Proc. Nat. Acad. Sc,. USA 72 (1975) 2833

Correction. In the article, "Active Transport of Calcium inInverted Vesicles of Escherichia coli," by Barry P. Rosenand John S. McClees, which appeared in the December 1974issue of Proc. Nat. Acad. Sci. USA 71, 5042-5046, the au-

thors have requested the following change. On page 5043, inthe left-hand column, the concentration of calcium in trans-port assays was given as 1 mM. Subsequent analysis of thecalcium solution showed that the actual concentration was

0.5 mM. Thus, the specific activities of calcium transportpresented in the Results section must be divided by a factorof two. The kinetic parameters were determined with sepa-

rate solutions and are correct as stated in the text.

Correction. In the article "Immunoreactive Somatostatin IsPresent in Discrete Cells of the Endocrine Pancreas" by M.P. Dubois that appeared in the April 1975 issue of Proc. Nat.Acad. Sci. USA 72, 1340-1343, the structure of somatostatinwas omitted by the printer. Line two of the left-hand col-umn on page 1340 should be:

H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thi-Phe-Thr-SrCys-OH.

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Proc. Nat. Acad. Sci. USAVol. 71, No. 12, pp. 5042-5046, December 1974

Active Transport of Calcium in Inverted Membrane Vesicles of Escherichia coli(membrane orientation/energy coupling/calcium uptake)

BARRY P. ROSEN AND JOHN S. McCLEES

Department of Biological Chemistry, University of Maryland, School of Medicine, Baltimore, Md. 21201

Communicated by Albert L. Lehninger, October 2, 1974

ABSTRACT Accumulation of 45Ca++ was found tooccur in membrane vesicles of E. coli prepared by lysis witha French pressure cell. The uptake occurs by active trans-port, requiring an energy source. Substrates of the elec-tron transport chain, including D-lactate, reduced phen-azine methosulfate, and NADH, stimulated accumu-lation, but this effect was blocked by the addition ofcyanide. ATP could also stimulate accumulation, and thiseffect was blocked by dicyclohexylcarbodiimide. Un-couplers of oxidative phosphorylation inhibited theaccumulation driven by either type of energy source.Accumulation of calcium is rapid, reaching the steady-

state plateau within 1 min. Addition of phosphate to theassay buffer results in a prolongation of the reaction, allow-ing for the time-dependent accumulation of calcium foras long as 30 min.

Vesicles prepared by lysis with a French pressure cellexhibit almost no ability to accumulate proline, whilevesicles prepared by the method of Kaback transportproline but exhibit little energy-dependent transport ofcalcium. It is suggested that the accumulation of calciumin these vesicles, which are believed to be inverted, reflectsa system that in vivo is responsible for the active extrusionof calcium from the cells.

The ability to transport calcium exists almost universally innature. Several microorganisms have been shown to accumu-late calcium. Several strains of Bacilli transport calciumduring sporulation (2, 3), and vesicles of both Bacillus mega-terium (4) and Azotobacter vinelandii (5) exhibit respiratory-driven accumulation of calcium. In eukaryotic organisms,most mitochondria exhibit calcium transport ability that iscoupled to either respiration or ATP hydrolysis (6). Cells ofcertain specialized tissues, however, actively pump calciumfrom their cytosol. Calcium is transported out of the sarco-plasm of muscle cells into the sarcoplasmic reticulum, a pro-cess believed to be involved in muscular contraction (7). Inthe intestinal absorption of calcium, the ion is believed to beactively pumped out of the serosal side of epithelial cells intothe plasma (8). It has been suggested that human erythro-cytes have an ATP-dependent calcium extrusion mechanismas well (9). Treatment of mitochondria with digitonin, Lubrol,or sonication results in the production of submitochondrialparticles. Calcium transport has been found in all three typesof vesicles (10-13), even though vesicles prepared by soni-cation or Lubrol treatment have been reported to be inverted(11, 12). However, the sidedness of submitochondrial vesiclesprepared by digitonin treatment or sonication has been ques-tioned (14).No calcium transport systems have been reported to exist in

Escherichia coli. Silver and Kralovic (15) reported that more

calcium is found in whole cells at 40 than at 230 or 370,leading to the suggestion that there may exist in E. coli atransport system that is responsible for the active exclusion ofcalcium from whole cells.

