Vol. 140, No. 1
JOURNAL OF BACTERIOLOGY, Oct. 1979, p. 197-205
0021-9193/79/10-0197/09$02.00/0
Membrane H+ Conductance of Streptococcus lactis PETER C. MALONEY Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Received for publication 22 June 1979
Membrane conductance to H+ was measured in the anaerobic bacterium Streptoccus lactis by a pulse technique employing a low driving force (0.1 pH unit; 6 mV). Over the pH range of 3.7 to 8.5, a constant value for passive H+ conductance was observed, corresponding to 0.2 ,umol of H+/s per pH unit per g, dry weight (1.6 IS/cm2 of surface area). The pH insensitivity of this low basal H+ conductance supports the idea that a circulation of protons can mediate highly efficient energy transductions across the membranes of bacteria. The circulation of protons across bacterial glycolysis (22) after addition of proton conducmembranes allows an indirect coupling between tors is best explained by assuming that low memcertain exergonic and endergonic reactions. For brane conductance to H+ had allowed a near example, in oxidative phosphorylation the initial equilibrium condition to be attained before adoutward movement of protons during electron dition of the ionophores. A few direct estimates transport establishes a difference in the electro- of H+ conductance appear to confirm this expecchemical potential for H+ across the membrane. tation for Escherichia coli (35), Paracoccus deSubsequently, this electrochemical H+ gradient nitrificans (37), and Streptococcus lactis (22). drives ATP synthesis, as protons reenter by way In these instances, estimates obtained at about of the membrane-bound Ca2", Mg2"-stimulated pH 7 (35, 37) or pH 6 (22) indicated that bacteria adenosine 5'-triphosphatase (E.C. 3.6.1.3; display low membrane conductance to H+. The experiments reported here provide quanBF0FI). Alternatively BFoF1 itself can initiate a proton circulation by coupling the extrusion of titative estimates of membrane conductance to H+ to the hydrolysis of ATP, as in certain an- H+ over a wide range of internal and external aerobes. In both cases, a variety of other path- pH (3.7 to 8.5). In S. lactis passive membrane ways are also available for H+ reentry, allowing conductance to H+ is low and invariant within the inward movements of protons to drive the the pH range explored. It is concluded that reactions of substrate accumulation, ion extru- energy transductions in bacteria can take place at high efficiencies when based on the circulation sion, and motility (10, 11, 36). Such events reflect the sequential intercon- of protons. version of various forms of potential energy, and MATERIALS AND METHODS the overall efficiency of these energy transductions is determined by the relative rates at which Bacterial strain and growth conditions. S. lacprotons move inward by specific (coupled to tis (ATCC 7962) was used in these experiments. Cells work performance) or nonspecific (passive, or were grown to early stationary phase (10 to 12 h) in (23) and then "leak") pathways. High efficiencies are achieved the complex medium described earlier washed twice and suspended using 300 mM KCI. Cell only when the major fraction of the proton cir- density was measured turbidimetrically by using a culation passes back into the cell by the coupled, Klett-Summerson colorimeter (no. 42 filter). One milrather than the passive, route. Accordingly, one liliter of a cell suspension at 100 Klett units contains expects that the plasma membranes of bacteria about 165 jig (dry weight) of cells and 0.3 j.I of intrawill be characterized by a relatively low passive cellular water (19, 22). The density of the cells finally permeability to H+, as are the membranes of suspended in 300 mM KCI was about 8,000 Klett units (13 mg [dry weight] per ml). Except for initial centrifmitochondria and chloroplasts (15, 18, 26). Indirect tests support the idea that basal H+ ugations (4°C), all experimental procedures were perconductance of bacterial membranes is low. formed at 25°C. Chemicals. Valinomycin was purchased from Thus, after membrane permeability to H+ is Calbiochem, and N,N'-dicyclohexyl carbodiimide artificially increased (16,33), the collapse of both (DCCD) was obtained from Schwarz/Mann Co. Carmembrane potential and pH gradient indicates bonyl cyanide-p-trifluoromethoxyphenylhydrazone that the prior maintenance of both depended (FCCP) was a gift of P. L. Pedersen (The Johns upon an initially low value of H+ permeability. Hopkins University School of Medicine, Baltimore, Similarly, the stimulation of respiration (37) or Md.). Valinomycin was used at a final concentration 197
