µM CCCP, using
the conditions defined in Fig. 1. The results in Fig. 3
show that the ATP level in cells treated with the ionophore for 10 min
was the same as the level in control cells. Because the results in Fig.
1 and our previous data (15) show that within this period, control
cells secreted proaerolysin but CCCP-treated cells did not, we can
conclude that secretion was blocked because of the decline in
,
not because the protonophore indirectly lowered energy levels below
those in untreated cells.
Effect of Changes in Cellular ATP on Secretion
Recent results
have suggested that ExeB, a TonB-like protein, and ExeA, a second inner
membrane protein containing a P type ATP binding site, may regulate the
rate of secretion of proaerolysin across the outer membrane (16). To
determine whether aerolysin secretion from the periplasm also depends
on high energy phosphate as this would predict, we studied release from
the periplasm in cells treated with arsenate to reduce cellular ATP
levels. We found that the ATP concentrations in A. hydrophila pKW206 were 7.2, 3.2, 4.0, 1.7, and 0.5 mM
when cells were treated with 0, 1, 2.5, 5, and 10 mM
arsenate, respectively. Fig. 4 shows that the decrease
in the ATP pool caused by arsenate treatment was accompanied by a
reduction in the secretion of proaerolysin. Only 40% of the initial
proaerolysin pool was secreted when the ATP level decreased below 1.7 mM. was measured in EDTA-treated cells incubated for
5 min in the presence of the same concentrations of arsenate used to
lower ATP levels, to determine whether the inhibitor also affected the
membrane potential in this time period. Fig. 4 shows that
remained practically constant between 158 and 150 mV, whatever the ATP
concentration in the cells. Thus we can conclude that secretion is
inhibited as a consequence of a decrease in cellular ATP independent of
any change in
.
Decreasing the pH of the Medium Results in a Decline in
Previous data from Wong and Buckley (15) have shown that
reducing the medium pH results in a decrease in proaerolysin secretion by A. salmonicida. It is possible that this is due to an
effect on , because it is known that decreasing pHout
decreases
in E. coli (28), as well as in other
bacteria (24). To determine whether this was the case,
was
measured with cells resuspended in buffers ranging from pH 5.5 to 8, containing glucose and chloramphenicol. The results are presented in
Fig. 5. It may be seen that when pHout fell
below 6.8,
was reduced to values near the threshold required for
secretion (see Fig. 2). Thus it is possible that lowering the medium pH
reduces secretion at least partly by decreasing
.
Hyperosmotic Conditions Inhibit Secretion
The osmotic shock
procedure used to obtain the periplasmic contents in the experiments
described above involves plasmolysing the cells by incubating them in
20% sucrose for 5 min and then diluting them into ice-cold distilled
water. We observed that proaerolysin does not leave the cells during
the plasmolysis step, which indicated that hyperosmotic conditions
somehow affect transport from the periplasm. To study this phenomenon
further, A. hydrophila pKW206 cells were incubated in PBS
buffer containing variable concentrations of sucrose or NaCl, and
proaerolysin release into the external medium was measured. The results
in Fig. 6 show that an increase in external osmolarity
from 0.28 osmol/liter (no addition of sucrose) to 0.75 osmol/liter (584 mM sucrose) almost totally prevented proaerolysin
secretion. Western blotting showed that the proaerolysin remained
cell-associated: there was no evidence that the trapped protein was
degraded (data not shown here). The hyperosmotic block was not due to a
decrease in below the 120 mV threshold value required for
secretion because
did not decrease significantly with increased
osmolarity (Fig. 6). Secretion was reduced in the same way when NaCl
rather than sucrose was used to increase osmolarity.
The experiments described in this paper extend the preliminary observations of Wong and Buckley (15) and allow a more precise description of the energetics of protein secretion from the periplasm via the general secretory pathway. As in the earlier work, we used A. salmonicida containing aerA from A. hydrophila, but in this study we also used A. hydrophila to minimize the risk that our observations would be peculiar to a specific strain or species. As before, we took advantage of the facts that virtually all of the proaerolysin associated with cells is in the periplasm and that in the presence of chloramphenicol, appearance of proaerolysin outside the cells represents the second step in secretion, namely transfer across the outer membrane.
The protonophore CCCP inhibited secretion from A. hydrophila as well as from A. salmonicida. The reduction in the apparent release of aerolysin appeared to be smaller in A. hydrophila; however, this is likely because it has a much smaller periplasmic pool of proaerolysin than A. salmonicida, which produces considerably more protein from the cloned gene. As a result, if the rate of secretion is approximately the same in both species, a larger fraction of the A. hydrophila pool leaves the periplasm during the time taken for the manipulations required in each experiment. Thus, within 5 min, 50% of the proaerolysin is released by A. salmonicida (Fig. 1), whereas A. hydrophila secretes all of its periplasmic pool in this time. If we consider that complete response to CCCP treatment may require at least 1-2 min, this could account for the fact that we measured 80% retention of proaerolysin in A. salmonicida following CCCP treatment, but only 50% in A. hydrophila under the same conditions.