If such is indeed the case, it should be possible to measurecalcium accumulation in membrane vesicles that have theopposite orientation to that of whole cells. Futai (16) inves-tigated the localization of enzymes in membrane vesicles ofE. coli prepared by lysis with a French pressure cell andfound that enzymes normally associated with the inner sur-face of the cytoplasmic membrane appear predominately onthe outer surface of such vesicles. Hertzberg and Hinkle (17),using vesicles prepared in a similar manner, reported thatsuch vesicles catalyze the oxidative phosphorylation of ADP.Moreover, the vesicles translocate protons inwards duringrespiration or ATP hydrolysis. Since whole cells (18) andright-side-out vesicles (19) catalyze the extrusion of protons,they suggested that membranes prepared with a Frenchpressure cell are inverted. Freeze-cleave electron microscopyof such vesicles confirms the fact that at least 60-80% of thesevesicles are inverted (20).

In this communication we have investigated the ability ofmembrane vesicles of E. coli prepared by lysis with a Frenchpressure cell to accumulate 4"Ca++. Such vesicles accumulatecalcium with a Km of about 0.1 mM. The process is energy-requiring and can be driven by various respiratory sub-strates. In addition, ATP can serve as an energy donor, sug-gesting that it is indeed the inverted vesicles and not con-taminating right-side-out vesicles that accumulate calcium.Moreover, these vesicles show little ability to transportproline, while right-side-out vesicles prepared by the methodof Kaback (1) transport proline but not calcium.

MATERIALS AND METHODS

Growth of Cells. Escherichia coli strain 7 (21) cultures were

grown in the basal salts medium described by Tanaka et al.(22) supplemented with 68 mM glycerol as a carbon source.

Chemicals. 45CaC12 (1.3-1.4 Ci/mmol) and i[G-3H]proline(4.75 Ci/mmol) were purchased from New England NuclearCorp. 45CaC12 was diluted with nonradioactive CaCl2 to 20mCi/mmol for kinetic determinations and 5 mCi/mmol forother assays. [3H]Proline was diluted with nonradioactiveproline to 1.65 Ci/mmol. All other compounds were analyticalgrade. Solutions of dicyclohexylcarbodiimide (DCCD) andcarbonylcyanide-m-chlorophenylhydrazone (CCCP) were pre-pared in ethanol.

Preparation of Membrane Vesicles. Cultures were harvestedin mid-exponential phase by centrifugation. The cells were

washed once with a buffer consisting of 10 mM Tris HCl(pH 7.2), 0.14 M KCl, 1 mM 2-mercaptoethanol, and 10%

5042

Abbreviations: PMS, phenazine methosulfate; DCCD, dicyclo-hexylcarbodiimide; CCCP, carbonylcyanide-m-chlorophenylhy-drazone; TKMG buffer, 10 mM Tris*HCl (pH 7.2)-0.14 MKCl-1 mM 2-mercaptoethanol-10% (v/v) glycerol.

Active Transport of Calcium 5043

(v/v) glycerol (TKAMG buffer) and resuspended to fivevolumes per gram of wet cells with TKMG buffer. The cellswere lysed by a single passage through a French pressure cell(American Instrument Co.) at 3000-4000 lbs./inch2. The sus-pension was centrifuged at 27,000 X g for 10 min. The pelletwas resuspended in a small volume of TKMG buffer and re-centrifuged. The supernatant solutions were centrifuged for 1hr at 105,000 X g. The pellet was washed once with TKMGbuffer, followed by resuspension in TKAIG buffer to 3-4 mgof protein per ml. Each step of the above procedure was per-formed at 40, and the suspension was kept on ice until use.Membranes were prepared and used within 1 day.