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of 1t utM, DCCD at 1 mM, and FCCP at 2 1M. These agents were added to cell suspensions as small volumes of concentrated stocks in ethanol; final ethanol concenitrations did not exceed 0.3%. Carbonic anhvdrase was from Sigma Chemical Co. Measurement of extracellular pH. External pH was nmeasured with a radiometer pH meter (PHM 64) equil)ped with a combined glass electrode (GK 232 IC). A variable voltage was stubtracted from the output of the pH meter so that the residual signal could be amiplified and displayed on a Linear Instruments recortder at 0.2 pH units full scale. Measurement of membrane conductance to H+. I)irect estimates of membranie conductance to ions are usually obtained by examining isotope exchange under steady-state or equilibrium conditions. However, such techniques cannot be used to follow proton nmovements because membranes are much more permeable to water than to H+ (or OH ). An alternative approach has been devised by Mitchell and Moyle (26), based on an earlier observation by Gilby and Few (9). In this method, an equilibrium distribution of H' is suddenly perturbed by addition of a small quantity of acid (or base) to the external phase. Conductanice to H4 is then calculated from measurements of the rate at which H+ returns to equilibrium. This technique is discussed in detail in other reports (26, 27, 37). Only a brief outline of general principles is given here, after which specific experimental conditions are described. In this assay, cells alre suspended in a lightly buffered medium at a cell density sufficiently high so that intracellular buffering power represents a substantial fraction of the total buffering power of the svstemn. After a preincubation period during which protons attain an equilibrium distribution across the membrane, a small quantity of acid is added, deflecting external pH by about 0.1 pH unit. If membrane permeability to H4 is relatively low, the initial (rapid) acidification records a titration of only outer buffering power (B,). With continue(d incubation, as protons eventuallv eniter cells, the final equilibriunm pH reflects titration of the total buffering power of the system (B,). Internal buffering power (B) is obtained as the difference between B, and B,. (B, = B, - B,,). From the measuLrements of buffering powers and the rate at which pH approaches final equilibrium, membrane conductance to H+ is calculated (26, :37):
(B,B
1/
where CM,' represents estinmated conductivity to H+ and T, is the half time with which external pH approaches final equilibrium. In practice, buffering powers may change considerably as pH changes (6, 26; see below). However, within the pH range (0.1 pH unit) caused by the acid pulse, variation of bufferinlg powers is limited to a few percent (10'% or less in the experiments reported here). This does not introduce significant error to either estimates of buffering powers or calculations yielding membrane conductance to H+. I'hese manipulations provide estimates of HE conductance only if the membrane is pernmeable to charged particles other than protons. Otherwise, net
miiovenmenits of H' mav be limited by formationi of an electric gradient (+ inside after an acid pulse) generated by proton diffusion. If this occurs, then the rate at which final equilibrium is approached would be determined by miiembrane conductance to other ions rather than to H+ itself. T'o avoid this kind of error in the experiments described here, cells were treated with valinomycin and suspended in medium containinlg external potassium about e(ual to initial internal potassiulm. ThUs, positivre charge moved as the proton coul(i be balanced by the opposite flow of potassium. In a(lditioni, lermeant anion was present (either Cl [211 or Cl and(i SCN [37]). The presence of the permiieant anion ensured that repeated acid pulses were not )aralleled by for-mationi of equilibrium cationi gra(ients, where internal K+ and H+ were substantially less than external K+ and H+ (and internal pH significantly more alkaline than external pH). Instead, continued acidification, especially to low external pH, would be accompanied by net entry of acid (e.g., HCI), rather than only K+ for H+ exchange. I'hus, external and internal pH were approximately equal over the pH range explored (see below). The following general procedures were used. One milliliter of washed cells in 300 nmM KCI (about 8,000 Klett units; 13 mg, dry weight) was placed in