INTRODUCTION

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Most microorganisms are neutrophiles, since they survive only at pH values ranging from 5 to 8.5 and exhibit maximum growth rates at pH 7.4 (24). There is, however, a diverse group of bacteria that thrive in highly alkaline environments (11). Bacillus alcalophilus is an obligate alkalophile that can grow at pH values ranging from 8.5 to 11.5, and optimum growth occurs at pH 10.6 (12). It has been suggested that the obligate alkalophiles fail to grow at neutral pH because their membranes become leaky (2). In addition, Krulwich et al. (13) encountered difficulties when they measured the buffering capacities (as determined with suspensions of cells permeabilized with Triton X-100 or n-butanol) of two alkalophilic bacteria, B. alcalophilus and Bacillus firmus RAB, at pH values below 6.5 due to loss of cell integrity.

The work presented here is the last part of an extensive study of the buffering capacity and membrane H⁺ conductance of gram-negative and gram-positive bacteria (17-22). We used a method in which the decay of an acid pulse is used to determine both parameters (15). By using this approach, we avoided the technical problems of the method involving permeabilizing cells, as described by Krulwich et al. (13). Here we report buffering capacity and membrane H⁺ conductance values for the following gram-positive bacteria: two mesophilic neutrophiles, Staphylococcus aureus and Bacillus subtilis, and the obligately alkalophilic bacillus B. alcalophilus. We measured both parameters in B. alcalophilus cells grown in two media at pH 8.5 and 10.5.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. The bacterial strains, media, and growth conditions used in this study are listed in Table 1. All strains were grown to the early stationary phase (13 h for neutrophiles and for the alkalophile grown in L-malate-carbonate medium and 24 h for the alkalophile grown in H.A. medium) and then washed three times in 300 mM KCl. The washed cells were centrifuged and resuspended in 300 mM KCl to final concentrations of 0.5 to 16 mg of cell protein per ml (1 × 10⁹ to 6 × 10⁹ cells per ml).

Protein determination. Protein concentration was determined by the method of Lowry et al. (14). Bovine serum albumin was used as the standard.

Buffering capacity and membrane H⁺ conductance. The buffering capacity and the membrane H⁺ conductance of the bacteria studied were measured by an acid pulse technique, as described elsewhere (15-23). Experiments were conducted in 10-ml glass vials with 7-ml samples of cell suspensions which were stirred magnetically at room temperature. The pH values of these suspensions were 6.2 (neutrophiles), 8.0 (alkalophile grown in H.A. medium), and 7.0 (alkalophile grown in L-malate medium). Valinomycin (final concentration, 10 µM; Sigma Chemical Co.) was then added from small volumes of concentrated stock solutions in acetone; the final acetone concentrations did not exceed 0.2%. The cells were allowed to equilibrate for about 2 h with intermittent mixing. Immediately before the assay, 0.23 ml of freshly prepared carbonic anhydrase (20 mg ml¹ in 300 mM KCl; Sigma) was added. Vigorous mixing with a small magnetic flea was performed after insertion of the pH electrode. After 5 to 10 min, an acid pulse was added, usually as a 50- to 150-µl portion of 100 mM HCl in 300 mM KCl, and changes in external pH were recorded for 3 min.

	RESULTS

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The buffering capacity and membrane H⁺ conductance of Staphylococcus aureus ATCC 9144 and B. subtilis ATCC 6633 were measured at pH 4.04 to 7.12 and at pH 3.74 to 7.82, respectively. The pH ranges studied for titrations of B. alcalophilus ATCC 27647 were 5.62 to 8.07 and 6.92 to 8.72 (cells grown in H.A. medium at pH 8.5 and 10.5, respectively), as well as 3.93 to 7.98 and 4.99 to 8.39 (cells grown in L-malate-carbonate medium at pH 8.5 and 10.5, respectively).