Spheroplasts lysed in Tris buffer showed no transport ofproline. Thus, the preparation of right-side-out vesicles wasperformed by the method of Kaback (1). Half of the vesicleswere washed twice with and resuspended in TKMG buffer forthe assay of calcium transport. The other half were washedtwice with 0.1 M potassium phosphate buffer (pH 6.6) con-taining 10 mMN EDTA, followed by resuspension of 0.1 Mpotassium phosphate buffer (p1l) 6.6) for the assay of prolinetransport. In order to demonstrate that lysis in phosphatebuffer does not affect the calcium transport system, the fol-lowing procedure was used. Cells were washed with 10 mMTris - HCl (pH 7.2), containing 30 mM NaCl, followed bysuspension in 0.1 A\I potassium phosphate buffer (pH 6.6).The cells were lysed with a French pressure cell at 3000-4000lbs./inch2 and centrifuged at 27,000 X g for 10 min. Thesupernatant solution was divided into two portions and cen-trifuged at 105,000 X g for 1 hr. The pellet from one portionwas washed twice with TKMG buffer and resuspended in thesame buffer at 3-4 mg of protein per ml. The other portionwas washed twice with 0.1 M\ potassium phosphate buffer(pH 6.6), containing 10 mMi EDTA, and resuspended in 0.1Mlpotassium phosphate buffer (pH 6.6) at 3-4 mg/ml of pro-tein. The Tris-washed suspension was used for the measure-ment of calcium transport, while the phosphate-washedportion was used to measure proline transport.

Transport Assay. The following procedure was adopted forthe measurement of 45Ca++ transport. The membranes werediluted 10-fold in a buffer consisting of 10 mM Tris HCl(pH 8.5), containing 0.14 AI KCI, at 230. When phosphatewas used in the assay, it was added to the Tris buffer in theform of potassium phosphate (pH 8.5). After a 15-min in-cubation at 230, an energy source, if used, was added, fol-lowed by the immediate addition of 45Ca++ at 1 mM finalconcentration. At various times after addition of 45Ca++,0.2-ml portions of the reaction mixture were filtered on 25-mmnitrocellulose filters (0.45 um pore size, Matheson-HiggensCo.) and washed with 5 ml of the assay buffer.The filters were dried, and the radioactivity was measured

by liquid scintillation counting. Proline transport was mea-sured by the method of Kaback (1), with 0.36, M [3H]prolinefinal concentration.

Other A ssays. Protein contents were determined by a micro-modification of the method of Lowry et al. (23), with bovineserum albumin as a standard. Kinetic parameters were cal-culated with a Univac 1108 computer by a program designedto give a least-squares fit to the data.

RESULTS

Conditions for Preparation of Membranes and Assay ofCalcium Transport. Initial experiments centered on the con-

20 Gz

1a:

U.~ ~ ~ ~ ~ N

Co.

Md s 10w

0

070

1 ~~~2TiME (MIN)

FIG. 1. Effect of pH on calcium accumulation. Vesicles wereprepared in TKMG buffer as described under Materials andMethods. The vesicles were diluted 10-fold into the assay bufferfollowed by the addition of 20 mM D-lactate. The filters werewashed with the same buffer used for the assay. The 10 mMTris HCl, 0.14 M KCI buffers were adjusted to the followingpH values: 0, pH 7.0; A, pH 7.5; A, pH 8.0; X, pH 8.5; *, pH 9.0.