a 1-dram glass vial aloing with 1.7 ml of 300 mM KCI and 0.1 ml of 1 M KSCN. T'he pH of such suspensions was between pH 5.8 and 6.0. Valinomycin was then added (10 MtM, final concentration), and cells were allowed to equilibrate for about 2 h with intermittent mixing. Immediately before assay, 0.1 ml of freshly prepared carbonic anhydrase (20 to 30 mg/ml in 300 mM KCI) was added. The glass vial was then placed on the surface of a 2-cm-thick Lucite block fixed to the surface of a magnetic stirrer. A vigorous mixing with a small magnetic flea was begun after insertion of the pH electrode. After 5 to 10 min, an acid pulse was given, usually as a 20-ul portion of standardized HCI (sometimes H2S0) from a 1.0-ml Hamilton syringe equipped with an automatic "stepper" control. The strength of the acid varied in different preparations (as outer buffering power varied) so that the initial acidifications were about 0.1 pH units. Although the acid pulse was directed at the bottom of the vial away from the electrode tip, some mixing artifact was unavoidable, and the initial change in outer pH was often spuriously high (by 10 to 15%c). Consequently, this initial deflection was not used for calculation of B_. Instead, the initial deflection used for estimating B,, was calculated by back extrapolation to zero time from semilogarithmic plots of pH against time (cf. Fig. 2). Because of the mixing artifact and because the halftime for response of the pH recording system was 2 to :3 s, pH changes during the first 15 s were not generally used in these plots. Specific modifications of this procedure were required in some instances. When assays were performed signiificantly below or above pH 6, an initial pH adjustment was done during the 2-h preincubation period. In these cases, one to four additions of 25 Il of 100 mM HCI or KOH (in 300 mM KCI) were made at about 20-min intervals until the desired pH was attained. By staggering the additions of acid or base, the pH "overshoot" during preincubation could be limited.
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When DCCD-treated cells were tested, the same procedures were followed except that cells were exposed to 1 mM DCCD for 1 h (pH 6) before manipulation of pH during preincubation. In all cases, at least 1 h was allowed for equilibration at the final pH before the experiment was started. Graphical analysis of the pH records was performed as described elsewhere (26, 37). After an acid pulse and after correction for base-line drift (if any), the difference between initial pH and final equilibrium pH was used to estimate B,. To estimate the half-time of approach to equilibrium, the difference between pH at any time and final equilibrium pH was plotted on a logarithmic scale against time. Usually, such plots were linear for three half-times (with one important exception, discussed below), and back extrapolation gave the size of the pH overshoot at zero time. The pH overshoot, when added to the difference between initial pH and final pH, allowed calculation of B,,. Measurements of B, and B,, along with the observed half-time of approach to final equilibrium, were used in calculation of membrane conductance to H+ according to the equation given above. These experiments were performed under conditions of high ionic strength. Nevertheless, estimates of buffering powers (and H+ conductances) have not been corrected for the lowered activity of H+ under these conditions because the data of Kielland (20) indicate that the activity coefficient for H+ would be reduced only slightly, from 1.0 to 0.78. Preliminary experiments performed at pH 4.5 or below revealed both a fast and a slow component to H+ entry after an acid pulse. Because the fast component disappeared when KSCN was omitted from the suspension medium, it was assumed that nonspecific effects of internal SCN- could lead to a serious overestimate of H+ conductance at low pH. Thus, in the work given here, KSCN was replaced by KCI for data collected at pH 5 or below. Several control experiments indicated that at pH 5 to 6 the presence or absence of KSCN had no effect on measurements of either buffering powers or membrane conductance to H+. Eliminating this artifact was especially important to the analysis of H+ conductance in control and DCCD-treated cells at low pH. Near pH 4, the low H+ conductance of DCCD-treated cells was not readily observed when KSCN was present, but was apparent when KSCN was absent. Three observations indicate that these techniques give satisfactory estimates of both membrane conductance