(Received for publication, September 29, 1994; and in revised form, December 5, 1994)
From the
Halocins H6 and H4 are bacteriocin-like substances capable of
killing sensitive halobacterial cells by affecting the bioenergetic
steady state across the membrane. The effect of either halocin on
living cells is similar, but the primary target of each is different.
Halocin H6 inhibited light-induced Na out-flow in
membrane vesicles of Halobacterium halobium. H4 did not, but
instead slowed the H
return-flow in the dark after
illumination. Halocin H6 adsorbs firmly to both whole cells and
membrane vesicles. The conclusion is that the primary target of halocin
H6 is the Na
/H
antiporter. This is
important not only insofar as it outlines the mechanism by which a
halocin works, but also for the fact that it is the first specific
physiological inhibitor of the halobacterial
Na
/H
antiporter to be described.
Halobacterium halobium is an extremely halophilic
archaebacterium. Halobacteria grow in the presence of NaCl levels as
high as 4 M while maintaining intracellular Na concentrations between 0.3 and 2 M (Christian and
Waltho, 1962; Ginzburg et al., 1970). Under such conditions
the problems of Na
exclusion and maintenance of
µ
Recently, halobacteria were shown to cause
antagonistic interactions due to the production of bacteriocin-like
substances named halocins
(Rodríguez-Valera et al., 1982;
Meseguer et al., 1986). Halocins H4 and H6 are produced by the
archaebacteria Haloferax mediterranei and Haloferax
gibbonsii, respectively. The effect of both is similar (Meseguer
and Rodríguez-Valera, 1985; Torreblanca et al., 1989): the halocins adsorb to sensitive cells,
producing deformation and lysis, leaving empty ``ghosts'' in
which the cell envelopes seem to be intact (Meseguer and
Rodríguez-Valera, 1986; Torreblanca et
al., 1990). To specify the mechanism of action of the
bacteriocins, it is important to identify the sequence of effects after
addition of them. Previous work with these halocins has shown that
several functions are affected in sensitive cells, but only those
related to the cytoplasmic membrane take place shortly after adding the
halocin (Meseguer and Rodríguez-Valera,
1986; Torreblanca et al., 1990; Meseguer et al.,
1991). These are inhibition of uptake and enhancement of release of
-aminoisobutyric acid (a non-metabolizable amino acid) and
alteration of light/induced pH changes mediated by bacteriorhodopsin.
We have already observed that the halocins' effects were quite
similar to those of dicyclohexylcarbodiimide (DCCD), (
)an
ATPase inhibitor, recently shown to also inhibit the
Na
/H
antiporter (Murakami and
Konishi, 1988). Under illumination, protons are pumped out of the cell
and re-enter fundamentally through ATPase and
Na
/H
antiporter; like halocins, DCCD
intensifies the light-dependent acidification of the medium. We showed
in a previous study that halocin H4 did not affect intracellular ATP
levels (Meseguer and Rodríguez-Valera,
1986; Torreblanca et al., 1990). Further, we attempted to
detect any effect of H6 and H4 on the enzyme in a specific assay with
reconstituted/ATPase vesicles and found that the activity was totally
unaffected by either halocin. These findings suggested that the
Na
/H
antiporter could be the target
for H4 and H6. Due to the significance of this hypothesis and future
applications we have studied the effects of H6 and H4 on H.
halobium at two levels: (i) indirectly on whole cells by
analyzing parameters related to Na
/H
antiporter activity: (ii) directly on
Na
/H
exchange in membrane vesicles.
An arbitrary unit (AU) is a measure of halocin activity evaluated in a 2-fold dilution method previously described (Meseguer and Rodríguez-Valera., 1986). AU/P = arbitrary units of halocin per milligram of sensitive cell protein.
Protein was determined by Lowry's method with bovine serum albumin as standard.