ditions for preparation and assay of vesicles. Vesicles preparedfrom late exponential phase and stationary phase culturesshowed little or no calcium transport under any conditionstested. Early and mid-exponential phase cultures, when pre-pared and assayed in 0.1 M potassium phosphate buffer(pH 6.6), yielded membranes that did not transport calcium.Membranes prepared in TKMG buffer exhibited the abilityto retain 2-5 nmol of 45Ca++ per mg of protein in the pres-ence of D-lactate, but with little or no time-dependency. Asshown in Fig. 1, the uptake of calcium is dependent on thepH of the assay medium, with the most rapid uptake andgreatest accumulation found in 10 mM Tris HCI (pH 8.5),containing 0.14 M KCl. KCl decreased the energy-inde-pendent binding of calcium to the membranes and filterssomewhat, although large energy-independent calcium bind-ing was still observed. Vesicles prepared in TKMG buffer ad-justed to pH 8.5 were capable of transporting calcium, butlost most of this activity within a few hours. Vesicles pre-pared in TKMG buffer at pH 7.2 were stable for up to 8 hr.

Kinetics of Calcium Transport. The transport was time-dependent, but the period of active accumulation was brief; asteady-state plateau was attained in less than 1 min, oftenfollowed by rapid exit of calcium, presenting difficulties in themeasurement of the initial rate of entry. For the determina-tion of the transport K, assays were performed at a series oftimes from 10 to 30 see at various concentrations of calcium.The results of one such experiment are shown in Fig. 2. Eachseries of points was fitted to a line which, in each case, yieldeda Km of between 0.125 and 0.154mM calcium, with an averageof 0.13 mM i 0.02 (SD). As expected, the apparent max-imal velocity decreased with increasing times. Extrapolationsto zero time yield an apparent maximal velocity of 33 nmol/min per mg of protein, but this varied from one preparation toanother.

Effect of Phosphate on Calcium Accumulation. Phosphateincreases the extent of calcium uptake in mitochondria (6).This effect is thought to occur by the formation of insolublecalcium phosphate salts inside the mitochondrion. The con-centration of free calcium remains constant after an initialaccumulation, so that the rate of exit of free calcium neverequals the rate of entrance. The addition of 2 mM potassiumphosphate (pH 8.5) to the Tris assay buffer resulted in an in-

Proc. Nat. Acad. Sci. USA 71 (1974)

5044 Biochemistry: Rosen and McClees

[Ca+'l mM

FIG. 2. Determination of the kinetic parameters of calciumtransport. Vesicles prepared in TKMG buffer were diluted 10-

fold into assay buffer, D-Lactate (20 mM) was added, and thereactions were initiated by the addition of 4rCaCI2 at variousconcentrations. Portions were filtered at 10 see (0), 20 see (0),and 30 see (U). The points are experimental; the lines are least-square calculations from the data.

crease in the extent of the uptake reaction. When the phos-phate concentration was raised to 10 mM, more calciumradioactivity was accumulated. Since the vesicles might havea permeability barrier to phosphate, an experiment was per-

formed in which vesicles were incubated at 390 for 30 min inthe presence of 2 mM and 10 mM phosphate and allowed tocool to room temperature before assay. The membranes in-cubated in 2 mM phosphate showed an enhancement of ac-

cumulation ability, but there was no significant differencesbetween the vesicles that were heated in 10 mM phosphateand those incubated at room temperature in the same mixture(data not shown). Fig. 3 shows the effect of 10 mM potassiumphosphate on calcium accumulation. Lysis of the cells in 0.1 Mpotassium phosphate buffer followed by washing with TKMGbuffer produced vesicles that showed comparable ability toaccumulate calcium.

Dependence of Calcium Transport on an Energy Source. Theeffect of substrates of the electron transport chain and ofATP on calcium uptake was investigated (Table 1). NADH,D-lactate, reduced phenazine methosulfate (PMS), and suc-

cinate were all very effective in stimulating calcium trans-

TABLE 1. Effect of energy sources on calciumaccumulation

Calcium uptake(nmol/mg of protein

Addition per 30 min)

None 25.920 mM 1)-Lactate 120.420 mM Succinate 85.20.1 mM PMS + 20 mM ascorbate 64.75mM NADH 175.35mM ATP + 5mMMgCl2 80.95mMADP + 5mMMgCl2 12.1

Calcium transport was measured in the presence of 10 mMpotassium phosphate (pH 8.5) as described under Materials and

Methods.