to H+ and buffering powers as a function of pH. (i) In many cases FCCP (2 ,uM, final concentration) was added after three to five trials (pH 3.8 to 6.8). After FCCP there was no significant pH change (±0.01 pH units), indicating that H+ was at electrochemical equilibrium, as required. (ii) Further acid pulses (in the presence of FCCP at the appropriate pH) gave an immediate titration of B, due to greatly increased H+ conductance (17). In these cases, calculations of B, before and after FCCP agreed within about ±10%. Comparable agreement was also found below pH 5, where FCCP-mediated H' movements were sufficiently slow to allow estimates of both B, and B,. (iii) Estimates of both membrane conductance to H+ and buffering powers were independent of the
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pH attained during preincubation. For example, when titration with acid was started at pH 7 and extended downward to pH 6, the measurements at the lower pH were not significantly different from estimates obtained when titrations were done using cells first equilibrated at pH 6. This overlap also indicates that the preparations were stable over the several hours required for collection of data. Buffering power and membrane conductance to H+ are presented as functions of external pH. The following argument suggests that external pH and internal pH have approximately the same value in these experiments. If protons are at equilibrium, internal pH may be calculated from the membrane potential using the Nernst relationship. Although the membrane potential was not directly measured in these experiments, its magnitude could be estimated from considerations of a Donnan (equilibrium) distribution (7) of the permeant cations (K+) and anions (primarily Cl-). External pH was about pH 6 for cells suspended in 300 mM KCI without added acid or alkali. Assuming internal potassium of about 350 mM (22, 23) and internal fixed anions of the same equivalent concentration, the Donnan potential would be about -12 mV. Thus, internal pH would be about 0.2 pH units more acidic than external pH. Alkalinization during the preincubation period would be expected to increase the Donnan potential, as effective net entry of KOH occurs. Upon acidification, however, the Donnan potential is expected to decrease, as effective net entry of HCI takes place. It was possible to estimate these changes in the Donnan potential because measurements of B, indicated the approximate net changes required for K+ or Cl- increases upon alkalinization or acidification. Such calculations suggest that at pH 8, the membrane potential was about -18 mV, but about 0 mV at pH 4. Thus, over this range, internal and external pH should be closely matched, with internal pH at most about 0.3 pH units more acidic than external pH (for external pH of 8 or above).
RESULTS Two series of experiments were performed. In the first series, behavior in the region pH 5 to 8 was examined. The second series of experiments compared control and DCCD-treated cells near pH 4, with occasional trials at a more alkaline pH. Behavior at pH 5 to 8. The pH records given in Fig. 1 were selected from three separate experiments in which 0.1 fLmol of H+ was added to cell suspensions after an initial equilibration at pH 5.3, 6.8, or 7.9 (comparable records showing behavior near pH 4 and 6 are given below). In each case, addition of acid led to a rapid titration of B,,, followed by a slower titration of B,. Because similar initial deflections (0.101 to 0.122 pH units) resulted from addition of the same quantity of acid, it is clear that B,, was similar in the three trials (B,, was 69 to 73 ,Lmol of H+/pH unit per g, dry weight). However, after final equilibrium was reached, the net pH changes
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C pH 7.9
minutes .02
pH min
0 ~iMole H+
0. IMole HI
FIG. 2. Decay of the pH overshoot after addition of acid. The pH recordings shown in Fig. I were analyzed graphically, as described earlier (26, 37). At 15- to 30-s interuals, the difference between measured
0 15Mole H+
FIG. 1. Changes in external pH after addition of acid. Data from three separate experiments are given. A tracing of observed changes in external pH after addition of 0.1 jnmol of H+ to cells (0.012 to 0.014 g, dry weight) equilibrated at pH 5.28 (A), pH 6.80 (B), or pH 7.93 (C) is shown. Time and pH scales shown at the right are appropriate for each trial. Dotted lines represent the asymptotes approached as final equilibrium occurred. See text and Fig. 2 for further details.
