Streptococcus sanguis

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1 JOURNAL OF BACTERIOLOGY, Nov. 1986, p /86/1114-5$2./ Copyright 1986, American Society for Microbiology Vol. 168, No. 2 ATP-Driven Calcium Transport in Membrane Vesicles of Streptococcus sanguis HUO-SHU HOUNG, ANITA R. LYNN, AND BARRY P. ROSEN* Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 2121 Received 24 March 1986/Accepted 2 June 1986 Calcium transport was investigated in membrane vesicles prepared from the oral bacterium Streptococcus sanguis. Procedures were devised for the preparation of membrane vesicles capable of accumulating 45Ca2+. Uptake was ATP dependent and did not require a proton motive force. Calcium transport in these vesicles was compared with 45Ca2+ accumulation in membrane vesicles from Streptococcus faecalis and Escherichia coli. The data support the existence of an ATP-driven calcium pump in S. sanguis similar to that in S. faecalis. This pump, which catalyzes uptake into membrane vesicles, would be responsible for extrusion of calcium from intact cells. Maintenance of intracellular calcium ion at concentrations below the extracellular concentration is an almost universal property of living cells. The mechanisms for calcium homeostasis fall into two classes: secondary calcium-cation exchangers and primary calcium pumps (9). Both are frequently found in most eucaryotic plasma membranes, but procaryotes use mainly secondary calcium/proton or calcium/sodium antiporters (3, 9). A notable exception is the organism Streptococcus faecalis, which exhibits ATPdependent extrusion of calcium from intact cells. Kobayashi et al. (7) have shown that membrane vesicle preparations actively accumulate calcium by using ATP and that uptake is independent of the presence of a proton motive force, indicative of a primary calcium pump rather than a secondary calcium antiporter. Since a large fraction of the vesicles are everted, this activity most likely corresponds to the extrusion process observed in cells. In contrast, Driessen et al. (4) showed that calcium transport into membrane vesicles from Streptococcus cremoris did not require ATP and was dependent on the formation of an electrochemical proton gradient. In those experiments bacteriorhodopsin was incorporated into membrane vesicles from S. cremoris, and a proton motive force was generated by light-driven proton pumping. These results suggest that S. cremoris has a cytoplasmic membrane calcium/proton antiporter. If streptococci are unique in having a eucaryotelike calcium pump rather than a calcium/proton exchanger, they could provide a bacterial model for the class of eucaryotic cation-translocating ATPases (3). We are interested in a biochemical characterization of such a calcium pump and have chosen to examine another member of the genus Streptococcus, S. sanguis, for several reasons. First, if two different strains of streptococci exhibit different modes of calcium transport, it is logical to examine others to determine which mode predominates. Second, the availability of plasmids in and transformation procedures for S. sanguis offers an advantage over S. faecalis for future molecular biological studies (8). Third, S. sanguis is an oral streptococcus. A knowledge of calcium homeostatic mechanisms in oral streptococci may be of importance in understanding how they have adapted to the high-calcium environment produced by dissolution of tooth enamel. In this communi- * Corresponding author. 14 cation we demonstrate that membrane vesicles prepared from cells of S. sanguis transport 45Ca2+ in an ATPdependent process. Cultures of S. sanguis (Challis) strain V288 (obtained from F. L. Macrina) and S. faecalis ATCC 979 (obtained from M. Solioz) were grown in 2 liters of KTY medium (6) at 37 C to mid-exponential phase. Cells of S. sanguis are normally resistant to lysozyme. Cells were made lysozyme sensitive by adding 5% glycine to cultures at an OD6co of 1 and incubating for 1 h at 37 C just prior to harvesting (8). Membrane vesicles were prepared as follows. Cells were harvested by centrifugation at 4 C, washed twice with 6 ml of cold 2 mm MgSO4, and suspended at 5 ml/g of wet cells in a buffer consisting of 5 mm HEPES (N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid) and.25 M maleic acid adjusted to ph 7.4 with KOH and containing 5 mm MgSO4,.25 M sucrose, 1 mm 3-mercaptoethanol, and a mixture of protease inhibitors (.5 mm each p-aminobenzamidine, phenylmethylsulfonyl fluoride, and 1,1-phenanthroline). Lysozyme and DNase were added to 1 mg/ml and 1,ug/ml, respectively, and the suspension was incubated at 37 C for 1 h with occasional swirling. The protoplasts were lysed by a single passage through a French pressure cell at 1, lb/in2 at 4C. Although not used for the experiments described below, a modified procedure is now being used for the preparation of vesicles. No effect of protease inhibitors has been observed on the initial activity or stability of the calcium transport systems, so membrane vesicles are now prepared without protease inhibitors. The composition of the buffer has been changed to.1 M MOPS-KOH, ph 7., containing 1 mm MgSO4. The suspension volume has been decreased to 2 ml/g of wet cells. The cells are incubated with lysozyme and DNase at the original concentration but for 9 min at 37 C. Protoplasts are lysed by two passages through the French press at 15, lb/in2. Lysed-cell suspensions were centrifuged for 1 min at 14, x g, and the pellet was discarded. The suspension was centrifuged for 1 h at 1, x g, and the membrane pellet was washed once with the same buffer. All centrifugation steps were performed at 4 C. The membrane vesicles were suspended in 2 ml of buffer, quickly frozen in a -7 C ethanol bath, and stored in small portions at -7C until use. Activity was stable for at least several months. Frozen Downloaded from on July 26, 218 by guest