TIME (MIN)

FIG. 3. Effect of phosphate on calcium accumulation. Vesicleswere diluted 10-fold in the assay medium and incubated for 15min at 230 before addition of 5 mM NADH and 1 mM 45CaC12,final concentrations. 0, +5 mM NADH and 10 mM potassiumphosphate, pH 8.5; 0, +5 mM NADH, no phosphate; *, +10mM potassium phosphate; O, no phosphate.

port. Different preparations of vesicles responded differentlyto the various energy donors. All were effective, but withsome membrane preparations, ascorbate plus PMS was themost efficient, while other preparations used NADH or D-lactate more efficiently. The reason for this variability is notknown. ATP was likewise able to serve as an energy donor forcalcium transport (Table 1). ADP, on the other hand, was in-effective in stimulating the accumulation of calcium.

Cyanide inhibited the respiratory-driven calcium uptake tonearly the level of the energy-independent uptake (Table 2).The accumulation in the presence of DCCD was not sig-nificantly different from the ethanol control. The ATP-driven accumulation, on the other hand, was almost un-affected by cyanide, but was inhibited 88% by DCCD. Cal-

TABLE 2. Effects of inhibitors and uncouplers oncalcium accumulation

Calcium uptake(nmol/mg ofprotein per %

Energy source Addition 15 min) Control

None None 23.0

5mM NADH None 116.3 10050 AM KCN 50.5 43

5 ul of ethanol* 100.2 10050IOM DCCD 93.3 9325,uM CCCP 27.0 27

5 mM ATP +5mM MgCl2 None 57.6 100

50IOM KCN 53.6 93

5 MAl of ethanol* 61.1 10050 AM DCCD 7.0 1225,uM CCCP 3.5 6

Calcium transport was measured in the presence of 10 mMpotassium phosphate (pH 8.5) as described under Materials andMethods.

* DCCD and CCCP were added as ethanol solutions.

Proc. Nat. Acad. Sci. USA 71 (1974)

Active Transport of Calcium 5045

cium accumulation driven by either NADH or ATP was in-hibited by CCCP.

It is not clear why the ATP-driven accumulation of cal-cium in the presence of DCCD or CCCP, as seen in Table 2,or the accumulation in the presence of ADP, as seen in Table1, is less than the energy-independent value. .Mg++, presentin these assays, reduced neither the energy-independent northe energy-dependent rates. It is possible that the adeninenucleotides chelate some 45Ca++.

Comparison of Calcium and Proline Transport in VesiclesPrepared by Different Procedures. Vesicles were prepared inpotassium phosphate buffer and then divided into a Tris-washed portion for the measurement of calcium uptake and aphosphate-washed portion for the measurement of prolineuptake, as described under Materials and Methods. The Tris-washed portion (Fig. 4A) exhibited a rapid, energy-dependentuptake of calcium, while the phosphate-washed portionshowed almost- no proline transport (Fig. 4B). Conversely,vesicles were prepared by the method of Kaback (1) but splitinto two portions during the working stage, as describedunder Materials and Methods. Such vesicles, when washedwith TKMG buffer, showed no energy-dependent calciumtransport (Fig. 4C). The ability of the phosphate-washedportion to transport proline (Fig. 4D) was comparable topreviously reported values (1).