caused by acid were quite different in each case (for trials A, B, and C, the difference between initial and final pH was 0.034, 0.051, and 0.087 pH units, respectively). Thus, Bi made substantially different contributions to B, as pH was varied between pH 5 and 8 (in trials A, B, and C, B, was 220, 140, and 97 ,tmol of H+/pH unit per g, dry weight, respectively). For these same trials, Fig. 2 shows the apparent exponential nature of the approach of external pH to its final value (half-times of 54, 94, and 59 s, for trials A, B, and C, respectively). The half-times of this decay, along with the estimates of buffering powers, gave calculated H+ conductances of 0.63, 0.26, and 0.23 ,umol of H+/s per pH unit per g, dry weight, in trials A, B, and C. The results of other individual trials from these and three additional experiments are summarized in Fig. 3, where the central graph shows measurements of B,, and B, as a function of pH. Over this pH range, there was a considerable change in both B0 and Bt, with a marked tendency for each to increase as pH became more acidic. As pH was lowered, B, increased more rapidly than B0 so that there was also a tendency for B, to increase as pH fell. Figure 3 (inset) also gives the calculations of B1 over this range. The individual estimates of B, are not shown; instead, B, was calculated as the difference between the two smooth curves that described the behavior
pH and the asymptotic pH (dotted lines in Fig. 1) was plotted on a logarithmic scale against time. Back extrapolation gave the Lalue of the pH overshoot at zero time. The ordinate parameter, F, expresses the difference betuween pH at any time and final pH as a fraction of this zero time overshoot. To convert the ordinate scale to pH units, F should be multiplied by 0.067, 0.054, or 0.035 for samples (A), (B), or (C), respectively. For the three trials shown, the apparent exponential approach of pH to final equilibrium occurred with half-times of 54 s (A), 94 s (B) or 59 s (C).
of Bo and B,. Between pH 8 and pH 5, B, of S. lactis could be calculated accurately by an empirical formula: Bi = (-33.9) pH + 292. Thus, as pH fell from 8 to 4, Bi rose 5.6-fold, from 21 to 123 timol of H+/pH unit per g, dry weight. A similar tendency has been noted by others, who have measured B, over about this same range for P. denitrificans (37), E. coli (6), and Staphlococcus aureus (6). In quantitative terms, Bi of these organisms is about 50% greater than that reported here for S. lactis. (If measurements are expressed on the basis of internal volume, rather than mass, then E. coli and S. lactis have about the same B,.) Although buffering powers changed considerably over this pH range, the calculations of membrane conductance to H+ did not indicate a significant variability in this region (Fig. 5; see
below). Effect of DCCD. From the results of other experiments using S. lactis, it has been concluded that BFoFj catalyzes an obligatory coupling between the synthesis of ATP and the entry of protons (22-24). This conclusion depended upon two assumptions about the properties of cells treated with DCCD, an inhibitor of BFoF, (2, 8, 12, 32): (i) that DCCD had no effect on passive membrane conductance to H+; and (ii) that the inhibitor did not alter B,. It was important to test these assumptions directly,
H+ CONDUCTANCE IN BACTERIA
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300
Bt
A
201
B
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6
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8
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pH FIG. 3. B,, B,,, and B, of S. lactis. Data from six separate experiments are presented. Each pair of points in the central graph gives the results of a single trial of the kind shown in Fig. I and 2, in which 0.1 limol of H+ was added to suspensions of cells equilibrated at various pH's. Calculation of B,, and B, was performed as described in the text. (inset) Points indicate Bi as the difference between the smooth curves describing the behavior of B,, and B, for 0.2 pH unit intervals between pH 5 and 8. The line drawn represents the best fit to these points, obtained by the method of least squares (r2 = 0.99), and shows the equation: Bi = -33.9 pH + 292.