H. halobium was grown in a medium containing 0.5% (w/v) yeast extract and 25
µCi/ml [C]methionine in 25% SW, pH 7.2. At
the end of the exponential growth phase the culture was centrifuged,
washed twice in 25% SW, and suspended in the same solution at a final
concentration of 1 mg protein/ml. This suspension was divided into five
1-ml parts, one without halocin as control, and the other four with
410, 820, 1640, and 3280 AU/ml halocin, respectively, and incubated at
37 °C, shaking. Samples (0.1 ml) were taken at 3-min intervals,
filtered through 0.7-µm pore size Whatman filters, and washed twice
with 1 ml of 25% SW. The filters were then placed in vials with 6 ml of
Ready Microliquid scintillation mixture (Beckman) and counted in a
Beckman LS-2800 counter.
Each sample was examined in triplicate and the
experiment was separately reproduced twice. The internal water space
() was calculated as a percentage of total water content of the
pellet and is given by
where pK = the dissociation constant of dimethyloxazoline dione (DMO) = 6.32 at 37 °C.
1 ml of cell suspension at 1 mg protein/ml
was placed in a 37 °C bath and magnetically stirred throughout the
assay. After addition of halocins and 10 µCi/ml
[H]triphenylmethylphosphonium bromide (TPMP) at 2
mM final concentration, 100-µl samples were taken at 3-min
intervals for 21 min and filtered through Whatman GF/F (0.7-µm pore
size) filters which were previously wetted with a 20 µM TPMP solution to avoid unspecific absorbtion. The filters were
then washed with 1 ml of 25% SW three times and counted as above. Each
experiment was carried out six separate times.
[H]TPMP unspecific binding to the cells was
measured in the same way, but the cell suspension was heated for 10 min
at 80 °C before starting the assay. The average of the values thus
obtained were subtracted from the counts obtained for each assay.
The membrane potential () was calculated after the
appropriate correction for nonspecific absorption using the Nernst
equation:
where Z = 2.3 RT/F = 61.5 mV at 37 °C.
Na-free control experiments were
done using 3 M KCl loaded vesicles.
Figure 1:
Killing effect of halocins on H.
halobium cells. Upper panel, starved cells; lower
panel, energized cells; (,
) halocin H4; (
,
) halocin H6; (
,
) external pH 6.0; (
,
)
external pH 7.2.
CFU, colony forming
units.
As with intracellular volume
our data on untreated cells are similar to that obtained by Bakker et al.(1976) for H. halobium cells. As expected
pH increases as the external pH decreases, and pH
between 6.0 and 7.2 external pH was almost constant, only a
slight variation being discernable. The response to halocin treatment
was generally a drop in pH
, except with halocin H4 at
external pH 7.2, where pH
rose, indicating an essential
difference between the mechanisms of action of H4 and H6 (Table 1).
p was dependent on
external pH in all cases with treated and untreated cells, both being
higher at pH 6.0 than at 7.2. Our results show that both halocins cause
the
p to decrease. The effect of halocin H6 on
p was appreciably greater than that of H4.
Figure 2: Typical light-dependent pH change trace of membrane vesicle suspension of H. halobium after halocin treatment. The membrane vesicle equilibrated in 2.9 M KCl, 0.1 M NaCl, 1 mM PIPES (pH 6.8) was illuminated with an actinic light, then pH change of the suspension was measured by a glass electrode using a pH meter. The magnitude of pH changes was determined by 0.01 N HCl pulse. The membrane vesicles were treated with 1000 AU H4 or 2000 AU H6.
In K-loaded vesicles, the
acidification occurred in a single saturation profile without the
Na
-dependent lag.
When the
Na-dependent H-influx activity was determined in
vesicles, the process was found to be markedly inhibited in H6-treated
vesicles (Fig. 3, upper panel). The inhibitory effect
of H4 was appreciably less. After washing treated membrane vesicles
with fresh salt by repeated centrifuging and resuspension (Fig. 3, lower panel), the effect of H4 was completely
eradicated, as shown by the fact that both the light-dependent pH
change profile and the dark-decay kinetics of
H
recovered to the control level. However, the washing treatment
could not remove the effect of H6 (Fig. 3, lower
panel). Inhibition was dependent on H6 concentration as shown in Fig. 4, and the Na
/H
exchange
process was completely suppressed at approximately 1500 AU of H6. At
less than 2000 AU, H4 only slightly restricted but did not quell the
process.