DISCUSSION

The net direction of most transport reactions among pro-karyotic organisms appears to be from the outside of the cellinwards, although right-side-out vesicles of E. coli have beenreported to extrude sodium during the uptake of rubidium(24). Systems with the opposite polarity have also beenshown to occur in eukaryotes (7-9). Silver and Kralovic (15)suggested that a system might exist that would be responsiblefor the prevention of calcium entry into cells of E. coli. Thisreport demonstrates that such a system for active transportof substrates does, indeed, exist for the accumulation ofcalcium into inverted vesicles of E. coli and may be that re-sponsible for the maintenance of a concentration gradient ofcalcium outside of whole cells. The calcium transport systemin these vesicles is capable of accumulating as much as 0.5,gmol/mg of protein in 30 min. This value is comparable to thevalue of about 1 jumol/mg of protein in 30 min for submito-chondrial particles from sonicated mitochondria (11) and thevalue of about 0.2 ,umol/mg of protein in 2 min found for sub-mitochondrial particles from Lubrol-treated mitochondria(12). Since the internal volume of the inverted E. coli mem-branes has not been determined and since the amount of freeinternal calcium is unknown, it is not now possible to expressthe uptake values as concentration ratios.The establishment of the direction of transport by this

system relies on the orientation of the membranes that areable to transport calcium. Several enzymes have been lo-calized on the inner surface of the inner membrane of E. coli.These include the dehydrogenase of the electron transportchain and the Mg++-ATPase (16). When cells are lysed bysonication or by a French pressure cell, those enzymes be-come completely accessible from the outer surface of the re-sultant inner membrane vesicles. This suggests an inversionof the inner membrane. But inner membrane vesicles pre-pared by the method of Kaback also have these enzymespartially accessible from the outside (16) and yet have been

zI-0cc

0lLLCo

-J0

w

I-U-Y

0r-

zc

mK

-n

-m

S0m

0~1

m

TIME (MIN)

FIG. 4. Transport in vesicles made by different procedures.The vesicles used in A and B were prepared by lysis with aFrench pressure cell; those used in C and D were prepared by themethod of Kaback (1). A and C show the accumulation of 45Ca++with 10 mM potassium phosphate (pH 8.5) added to the assaybuffer. B and D show the accumulation of ['H]proline by thesetwo types of membrane preparations. 0, No energy source; *,+ 0.1 mM PMS and 20 mM ascorbate.

shown by electron microscopy to be predominately right-side-out (20). Another feature of whole cells and right-side-out vesicles is respiratory-driven proton extrusion (18, 19).But vesicles prepared by lysis with a French pressure cellshow a respiratory-driven proton uptake (17), suggesting in-version. Moreover, populations of such vesicles have beenshown to consist primarily of inside-out inner membranevesicles by electron microscopy (20).Thus, it would appear that thepreparationsused in thisstudy

contain a predominance of inverted vesicles. It is possible thata small amount of right-side-out vesicles [estimated variouslyas less than 5% (16) to as much as 40% (20) ] are responsiblefor the observed calcium uptake. But vesicles known to beright-side-out do not transport calcium (Fig. 4). If the uptakeof calcium by these vesicles is a function of the fraction withthe right-side-out orientation, then this population is inca-pable of transporting proline as well.

It might be argued that the lysozyme-EDTA procedure insome way inactivates the calcium pump and that vesicles pre-pared by lysis with a French pressure cell have too small aninternal volume to show that they actually transport proline.Here the ability to utilize certain energy sources should beconsidered. Whole cells are capable of driving many transportreactions with glycolytically derived ATP via the Mg++-ATPase (25), but right-side-out vesicles cannot normally doso (1). Van Thienen and Postma (26) have suggested that thereason for this is that only the ATPase on the inner surface of

Proc. Nat. Acad. Sci. USA 71 (1974)