2 VOL. 168, 1986 NOTES Un) 5 - Al I& o S. fsecalis A S. sngusts IUM CONCENTRATION (ym) FIG. 1. Dependency of calcium transport on calcium concentration. Calcium transport in membrane vesicles from S. sanguis and S. faecalis were assayed as described in the text with an ATP-regenerating system. The concentration of 45CaCl2 was altered by addition of EGTA. Initial rates were estimated from 2- and 4-min time points. Data are plotted as substrate (S) divided by velocity (V). vesicles were thawed as quickly as possible in a 37 C water bath. Everted membrane vesicles were prepared from Escherichia coli HB11 as described previously (1). Calcium transport was assayed at 3 C in 1 ml of 25 mm HEPES-NaOH, ph 7.2, containing 5 mm MgSO4, 25 mm sucrose, 1 mm,-mercaptoethanol, 5,uM 45CaC12, 34,uM EGTA, and.3 mg of membrane vesicles. The free calcium concentration was calculated to be 16,uM before addition of ATP and 5,uM after addition of ATP. The assay was initiated by addition of 5 mm ATP. At intervals,.15-ml portions were filtered through.45-p.m-pore-size nitrocellulose filters, washed with 5 ml of assay buffer lacking sucrose, and dried, and radioactivity was measured by liquid scintillation counting. Experiments in which the calcium concentration was varied were done at constant specific radioactivity and constant total calcium. EGTA was added to change the free calcium concentration (5). In some experiments 25 mm phosphocreatine and 5 U of creatine phosphokinase were added as an ATP-regenerating system. Uptake of calcium was compared for membrane vesicles from S. sanguis and S. faecalis. Both accumulated 45Ca2+ with an apparent affinity of approximately.5 p.m for calcium (Fig. 1). Kobayashi et al. (7) reported a Km of. 15 to 1 mm, depending on the ph of the assay medium. Our considerably lower values are probably the result of our use of an EGTA buffer system for calcium, allowing more precise control over the free calcium concentration. This is especially important at very low calcium concentrations, at which the membranes themselves can bind a significant proportion of the added calcium. Without a calcium buffer the free concentration can be quite different from the total added calcium. Accumulation in both S. sanguis and S. faecalis was dependent on ATP, with an apparent affinity for ATP of approximately.1 mm (Fig. 2). Here too, an Downloaded from on July 26, 218 by guest 4-2; ATP CONCENTRATION (mm) FIG. 2. Dependency of calcium transport on ATP concentration. Calcium transport in membrane vesicles from S. sanguis and S. faecalis were assayed as described in the text. The initial concentration of ATP was varied as indicated. An ATP-regenerating system was used. Data are plotted as substrate (S) divided by velocity (V).

3 142 NOTES J. BACTERIOL. -1: ạ- L n - c LLJ C- I L- _ C-), L- C-) c CL cn 3.- Il 3- Downloaded from on July 26, 218 by guest LLI cl dc 3-) L L-) TIME (MINUTES) FIG. 3. Energetics of ATP-dependent calcium uptake in bacterial membrane vesicles. Calcium transport was assayed as described in the text with membrane vesicles from E. coli (A), S. sanguis (B), and S. faecalis (C). Additions: *, control;, with 1 FM FCCP;, with 25,uM DCCD; O, no ATP added.