Figure 3:
Effect of halocins H4 and H6 on the
Na/H
exchange in the membrane vesicle
of H. halobium. The membrane vesicles were treated with 1000
AU H4 or 1500 AU H6 (upper panel). The vesicles treated with
halocins and also the control vesicles were washed by repeating
centrifugation and resuspension in the fresh buffered salt medium, then
the light-dependent pH changes were measured as above (lower
panel).
, untreated control;
, H4;
,
H6.
Figure 4:
Concentration dependence of halocin
effect. The membrane vesicles were treated with elevated concentrations
of halocins. The sample volume for measurement was adjusted to that of
halocin-added samples with the solution for each halocin preparation.
, vesicles treated with H4;
, vesicles treated with
H6.
The dark-decay kinetics of light-induced external
H
were found to be significantly enhanced in
H6-treated vesicles. On the other hand H4 significantly slowed the
H
back-flux (Table 3).
Figure 5:
Inhibition of light-dependent
Na extrusion in the membrane vesicles of H.
halobium by halocin H6. Intravesicular Na
was
determined at the indicated illumination time periods by an ion
chromatography. Each data point is the average of two independent
experiments, and the determinations were duplicated for each
experiment.
, untreated control;
, 1500 AU of halocin
H6.
In a general sense, it is not easy to specify the mechanism of action or the primary target of a bacteriocin because of the difficulty in distinguishing primary lesions from secondary effects. In fact we show here an example of two substances whose effects on living cells are quite similar but whose mechanisms of action are different.
In previous reports (Meseguer and Rodríguez-Valera, 1986; Torreblanca et al., 1990), we showed that halocins H4 and H6 induce morphological changes and lysis in sensitive cells. However, here we detected a significant difference since lysis induction is twice as effective with H6 as with H4. H6 also showed greater effectiveness in killing sensitive cells. It was also clear from our results that both halocins are more lethal on energized cells, and most potent at an external pH of 7.2, which is optimal for growth. This indicates that the halocins' primary targets are more susceptible when the membrane is energetically more active or its mechanisms are actively functioning.
The determination of intracellular volume of
halocin-treated cells was essential for pH and
calculation. The observed increase of V
as a
consequence of halocin treatment was not surprising on the basis of the
effects of H4 and H6 on the morphology of sensitive cells; after
halocin addition the cells are progressively transformed into
spheroplasts and ghosts. Although these changes affect final
calculations of
pH and
, this is not the reason for the
data obtained. In fact, dimethyloxazoline dione and TPMP distribution
across the membrane is not dependent on V
, and
radioactivity counting with these tracers showed significant
differences between treated and untreated samples. The variation of
and
pH consequently affects
p calculations. It is important to note that our results in V
,
pH,
, and
p for
untreated control cells are essentially the same as those obtained by
Bakker (Bakker et al. 1976; Helgerson et al., 1983)
for H. halobium and equivalent to those for other neutrophilic
bacteria (Padan et al., 1981).
Several bacteriocins and
killer toxins which act on the membrane of sensitive cells have been
shown to affect . Such is the case of colicin K (Weiss and
Luria, 1978), staphylococcin Pep5 (Sahl, 1985), colicin V (Yang and
Konisky, 1984), and the killer toxin from Pichia kluyveri (Kagan, 1983), all of which form ion-permeable channels in
sensitive cell membranes and immediately destroy the transmembrane
potential difference. Halocins, conversely, did not immediately destroy
the
of sensitive cells, but induced a weaker decrease.
Moreover, the observed light-induced pH changes in halocin-treated
cells do not corroborate the formation of ion-permeable pores. As far
as we know, no bacteriocins or killer toxins have been reported which
behave in the same way as halocins H4 and H6. It is widely reported and
accepted that the Na
/H
antiporter is
closely involved in the regulation of intracellular pH and the control
of cell volume (Padan et al., 1981; Krulwich, 1983; Booth,
1985; Grinstein et al. 1989). Further, as we mentioned above,
some of the observed effects of the halocins were similar to those of
DCCD which inhibits halobacterial Na
/H
antiporter. Therefore, the effects of halocins on whole cells
strongly suggest their inhibitory effects on the
Na
/H
antiporter and/or other ion
transporters. Since H4 shows appreciable differences, the site of
action is different from that of H6. This fact was further corroborated
in membrane vesicles.