5046 Biochemistry: Rosen and McClees

such vesicles can couple the energy from ATP to transport,and that ATP cannot penetrate the membrane. Their pre-liminary results suggest that ATP can energize serine trans-port in such vesicles when ATP is allowed to reach the crypticATPase (26). Calcium transport is supported by ATP (Table1), and the sensitivity to DCCD and CCCP (Table 2) sug-gests that the energy is transduced through the Mg++-ATPase. Similarly, NADH is a poor energy source for trans-port in right-side-out vesicles of E. coli (1), presumably due tothe impermeability of the membrane to pyridine nucleotides.Yet NADH is more efficient than D-lactate in driving calciumtransport in vesicles prepared by lysis with a French pressurecell (Table 1). These results strongly suggest that vesiclesthat accumulate calcium are inverted.Can the energetics of the calcium transport system be ac-

commodated by the hypothesis of Mitchell (27)? In that hy-pothesis, either the electron transport during the oxidation ofcompounds or the ATPase during the hydrolysis of ATP gen-erates an electrochemical gradient of protons. In whole cellsthe gradient is positive and acid on the outside, negative andbasic on the inside of the cell. In inverted vesicles the gra-dient ought have the reverse polarity, as Hertzberg and Hinklehave suggested (17). It seems unlikely that an electrochemicalgradient of calcium could be established at the same time asthe electrochemical gradient of protons. It may be that thecalcium porter acts as a calcium-proton antiporter (or sy-nonymously, a calcium-hydroxyl symporter) as suggested byMitchell (27). Since inverted vesicles establish a proton gra-dient, acid inside, a flow of protons out of the vesicles via thecalcium porter could drive the uptake of calcium inwardsagainst its concentration gradient. The reverse situationwould occur in whole cells, with an influx of protons coupledto an efflux of calcium. This could produce an electroneutralaccumulation of calcium driven by the protonmotive force.Such anl explanation would explain the unusual requirementfor an alkaline pH (Fig. 1). Another possibility is a calcium-phosphate symport, again preventing an electrogenic uptakeof calcium.The analogy between the calcium transport system of E.

coli and the calcium pumps of eukaryotic organisms is strik-ing. Yet, those systems have proven functions. For example,the active exclusion of calcium from the sarcoplasmic retic-ulum may well be a major factor in muscular contraction.What, then, is the function in E. coli of a system for the activeexclusion of calcium from the cell? A trivial possibility is thatcalcium would interfere with magnesium-requiring reactionsor in some other way upset metabolism. Calcium concentra-tions in the lumen of the intestine may at times reach valuesapproaching that of milk (5-10 mM), especially during in-fancy. A low affinity, high capacity system for the exclusion ofcalcium could be beneficial to enteric bacteria. In fact, Silverand Toth have recently reported that the internal calciumconcentration in growing E. coli is only about 10% that of theexternal medium [Silver, S. & Toth, K. (1974) Abstr. Annu.Meet. Amer. Soc. Mficrobiol., p. 151]. A more interesting pos-sibility is that calcium fluxes might be related to flagellarmovement. Larson et al. (28) have recently shown that theresponse to a chemotactic signal, that is, movement of the

flagella, is energized by the same coupling mechanism as isactive transport and oxidative phosphorylation. How thesignal is transmitted from the chemoreceptor to the flagella isunknown. It is interesting to speculate that the calcium pumpmight be involved. Just as the signal from the nervous systemcauses the inactivation of the Ca++-ATPase of the sarco-plasmic reticulum, and a transient influx of calcium into thesarcoplasm, resulting in an activation of myosin and muscularcontraction, it is possible that the signal of the chemoreceptorof E. coli causes an inactivation of the calcium pump, al-lowing calcium to enter the cell and initiate flagellar move-ment.

We thank Dr. S. Silver for his valuable discussion. The com-puter time for this project was supported in full through thefacilities of the Computer Science Center of the University ofMaryland. This research was supported by Grant GB-35831from the National Science Foundation.

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3. Bronner, F. & Freund, T. S. (1972) in Spores, V, eds. Halvor-son, H. O., Hanson, R. & Cambell, L. L. (American Societyfor Microbiology, Washington, D.C.), pp. 187-190.

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Proc. Nat. Acad. Sci. USA 71 (1974)