using the techniques described here, because other reports have shown an unexpected increase in apparent membrane H+ conductance for certain strains of E. coli after exposure to DCCD (21, 35). The two experiments shown in Fig. 4 compare the behavior of control and DCCD-treated cells at about pH 4 and 6. At either pH, control and treated cells showed comparable B,, B0, or B, (legend to Fig. 4). Other experiments (pH 3.6 to 6.8) also indicated that the two cell types had similar buffering powers (not shown), and for the region of overlapping pH, measurements in this series of experiments agreed well with those found in the earlier trials (Fig. 3). Moreover, below pH 5, observed Bi was only about 10 to 30% greater than that predicted by the empirical relationship between pH and Bi noted before (inset to Fig. 3). (When experiments were performed at low pH [near pH 4], B0 and B, included contributions made by the aqueous phase as well as by cells. For example, at pH 4 [e.g.,
0.08
fLMole H+
2 min
FIG. 4. Effect of DCCD on H+ conductance at pH 4 and 6. Changes of external pH after addition of acid are shown for two separate experiments comparing cells treated with 1 mM DCCD (in ethanol) or ethanol alone. Dashed lines show the asymptotes
approached during final equilibration; graphical analysis was performed as described in the text (see also Fig. I and 2). (A) 0.4 [Lmol of H+ was added to DCCD-treated cells (pH 3.77) and control cells (pH 3.98). For DCCD-treated and control cells, respectively, values for buffering powers were 406 and 391 (B,), 249 and 215 (B,,), 157 and 176 (B,) ,umol of H+/ pH unit per g, dry weight. H+ conductances for treated and control cells were 0.33 and 3.3 Mmol of H+/s per pH unit per g, dry weight, respectively. (B) 0.08 ,umol of H+ was added to DCCD-treated (pH 6.21) and control (pH 6.19) cells. For DCCD-treated and control cells, respectively, calculated buffering powers were 148 and 135 (B,), 43 and 39 (B,,), 105 and 95 (Bi) tLmol of H+/pH unit per g, dry weight. H+ conductances for treated and control cells were 0.25 and 0.16 ,umol of H+/s per pH unit per g, dry weight, respectively.
Fig. 4A] the iincrease in H+ concentration required for the 0.1 pH unit of acidification accounted for about 20% of the observed B0. Because intracellular water was only a small fraction of total water, less than 1% of Bi could be accounted for by changes of internal H+ concentration at this pH.) Although DCCD did not the alter buffering
power of S. lactis, the presence of the inhibitor did have a marked effect on membrane conductance to H+ under some conditions. This is also illustrated by the experiments shown in Fig. 4. Because control and treated cells showed similar values for buffering powers, relative H+ conductance was indicated directly by the ratio of
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the half-times at which final pH was approached after the acid pulse. At pH 4 (Fig. 4A), the halftime of approach to equilibrium differed by about 10-fold in the two preparations, corresponding to a significant elevation of H+ conductance in control cells (3.2 [imol of H+/s per pH unit per g, dry weight) with respect to DCCD-treated cells (0.33 imol of H+/s per pH unit per g, dry weight). However, at pH 6 (Fig. 4B) these half-times were only slightly different, indicating H+ conductances of 0.25 and 0.16 [imol of H+/s per pH unit per g, dry weight, respectively. This latter difference was not considered significant because in a large number of other trials mean H+ conductance for control cells was about 0.2 [tmol of H+/s per pH unit per g, dry weight (see below). Comparisons at more alkaline pH also indicated similar H+ conductance in control and treated cells (Fig. 5). Membrane conductance to H' as a function of pH. Figure 5 presents the measurements of membrane conductance to H+ made in the two series of experiments discussed above, using control (solid symbols) or DCCD-treated (open symbols) cells. Each point on the graph represents the mean of two to seven trials centering about the pH values given on the abscissa. Several conclusions follow from these measurements. One is that over a wide range (pH 5.5 to 8.5), membrane conductance to H+ was insensitive to the absolute concentration of protons.
Thus, despite a 1,000-fold variation in the concentration of H+, membrane conductance was constant at 0.20 ± 0.02 timol of H+/s per pH unit per g, dry weight (mean ± standard error of the mean, 14 measurements). Moreover, within this range, the presence of DCCD had no effect on H+ conductance (0.24 ± 0.09 timol of H+/s per pH unit per g, dry weight). It is also clear that as pH fell below pH 5, there was an increase in H+ conductance of control cells; for these cells apparent membrane conductance to H+ rose as high as 25-fold over the basal value. In the cases centering about pH 4, mean H+ conductance of control cells was 3.3 ± 0.5 yumol of H+/s per pH unit per g, dry weight, while H+ conductance of cells treated with DCCD was 0.52 ± 0.17 ymol of H+/s per pH unit per g, dry weight. Thus, the elevation seen in untreated cells was largely blocked (90%) by prior exposure to DCCD.