The Na/H
antiporter activity is clearly demonstrable when a membrane
vesicle suspension of H. halobium loaded with a small amount
of Na
is illuminated. It has been shown that the
initial suppression of the pH change is intravesicular Na
dependent and is caused by the Na
/H
antiporter activity (Murakami and Konishi, 1987, 1988). As
intravesicular Na
decreases, the
Na
-coupled H
influx becomes
inhibited, and thus the acidification of the medium is enhanced. Since
the passive Na
influx is quite slow, the
intravesicular Na
concentration remains low for
30-60 min in the dark, so that a marked enhancement of the
external pH change occurs when illumination is repeated (upper
traces in Fig. 2). Therefore, the
H
between the initial and second illumination is attributed to the
Na
-dependent H
influx process and is
the direct indicator of the Na
/H
antiporter activity.
The increase of the initial acidification
induced by H6 shows a significant effect of this halocin on
Na/H
antiporter activity, indeed, the
Na
-dependent H
influx activity was
strongly inhibited by H6. Since DCCD produces the same pH change
profile as H6, and H6 did not further affect the pH profile in
DCCD-treated vesicles, H6 seems to inhibit the same
Na
/H
antiporter we previously
reported (Murakami and Konishi, 1989).
The dark decay kinetics of
light-induced external H
were found to be
significantly enhanced in H6-treated vesicles (Table 3). This is
logical because in the control vesicle the electrochemical potential
generated by the primary light driven pumps are accumulated in the
forms of
pH,
pNa
, and
as a
result of antipoter functioning (Konishi and Murakami, 1992).
Furthermore, in H6-treated vesicle,
pNa
is not
created because of an inhibition of Na
/H
antiporter. Thus,
pH which could be the major driving force
for the inward flux of proton after turning off the light will be
greater in H6-treated vesicle than in control. In addition, the passive
proton influx through the antiporter does not occur, since our previous
study showed that neither
pNa
nor
pH
activates the Na
/H
antiporter even if
the antiporter is intact (Murakami and Konishi, 1989, 1990). On the
other hand, H4 rather slowed the
H
dissipation
rate. This is another indication that the site of action of H4 is
different from H6.
The H6-dependent inhibition of the halobacterial
Na/H
antiporter was further
demonstrated by measuring the intravesicular Na
after
illumination. As previously shown by
Na
tracer (Murakami and Konishi, 1988), intravesicular Na
was depleted within 10 min of illumination in untreated vesicles.
However, Na
is retained in the H6-treated vesicles.
This is in accordance with the inhibition of uptake of
-aminoisobutyric acid produced by halocin H6 (Meseguer et
al., 1991). The amino acid transport in these microorganisms has
been described as a Na
gradient-dependent symport
system (McDonald et al., 1977), which is itself generated by
the Na
/H
antiporter. Moreover, some
authors have theorized that this exchanger, and the AIB transport
system may have a common subunit (Krulwich, 1983).
The specific
target of halocin H4 is still not known. Results indicate a possible
effect on passive H permeability of the membrane, but
more experiments will be necessary to pinpoint its mechanism of action.
In contrast, in the case of halocin H6, the evidence was clear. The
strong binding of H6 to vesicles shown here, and to whole cells shown
previously (Torreblanca et al., 1989), suggests that H6
inhibits the Na
/H
antiporter by
specifically binding to it. So far DCCD is the only inhibitor for the
halobacterial antiporter, but DCCD (a carboxyl modifying reagent) also
reacts with other membrane components such as
H
-ATPase. The lack of any specific inhibitor for the
halobacterial antiporter hinders analyses of physiological reaction or
mechanism of action of Na
/H
exchange
in halophiles. The marked inhibitory effect on the
Na
/H
antiporter observed here with H6
is quite gratifying, since it not only proves the exemplary mode of the
bacteriocin's action in hitting the central device utilized by
halobacteria to adapt to highly saline environments, but also provides
a new tool for analyzing the molecular mechanisms of halobacterial
Na
/H
antiporter.