Figure 1 summarizes the values obtained for B_o and B_t as a function of pH for each bacterium studied. Over the pH ranges used, there were considerable changes in B_o, B_t, and B_i. Cells of the alkalophilic bacterium B. alcalophilus grown in H.A. medium at pH 8.5 and 10.5 exhibited higher buffering capacity values than cells of the neutrophilic bacteria Staphylococcus aureus and B. subtilis. The neutrophilic bacteria studied had maximum B_o and B_t values at low pH values, and B. alcalophilus grown under the same conditions exhibited maximum B_o and B_t values at a pH near neutrality. Staphylococcus aureus exhibited maximum B_o and B_t values of 2,325 nmol of H⁺/pH unit per mg of protein at pH 4.04 and 3,697 nmol of H⁺/pH unit per mg of protein at 4.4, respectively (Fig. 1A). The suspensions of B. subtilis exhibited maximum B_o and B_t values of 1,620 nmol of H⁺/pH unit per mg of protein at pH 3.74 and 2,657 nmol of H⁺/pH unit per mg of protein at pH 4.3, respectively (Fig. 1B). B. alcalophilus grown in H.A. medium at pH 10.5 exhibited B_o and B_t values that were 3- to 10-fold greater than those of cells grown at pH 8.5 (Fig. 1C and D). Cells grown at pH 8.5 exhibited maximum B_o and B_t values of 5,672 nmol of H⁺/pH unit per mg of protein at pH 6.6 and 9,043 nmol of H⁺/pH unit per mg of protein at pH 6.8, respectively. B. alcalophilus cells grown at pH 10.5 exhibited maximum B_o and B_t values of 16,603 nmol of H⁺/pH unit per mg of protein at pH 6.9 and 92,792 nmol of H⁺/pH unit per mg of protein at pH 7.4, respectively.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Bo, Bt, and Bi values for Staphylococcus aureus ATCC 9144 (A), B. subtilis ATCC 6633 (B), B. alcalophilus ATCC 27647 grown in H.A. medium at pH 8.5 (C) and pH 10.5 (D), and B. alcalophilus grown in L-malate-carbonate medium at pH 8.5 (E) and pH 10.5 (F). prot, protein.

To determine whether the high buffering capacity of B. alcalophilus was dependent on the culture conditions, B. alcalophilus was cultivated aerobically in L-malate-carbonate medium. We found again that B. alcalophilus cells grown at pH 10.5 had a higher buffering capacity than cells grown at pH 8.5. As shown in Fig. 1E and F, cells of the alkalophilic bacterium B. alcalophilus grown aerobically in L-malate-carbonate medium at pH 8.5 and 10.5 exhibited lower buffering capacity values than B. alcalophilus cells grown in H.A. medium. Cells grown at pH 8.5 exhibited maximum B_o and B_t values of 488 and 862 nmol of H⁺/pH unit per mg of protein, respectively (both at pH 3.93). B. alcalophilus cells grown at pH 10.5 exhibited maximum B_o and B_t values of 661 nmol of H⁺/pH unit per mg of protein at pH 6.4 and 1,266 nmol of H⁺/pH unit per mg of protein at pH 6.12, respectively.