DISCUSSION The major goal of these experiments was to provide a quantitative description of membrane conductance to H+ over a wide range of pH. The results (Fig. 5) indicate that for S. lactis passive H+ conductance is constant at 0.20 fimol of H+/ s per pH unit per g, dry weight, between pH 5.5 and 8.5. Below pH 5 apparent H+ conductance was greatly increased, and it is likely that this reflects a functional dissociation of the F,, and F, sectors of BFOF, at low pH. The F, portion of
5r
.
3' 3
-DCCD
>1.
w)
E
Cu 0N 3 ° I: 1) o0 C
+
2
I 2
I
0
+DCCD
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0
1
3
4
5
WC 6
'I
7
lo
8
1
9
pH FIG. 5. Membrane conductance to H+. Membrane conductance to H+ was calculated (.see text) from acidi pulse experiments of the kind deseribed in Fig. 1, 2, and 4 for control cells (0) or cells pretreated with 1 mM DCCD (0). Data were collected in 12 separate experiments. Each point shown represents the mean value of all trials (tuwo to setven) conducted on a single sample. The pH shown gites the average initial pH in such trials (the pH range explored in any one sample uas less than 0.3 pH units). In calculation of membrane conductance, the estimates of B,, B,,, and B, made in the individual trials were u.sed. When experiments uwere performred belouw pH 5, KSCN was omitted from the incubation medilum (see text).
VOL. 140, 1979
this enzyme is released from isolated membranes during incubation at low ionic strength in the presence of chelators of divalent cations; readdition of divalent cation is usually necessary for reconstitution of the functional complex (1, 34, 39). These observations suggest that salt bridges can be important to a functional association between Fo and F,. If so, then dissociation of Fo and F, might also occur at higher ionic strength, without chelators, when the pH approaches the pK's of the groups participating in salt bridge formation. Because membranes containing only Fo show high H+ conductance that is sensitive to DCCD (2, 31, 32, 35), the results given in Fig. 5 would be expected if critical cation binding groups on either Fo or F1 have pK's of about 4. Other, more complex mechanisms might be suggested, but this appears to be the simplest explanation at the present time. With this assumption, it also follows that H+ conductance of the membrane as a whole (excluding the DCCDsensitive, Fo-mediated component) remained stable over the entire pH range explored (pH 3.7 to 8.5). In the experiments reported here, estimates of H+ conductance were obtained after protons were moved out of equilibrium by about 6 mV (0.1 pH unit). It is of interest to compare these estimates with measurements made when the total driving force on H+ movements was about 200 mV because H+ can be maintained out of equilibrium to this extent in bacteria (3, 6, 10, 33, 36, 40) as well as in mitochondria (4, 28, 29) and chloroplasts (18). Such a comparison is possible for S. lactis because other work (22) has examined H+ movements in response to an artificially imposed electrochemical H+ gradient of 200 mV. In the earlier experiments (22), a membrane potential was generated by addition of valinomycin to cells suspended in low potassium medium; H+ inflow was calculated from measurements of changes in external pH. In four experiments with cells exposed to DCCD, the initial rate of H+ entry was 15 ± 2.3 mmol of H+/min per liter of cell water. For the initial driving force of 200 mV (to which the membrane potential contributed about 180 to 190 mV), this H+ influx would correspond to 0.12 ± 0.02 Mmol of H+/s per pH unit per g, dry weight. It is likely that such H+ movements reflected the passive inflow of protons, solely under the influence of the electrochemical H+ gradient, because the presence of DCCD prevented H+ inflow by way of BF,F, and because net ion movements were restricted to the exchange of H+ and K+ (in the presence of valinomycin). A similarly low estimate of H+ conductance (0.17 ,umol of H+/s per pH unit per g, dry weight) comes from other
H+ CONDUCTANCE IN BACTERIA