4 VOL. 168, 1986 NOTES 143 C - L CL ol SE L) gz 4-3, 2 I1- L.)E FIG. 4. Effect of ionophores and vanadate on ATP-dependent calcium transport in membrane vesicles of S. sanguis. Calcium transport was measured as described in the text. Valinomycin and nigericin were added at 2 and 6.5 F±M, respectively. ATP-regenerating system is important at the lowest concentrations of ATP. To determine whether calcium transport in S. sanguis was catalyzed by a primary pump or secondary exchange system, we compared the effect of ionophores and inhibitors on ATP-driven 45Ca2+ accumulation in membrane vesicles from E. coli, S. faecalis, and S. sanguis. Bacterial H+-translocating ATPases (FoFj) catalyze ATPdependent proton pumping, establishing electrochemical proton gradients (1). In membrane vesicles from E. coli the proton gradient drives calcium accumulation via calcium/proton antiporters (1). N,N'-Dicyclohexylcarbodiimide (DCCD), an inhibitor of the FoF, (1), completely eliminated calcium uptake into E. coli vesicles, whereas calcium uptake in vesicles of S. sanguis was insensitive to DCCD (Fig. 3). In membrane vesicles of S. faecalis DCCD was partially inhibitory but even at concentrations in excess of.1 mm inhibition was never complete (data not shown). Considering the reactivity of DCCD, inhibition may be nonspecific. Alternatively, there may be two calcium transport systems, one of which is DCCD sensitive and the other resistant, as has been shown for E. coli (1). This may also explain the contrasting results of Driessen et al. (4), who found only calcium/proton antiporter activity in membrane vesicles of S. cremoris. It may be that each streptococcal strain has both an ATP-driven calcium pump and a calcium/proton antiporter, but that the proportions of the two systems differ in the various strains. In membrane vesicles of S. faecalis calcium accumulation was insensitive to the protonophoric action of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), which would be expected to collapse the proton gradient (Fig. 3). In contrast, calcium uptake into membrane vesicles of E. coli was completely inhibited by FCCP. Calcium accumulation in membrane vesicles from S. sanguis was partially inhibited by FCCP, but substantial uptake still occurred at levels of FCCP which should have been sufficient to collapse the proton motive force. The combination of the ionophores nigericin and valinomycin, which also acts to collapse the electrochemical proton gradient in the presence of K+, did not affect calcium uptake into vesicles of S. sanguis (Fig. 4). Thus, the inhibition by FCCP cannot have been due to its action as a protonophore. Vanadate, an inhibitor of many eucaryotic ion motive ATPases (3), effectively inhibited calcium transport in vesicles of S. sanguis (Fig. 4), with half-maximal inhibition occurring at a concentration of less than 5,uM. Calcium transport in everted membrane vesicles of E. coli was not inhibited by vanadate (data not shown). Our results indicate that neither the FoF, nor a proton motive force is necessary for calcium transport into membrane vesicles of S. sanguis, in agreement with the findings of Kobayashi et al. for S. faecalis (7). Since the calcium/proton antiporter of E. coli requires a proton motive force and, when ATP is used as an energy source, the FoF1 (1), the major system for calcium uptake into membrane vesicles of the two streptococci cannot be a calcium-proton exchange system. The inhibition by vanadate suggests the existence of a Ca2+-translocating ATPase with similarities to the calcium pumps of eucaryotic plasma membrane and sarcoplasmic reticulum (3). However, we have not specifically examined the formation of a proton motive force in these vesicles and cannot eliminate the possibility of a calcium-proton antiporter which is quantitatively minor compared with the primary calcium pump. In a preliminary report, Burkler and Solioz (2) demonstrated solubilization and reconstitution of calcium pump activity in S. faecalis. Using a different method, we have recently solubilized and reconstituted the calcium pumps from S. sanguis, S. faecalis, and S. lactis (A. R. Lynn, B. P. Rosen, S. V. Ambudkar, and P. C. Maloney, Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, K65, p. 24) and have partially purified ATP-dependent calcium transport activity from S. sanguis (A. R. Lynn and EN. P. Rosen, Abstr. Annu. Meet. Am. Soc. Biol. Chem. 1986, abstr. 1615, Fed. Proc. 45:1758). In reconstituted proteoliposomes, calcium transport activity retains its sensitivity to vanadate and insensitivity to ionophores and DCCD, indicating that the transport observed in membrane vesicles reflects the catalytic activity of a calcium pump. This work was supported by Public Health Service grant DE671 from the National Institutes of Health. Downloaded from on July 26, 218 by guest

5 144 NOTES J. BACTERIOL. LITERATURE CITED 1. Ambudkar, S. V., G. W. Zlotnick, and B. P. Rosen Calcium efflux from Escherichia coli. Evidence for two systems. J. Biol. Chem. 259: Burkler, J., and M. Solioz The ATP-dependent Ca2lpumping system of Streptococcus faecium. Ann. N.Y. Acad. Sci. 42: Carafoli, E., G. Inesi, and B. P. Rosen Calcium transport across biological membranes, p In H. Sigel (ed.), Metal ions in biological systems. Marcel Dekker, Inc., New York. 4. Driessen, A. J. M., K. J. HeWngwerf, and W. N. Konings Light induced generation of a protonmotive force and Ca2+transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes. Biochim. Biophys. Acta 88: Fabiato, A., and F. Fabiato Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. (Paris) 75: Harold, F. M., E. Paviasova, and J. R. Baarda A transmembrane ph gradient in Streptococcus faecalis: origin, and dissipation by proton conductors and N,N'-dicyclohexylcarbodiimide. Biochim. Biophys. Acta 196: Kobayashi, H., J. Van Brunt, and F. M. Harold ATPlinked calcium transport in cells and membrane vesicles of Streptococcus faecalis. J. Biol. Chem. 253: Macrina, F. L., P. H. Wood, and K. R. Jones Simple method for demonstrating small plasmid deoxyribonucleic acid molecules in oral streptococci. Appl. Environ. Microbiol. 39: Rosen, B. P Calcium transport in microorganisms, p In E. Carafoli (ed.), Membrane transport of calcium. Academic Press, Inc., New York. 1. Rosen, B. P., and E. R. Kashket Energetics of active transport, p In B. P. Rosen (ed.), Bacterial transport. Marcel Dekker, Inc., New York. Downloaded from on July 26, 218 by guest