B_i values were calculated from smooth curves that described the behavior of B_o and B_t (Fig. 2). Staphylococcus aureus and B. subtilis had comparable B_i values at external pH values below 7.0. Staphylococcus aureus exhibited a maximum B_i value of 1,594 nmol of H⁺/pH unit per mg of protein at pH 4.5, and B. subtilis exhibited a maximum B_i value of 1,533 nmol of H⁺/pH unit per mg of protein at pH 4.5. The suspensions of B. alcalophilus grown in H.A. medium at pH 10.5 exhibited B_i values that were 20-fold greater than those of cells grown at pH 8.5. Cells grown at pH 8.5 exhibited a maximum B_i value of 3,845 nmol of H⁺/pH unit per mg of protein at pH 7.1. B. alcalophilus cells grown at pH 10.5 exhibited a maximum B_i value of 82,233 nmol of H⁺/pH unit per mg of protein at pH 7.4. Clearly, the B_i values of B. alcalophilus grown aerobically in L-malate-carbonate medium were far lower than those of B. alcalophilus grown in H.A. medium, both at pH 8.5 and at pH 10.5, over the pH range studied. The B_i of cells grown at pH 8.5 declined as the pH increased from 3.93 to 7.98, and the maximum B_i value was 373 nmol of H⁺/pH unit per mg of protein. B. alcalophilus cells grown at pH 10.5 exhibited B_i values that were somewhat higher than those of cells grown at pH 8.5. The suspensions of B. alcalophilus grown at pH 10.5 exhibited a maximum B_i value of 667 nmol of H⁺/pH unit per mg of protein at pH 5.77. 

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Bi values for Staphylococcus aureus, B. subtilis, and B. alcalophilus. Bi values were calculated from smooth curves that described the behavior of Bo and Bt. prot, protein.

The passive H⁺ conductance of the species studied and the buffering capacity were sensitive to the proton concentration at the external surface over the pH range studied (Fig. 3). Staphylococcus aureus had a maximum membrane H⁺ conductance of 10.29 nmol of H⁺/s per pH unit per mg of protein at pH 4.4 and a minimum membrane H⁺ conductance of 1.97 nmol of H⁺/s per pH unit per mg of protein at pH 7.1. B. subtilis had a maximum membrane H⁺ conductance of 6.73 nmol of H⁺/s per pH unit per mg of protein at pH 4.4 and a minimum membrane H⁺ conductance of 0.76 nmol of H⁺/s per pH unit per mg of protein at pH 7.8. B. alcalophilus grown in H.A. medium at pH 10.5 exhibited a maximum membrane H⁺ conductance of 130 nmol of H⁺/s per pH unit per mg of protein at pH 6.9 and a minimum membrane H⁺ conductance of 28.5 nmol of H⁺/s per pH unit per mg of protein at pH 8.7. B. alcalophilus grown at pH 8.5 had a maximum membrane H⁺ conductance of 28.4 nmol of H⁺/s per pH unit per mg of protein at pH 7.0 and a minimum membrane H⁺ conductance of 4.5 nmol of H⁺/s per pH unit per mg of protein at pH 8.0. B. alcalophilus grown aerobically in L-malate-carbonate medium at pH 10.5 exhibited a maximum membrane H⁺ conductance of 3.42 nmol of H⁺/s per pH unit per mg of protein at pH 6.27 and a minimum membrane H⁺ conductance of 0.52 nmol of H⁺/s per pH unit per mg of protein at pH 8.39. Cells grown at pH 8.5 had a maximum membrane H⁺ conductance of 2.31 nmol of H⁺/s per pH unit per mg of protein at pH 3.93 and a minimum membrane H⁺ conductance of 0.03 nmol of H⁺/s per pH unit per mg of protein at pH 7.98 (data were obtained from smooth curves that described the behavior of passive proton conductance).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Membrane H+ conductance of Staphylococcus aureus (A), B. subtilis (B), B. alcalophilus grown in H.A. medium at pH 8.5 (C) and pH 10.5 (D), and B. alcalophilus grown in L-malate-carbonate medium at pH 8.5 (E) and pH 10.5 (F). prot, protein.