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experiments (unpublished) in which an imposed pH gradient represented the dominant driving force (120 to 240 mV) across the membranes of DCCD-treated cells. Thus, there is excellent agreement among the results of several different experimental systems, each of which allows an independent estimate of passive membrane conductance to H+. Considered together, these comparisons suggest that membrane H+ conductance is constant over a considerable range of total driving force, whether an electrical or chemical gradient, and over a wide range of absolute H+ concentration. Moreover, these comparisons provide no support for the idea that in bacteria membrane H+ conductance would deviate from ohmic behavior under physiological conditions; this possibility has been suggested by the results of indirect tests in mitochondria (29). The measurements reported here agree well with similar data available from other studies. Using the estimate (Fig. 5) of 0.2 ,umol of H+/s per pH unit per g, dry weight, and assuming that the early stationary-phase cells (S. lactis) are spheres of 0.5 ,um diameter (5), one may express H+ conductance in electrical units (microsiemens) (26). For S. lactis this yields a conductance of about 1.6 pS/cm2 of membrane surface, a value that compares favorably with the estimates made for P. denitrificans (0.4 MS/cm2 at pH 7; 37); mitochondria (0.4 MuS/cm2 at pH 6; 26); and submitochondrial particles (1.6 MS/cm2 at pH 6; 15). As noted by Mitchell and Moyle (26) these low conductances approach the maximal values obtained for total ion conductance of certain artificial membranes free of protein (14). However, assays of H+ conductance employing this pulse technique must yield measurements representing the net balance between movements of both H+ and OH- ions. In contrast, the studies initiated by Nichols and Deamer (30) have assessed individual H+ and OHfluxes across liposome membranes. The analysis given in that work indicates that the low effective H+ conductance of biological membranes must arise from opposing H+ and OH- fluxes that occur at very high rates. With the measurements of H+ conductance given here, it is also possible to estimate the probable efficiency of energy transductions initiated by BF0Fj in S. lactis. If the electrochemical H+ gradient established during glycolysis is about 200 mV (3, 10, 13), then passive H+ inflow should correspond to about 26 mmol of H+/min per liter of cell water. In turn, this would demand a necessary basal rate for glycolysis of 13 mmol of lactic acid produced per min per liter of cell water, assuming BFoFj extrudes 2H+ per mole-
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cule of ATP hydrolyzed. Maximal rates of glycolysis attributable to turnover of BF(F, in S. lactis appear to be about 180 mmol of lactic acid produced per min per liter of cell water (pH 6; 22; unpublished data). Thus, the passive H+ conductance measured here suggests an overall efficiency of as high as 93% for the interconversion of potential energy available in chemical form (ATP) and that represented by the electrochemical H+ gradient. The chemiosmotic theory assumes (25, 26) that the low H+ conductance measured by acidpulse experiments gives a reliable estimate of passive H+ flow when the electrochemical H+ gradient attains maximal size (200 mV and above). Although indirect tests (26, 29) support this idea, it has not yet been possible to test this directly. In bacteria (10, 33, 36, 40), mitochondria, (28, 29) and chloroplasts (18), the electrochemical H+ gradient can take the form of a membrane potential, a pH gradient, or both. Thus, it is also assumed that passive membrane H+ conductance is not significantly altered by variation in these parameters. Similarly, because energy transductions occur with high efficiencies in everted preparations of bacterial or mitochondrial membranes (4, 38, 40), it might be expected that passive H+ conductance is relatively insensitive to H+ concentration at the (normally) external surface. The experiments and comparisons reported here represent an attempt to explore these predictions with direct measurements; the results obtained fully support these important assumptions.
J. BACTERIOL.
7.
8.
9.
10. 11. 12.
13. 14. 15. 16.
17.
18. 19.
ACKNOWLEDGMENT This work was supported by Public Health Service grant GM 24195 from the National Institute of General Medical Sciences.
20.
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