Our experiments show that inhibition of secretion by CCCP was likely
due to the decrease of that results from treatment with the
protonophore, rather than to an indirect effect on cellular high-energy
phosphate because during the time period of our experiments, ATP levels
in the CCCP treated cells were the same as levels in control cells
(Fig. 3). Conversely, our results with arsenate-treated cells indicate
that secretion also depends upon cellular ATP levels. The observation
that both ATP and
are required for secretion across the outer
membrane is comparable to that made by Marino et al. (29) in
a study of translocation of lipopolysaccharide from the inner membrane
to the outer membrane of E. coli. They found that
translocation is blocked by decreasing the ATP pool with arsenate,
under conditions where the protonmotive force is not reduced, but they
also found that translocation could be prevented by decreasing
with a protonophore under conditions where the ATP level was not
modified.
Because both ExeA and ExeE contain ATP binding sites in their primary
structures, they are obvious candidates to explain inhibition of
secretion by reduced ATP levels. Howard et al. (16) recently pointed out a resemblance between ExeB and TonB, which opens gated ports for the inward movement of ligands across the outer membrane (30). The authors proposed that hydrolysis of ATP by ExeA may cause a
conformational change in ExeB leading to the opening of a secretion
port in the outer membrane. Ligand uptake involving TonB is known to
depend on in some unknown manner (30), and the
dependence
of secretion we observe here may extend the similarity between TonB and
ExeB further. Interestingly, in experiments comparable to ours, Pugsley
and coworkers2 have observed that secretion of
PulA by E. coli containing the cloned Klebsiella
pul genes is not affected by reducing cellular ATP, although as
with aerolysin secretion, it is inhibited by declines in
. In
contrast to secretion by Aeromonas spp., pullulanase secretion by K. oxytoca or by E. coli does not
appear to require a gene product comparable to ExeA. Thus, one way to
rationalize our results with those of Possot et al.2
is to argue that it is the function of ExeA rather than ExeE that is
sensitive to the reductions in cellular ATP caused by the arsenate
treatment. This explanation is also in accordance with previous
proposals (based on homologies between pul genes and type IV
pilin assembly genes) that PulE and its homologs provide energy for the
assembly of the secretion apparatus rather than for the secretion
process itself (3, 5).
Experiments with other bacteria have shown that although increasing
pH by lowering the pH of the external medium results in a decrease
in
, the protonmotive force remains quite constant (180 mV) over
a large range of external hydrogen ion concentrations (24). If this is
the case with Aeromonas spp., then one explanation for the
inhibiton of secretion we observe with reduced medium pH is that the
process depends specifically on the
component of the
protonmotive force. However, it is also possible that the pH effect is
independent of the protonmotive force effect we have observed. This
might be more consistent with the recent observation made by Possot
et al.2 in their study of pullulanase secretion by
E. coli. They found that inhibiton of secretion by lowering
the pH of the medium is irreversible, in contrast to CCCP inhibition.
Perhaps in both E. coli and Aeromonas spp., the
structures of secreted proteins or of some critical component of the
secretory machinery are altered by low pH, reducing the apparent rate
of secretion.
Secretion was also prevented when cells were exposed to hyperosmotic
conditions. This was not a consequence of energy depletion, because
neither nor cellular ATP levels changed during the incubation.
The inner membrane contracts away from the outer membrane under
hyperosmotic conditions (31), and this physical effect may account for
the inhibition of secretion, either through a general disruption of the
secretion apparatus or by prevention of a necessary contact between the
TonB-like ExeB protein and the outer membrane. It is certainly
conceivable that contact between the inner and outer membranes is
required for secretion because there is evidence of such a requirement
both for incorporation of lipopolysaccharide into the outer membrane
and for colicin import by E. coli. (27, 32). However,
Houssin et al. (33) have shown that protonmotive
force-driven transport, facilitated diffusion, and ATP-driven transport
in E. coli are also inhibited by an increase in osmotic
pressure, although neither the membrane potential nor ATP is decreased.
The authors argued that inhibition may be due to conformational changes
in cytoplasmic membrane proteins induced by deformation of the
membrane. In the same way, structural changes in inner membrane
components of the Aeromonas secretory machinery could lead
to inhibition of secretion.
We are grateful to Tracy Lawrence and Vivian Ast for skilled technical assistance.