©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Specific Inhibition of the Halobacterial Na/H Antiporter by Halocin H6 (*)

(Received for publication, September 29, 1994; and in revised form, December 5, 1994)

Inmaculada Meseguer (1)(§) Marina Torreblanca (1)(¶) Tetsuya Konishi (2)

From the  (1)Department of Genética y Microbiología, Facultad de Medicina, Universidad de Alicante, Campus de San Juan, Apartado 374, 03080 Alicante, Spain and the (2)Department of Radiochemistry/Biophysics, Niigata College of Pharmacy, Kamishin/ei 5/13/2 Niigata, 950/21 Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 Deltaµ

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 alpha-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), (^1)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.


MATERIALS AND METHODS

Bacterial Strains

H. gibbonsii strain Ma 2.39 (Torreblanca et al., 1986) and H. mediterranei ATCC 33500 were used as sources of halocins H6 and H4, respectively. H. halobium NRC 817 was used as sensitive strain. Membrane vesicles were obtained from H. halobium R(1)M(1).

Culture Conditions, Purification, and Activity Assays

These were as described previously (Meseguer and Rodríguez-Valera, 1985; Torreblanca et al., 1989). Except when indicated, media and salt solutions contained a mixture of marine salts referred to as SW (Rodríguez-Valera et al., 1983).

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.

Cellular Lysis at Different Times and Doses of Halocin

It was important to use adequate doses of halocins to ensure that after 30 min the treated cells remained whole and not lysed. We used L-[methyl-^14C]methionine (New England Nuclear) an amino acid essential for the growth of H. halobium (Dundas et al., 1963).

H. halobium was grown in a medium containing 0.5% (w/v) yeast extract and 25 µCi/ml [^14C]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.

Killing Effect of Halocins on H. halobium Cells

This assay was done at two different metabolic states of sensitive cells, fresh cells in the exponential growth phase and starved cells. In the first case an exponential growth phase culture was centrifuged, suspended at 1 mg of protein/ml in 25% SW solution, and immediately assayed. In the second, the cells were suspended in the same way and then kept at 37 °C for 48 h. Both suspensions were diluted to give an approximate content of 10^4 cells/ml. Dilutions were made in two different buffers, 0.1 M MES in 25% SW, pH 6.0, and 0.05 M HEPES in 25% SW, pH 7.2. Similarly, 2-fold dilutions of the concentrated halocins were made in the same buffers, then 50 µl volumes of each dilution were mixed with 950 µl of each cell suspension. As a control, 950 µl of the cell suspensions were mixed with 50 µl of each buffer. After 18 h of incubation at 37 °C, 0.1 ml of each mixture was plated on solid medium. Colonies were counted after 1 week of incubation at 37 °C.

Effect of Halocins on Intracellular Volume of H. halobium Cells

1-ml fractions of H. halobium cell suspension in 25% SW at 1 mg protein/ml were placed into microtubes with halocins (H4 or H6) or 25% SW as controls. All the tubes were incubated for 20 min at 37 °C, then ^3H(2)O at 5 µCi/mg of cell protein and [^14C]dextran at 2.5 µCi/mg of cell protein were added to each tube, incubated for an additional 10 min and centrifuged for 10 min at 13,000 revolutions/min. Aliquots (10 µl) of the supernatant fluid were added to 4 ml of scintillation liquid and counted. The remaining supernatant fluid was eliminated and the pellet, dried with filter paper, was suspended in 100 µl of 1 M NaOH and gently shaken overnight at 37 °C. Aliquots (10 µl) were added to scintillation liquid and counted as above. To avoid differences in quenching, 10 µl of 1 M NaOH were added to the samples of supernatant fluid, and 10 µl of 25% SW were added to the samples of the pellets before counting the samples.

Each sample was examined in triplicate and the experiment was separately reproduced twice. The internal water space (alpha) was calculated as a percentage of total water content of the pellet and is given by

Effect of Halocins on Intracellular pH of H. halobium Cells

Intracellular pH was studied at two external pH values using buffers as in cellular lysis assay. Cell suspensions at 1 mg protein/ml were incubated for 30 min in presence of halocins and 8 µM [^14C]dimethyloxazoline dione. From this point onward the methodology was the same as in the V(i) study. Intracellular pH can be calculated using the Mitchell equation:

where pK = the dissociation constant of dimethyloxazoline dione (DMO) = 6.32 at 37 °C.