	DISCUSSION

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The results reported here provide estimates of the buffering capacity and membrane H⁺ conductance values for neutrophilic and alkalophilic gram-positive bacteria. The method used gave reproducible measurements of these parameters over a wide range of external pHs. The differences between our results and the results obtained by other workers for the same species (3, 13) could be due to the culture conditions used, the solutions employed to prepare bacterial suspensions, and the technical approaches used. In addition, buffering capacity and membrane conductance for protons vary from one strain to another, as has been shown for Serratia marcescens (17). Staphylococcus aureus exhibited greater buffering capacity and membrane H⁺ conductance values than the acidophilic bacterium Lactobacillus acidophilus (18). The buffering capacity values obtained for B. subtilis were somewhat lower than those obtained by Krulwich et al. (13) with suspensions of permeabilized cells. Moreover, at pH values ranging from 8 to 7, the B_o and B_t values for B. alcalophilus grown aerobically in L-malate-carbonate medium at pH 10.5 were comparable to those reported by Krulwich et al. (13) for the same bacterium grown under the same cultural conditions. The buffering capacity and membrane H⁺ conductance values for B. subtilis and B. alcalophilus grown aerobically were in the range found for other bacteria (13, 17, 18, 20).

We obtained quantitative estimates of the buffering capacity and membrane H⁺ conductance of B. alcalophilus at external pH values below 9.0. Under these pH conditions, imposition of a valinomycin-mediated potassium difussion potential is a way to energize ATP synthesis (5). The H⁺ translocation involved in ATP synthesis could increase the buffering capacity values obtained by the acid pulse technique. In order to avoid this possibility, cells were allowed to equilibrate for about 2 h with intermittent mixing after valinomycin addition. In this study we showed that the buffering capacity of B. alcalophilus cells grown at pH 10.5 was greater than the buffering capacity of cells grown at pH 8.5. Interestingly, the range of external pHs at which we could measure the buffering capacity and membrane H⁺ conductance of B. alcalophilus depended on the growth conditions. Suspensions of B. alcalophilus cells grown at pH 8.5 in H.A. medium and in L-malate medium were more stable under acidic conditions than suspensions of cells grown at pH 10.5 in the same media. Also of importance is the finding that B. alcalophilus cells grown in L-malate medium exhibited greater resistance to low external pH values than cells grown in H.A. medium. The resistance seemed to correlate with the pH values of the suspensions.

Our results show that the buffering capacity and membrane H⁺ conductance of B. alcalophilus are influenced by the pH of the medium and culture conditions. It has been shown that certain enzymatic activities can be induced by a change in external pH (6) and that surface characteristics of gram-positive bacteria depend on the pH of cultures and the energized state of the membrane. Aono et al. (1) demonstrated that cell walls of Bacillus lentus C-125, an alkalophile, grown at pH 10.0 were about 20% thicker than the cell walls of organisms grown at pH 7.0 and that they had about three times the negative charge density of the cell walls of strain C-125 cells grown in a neutral environment. Differences in the proton motive force between respiring and fermenting cells have been reported for gram-positive and gram-negative bacteria. Aerobically growing cells had a greater proton motive force than cells growing anaerobically, and these differences depended on the pH of the medium (8, 9). In a different study, it was reported that the cell wall of B. subtilis was less negatively charged when the bacteria were metabolizing a carbon source and creating a proton motive force than when the cells had deenergized membranes (10). Our buffering capacity results for B. alcalophilus agree well with these reports.

Membrane H⁺ conductance is the rate at which protons leak inward, and the balance among membrane H⁺ conductance, the proton motive gradient, and the rate of outward pumping determines whether a bacterial cell can sustain an appropriate pH gradient under acid or alkaline conditions. This study revealed that B. alcalophilus grown aerobically had membrane H⁺ conductance values comparable to those found for neutrophiles over the pH range studied (17-21). It has to be noted that we could not measure the membrane conductance for protons of B. alcalophilus at high external pH values. It was not possible to raise the pH of the suspensions to an initial alkaline pH of more than 9.0. The external pH values of the suspensions dropped quickly even when small and gradual titrations upward were conducted. The membrane H⁺ conductance of B. alcalophilus grown in H.A. medium was extraordinarily high. B. alcalophilus fermenting cells had membrane H⁺ conductance values comparable to those reported for Halobacterium halobium, an archaeobacterium with gated ion channels (22). It will be interesting to study the pH response or pH homeostasis of B. alcalophilus fermenting cells.