Effect of Halocins on Membrane Potential Difference

The membrane potential difference across the membrane was studied at two external pH values using the buffers referred to in the cellular lysis assay.

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 [^3H]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.

[^3H]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 (Delta) 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.

Proton Motive Force Calculations

The proton motive force (Deltap) consisting of a proton concentration gradient (DeltapH) and a membrane electrical potential (Delta) can be expressed as:

Membrane Vesicle Preparation

Membrane vesicles were prepared by a freeze-thawing method (Murakami and Konishi, 1989) and suspended in 2.9 M KCl, 0.1 M NaCl, 1 mM PIPES (pH 6.8) at a protein concentration of 1 mg vesicle protein/ml. The vesicle suspension was stored in a refrigerator for at least 3 days to attain equilibration of ion distribution before use. Protein was determined using BCA protein assay kit (Pierce Chemical Co. Ltd.) with bovine serum albumin as standard.

Halocin Treatment of the Membrane Vesicles

Halocins H4 or H6 were added to 2 ml of the vesicle suspension and stored at 4 °C in a refrigerator overnight. For the control, the same volume of the solvent was added instead of halocin solution.

Light-dependent pH Change and the Sodium-dependent pH Change Measurement

Light-induced pH changes of the vesicle suspension were measured by a glass microelectrode as described previously (Murakami and Konishi, 1985, 1988). The Na-dependent fraction of the pH change was determined by repeating illumination again at 5 min after measurement of the first light-dependent pH change trace of the suspension, and the difference between the first and second pH traces was evaluated as the Na-dependent H influx activity of the vesicles. The vesicles treated with halocins and also the control vesicles were washed by repeating centrifuging and resuspension in the fresh buffered salt medium, then the light-dependent pH changes were measured as above.

Na-free control experiments were done using 3 M KCl loaded vesicles.

Ion Chromatographic Determination of Light-dependent Na Efflux

The vesicle suspension prepared above was illuminated under the same conditions used for the pH change measurement. At the defined time period of illumination, 100 µl of the suspension was taken and diluted into 1.2 ml of 6 M glycerol containing 20 mM MgSO(4) in a centrifuge tube and subjected to centrifuging at 15,000 revolutions/min for 5 min at 4 °C using Hitachi model CRI5B centrifuge apparatus. The pellet thus obtained was rinsed with the same dilution solution and suspended in 1 ml of distilled water. After a brief sonication, an aliquot was injected into a Dionex 4500 l model ion chromatographic apparatus equipped with CSg-12 and CS-12 columns for determining Na, K, and Mg. The elution solution and the suppressor solution used were 20 mM methane sulfonate (1 ml/min) and 40 mM tetramethylammonium hydroxide (5 ml/min), respectively. The Na retained in the vesicles was calculated after subtracting the external contribution of each ion obtained from the Mg peak height and then compared as protein base. Each data point is the average of two independent experiments, and the determinations were duplicated for each experiment.


RESULTS

Cellular Lysis at Different Times and Doses of Halocins

Untreated cells retained ^14C-labeled methionine, which indicates their integrity. The results obtained in treated cells after 30 min of exposure to halocins demonstrated that while those treated at or below 1640 AU/P of H4 or 820 AU/P of H6 retained ^14C-labeled methionine, treatment at higher doses gave erratic values.

Killing Effect of Halocins on H. halobium Cells

Fig. 1shows first that H6 kills sensitive cells more effectively than H4, as the doses needed to kill all the cells are appreciably smaller in all cases. It was evident that both halocins are more effective on energized cells, since four to six times the quantity of either halocin was required to exterminate starved ones. The halocins were most efficient at pH 7.2, which is also optimal for H. halobium growth.


Figure 1: Killing effect of halocins on H. halobium cells. Upper panel, starved cells; lower panel, energized cells; (circle, box) halocin H4; (bullet, ) halocin H6; (circle, bullet) external pH 6.0; (box, ) external pH 7.2. CFU, colony forming units.



Effect of Halocins on Intracellular Volume and Intracellular pH

The V(i) (internal water space in cell pellets), was calculated from the differential distribution of ^3H(2)O and [^14C]dextran. Using the modified microcentrifuging technique, we obtained appreciably smaller standard deviations than those previously reported (Bakker et al., 1976; Helgerson et al., 1983), although the average V(i) value was quite similar. The percentage of internal space of the pellet (alpha) was 48% ± 2 on the basis of 24 determinations. Intracellular volume did not change between 6.0 and 7.2 external pH. After the addition of halocins, the intracellular volume increases, and the rise was dose-dependent. At the same dosage, 820 AU/P, with H6 the intracellular volume increased by up to 40%, while halocin H4 only produced a 5% increase (Table 1).



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 DeltapH increases as the external pH decreases, and pH(i) 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(i), except with halocin H4 at external pH 7.2, where pH(i) rose, indicating an essential difference between the mechanisms of action of H4 and H6 (Table 1).

Effect of Halocins on Membrane Potential Difference Across the Membrane and Proton Motive Force

Table 2presents mean values obtained for DeltapH, Delta, and Deltap. The halocins produced a dose-dependent drop in Delta, which reached the lowest level after 12-15 min of treatment. This decrease was more apparent at 7.2 external pH than at 6.0, and again halocin H6 was more effective than H4. When the values were compared as a percentage with respect to the controls, at 6.0 external pH halocin H4 (1640 AU/P) induced a Delta drop of 11%, and H6 (820 AU/P) a drop of 22%. At 7.2 external pH, the Delta dropped 14% with halocin H4 (1640 AU/P) and 35% with H6 (820 AU/P).



Deltap 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 Deltap to decrease. The effect of halocin H6 on Deltap was appreciably greater than that of H4.

Effect of Halocins on Light-dependent pH Change Trace of Membrane Vesicles

When the membrane vesicle of H. halobium loaded with a small amount of Na is illuminated with a 570 nm light, a stepwise pH change occurs in the external medium as shown in Fig. 2. Medium acidification is slightly reduced in the initial stage of illumination and then gradually enhanced. Halocin H4 did not change the pH profile significantly, but H6 considerably enhanced the initial acidification phase and the diphasic pH change disappeared.


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 DeltaH 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). box, untreated control; circle, H4; bullet, 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. circle, vesicles treated with H4; bullet, vesicles treated with H6.



The dark-decay kinetics of light-induced external DeltaH were found to be significantly enhanced in H6-treated vesicles. On the other hand H4 significantly slowed the H back-flux (Table 3).



Effect of Halocin H6 on Light-dependent Na Efflux

Fig. 5shows that under illumination intravesicular Na was quickly removed from vesicles. After 6 min only 20% was retained within the vesicles. When H6-treated vesicles were illuminated only a slight reduction of less than 10% occurred.


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. circle, untreated control; bullet, 1500 AU of halocin H6.




DISCUSSION

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 DeltapH and Delta calculation. The observed increase of V(i) 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 DeltapH and Delta, this is not the reason for the data obtained. In fact, dimethyloxazoline dione and TPMP distribution across the membrane is not dependent on V(i), and radioactivity counting with these tracers showed significant differences between treated and untreated samples. The variation of Delta and DeltapH consequently affects Deltap calculations. It is important to note that our results in V(i), DeltapH, Delta, and Deltap 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 Delta. 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 Delta 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 DeltaH 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 DeltaH 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 DeltapH, DeltapNa, and Delta as a result of antipoter functioning (Konishi and Murakami, 1992). Furthermore, in H6-treated vesicle, DeltapNa is not created because of an inhibition of Na/H antiporter. Thus, DeltapH 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 DeltapNa nor DeltapH activates the Na/H antiporter even if the antiporter is intact (Murakami and Konishi, 1989, 1990). On the other hand, H4 rather slowed the DeltaH 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 alpha-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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 34-6-5659811.

Recipient of a fellowship from the Generalitat Valenciana.

(^1)
The abbreviations used are: DCCD, dicyclohexylcarbodiimide; AU, arbitrary unit(s); MES, 4-morpholineethanesulfonic acid; TPMP, triphenylmethylphosphonium bromide; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. Kikuo Oikawa and Noriko Imaizumi of the Department of Hygiene Chemistry, Niigata College of Pharmacy for their helpful guidance in using Ion Chromatography for Na analysis. The secretarial assistance of K. Hernández is gratefully acknowledged.


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