Vol. 169, No. 12JOURNAL OF BACTERIOLOGY, Dec. 1987, p. 5761-57650021-9193/87/125761-05$02.00/0Copyright © 1987, American Society for Microbiology

Bioenergetic Properties of Alkalophilic Bacillus sp. Strain C-59 onan Alkaline Medium Containing K2CO3

MAKIO KITADA* AND KOKI HORIKOSHIThe Riken Institute, Wako-shi, Saitama 351-01, Japan

Received 4 May 1987/Accepted 29 August 1987

Alkalophilic BaciUus sp. strain C-59 could grow well on an alkaline medium containing K2CO3, as well asNa2CO3, but did not grow on K+-depleted medium. Right-side-out membrane vesicles, energized in the absenceof Na+, however, could not take up [14C]methylamine actively, while vesicles equilibrated with 10 mM NaCIactively took up [14CJmethylamine. The uptake of [14C]serine was also stimulated by the addition of Na+, andthe imposition of a sodium gradient caused transient uptake. These results indicated that an Na+/H+ antiporterwas involved in pH homeostasis and generation of an electrochemical sodium gradient in strain C-59 eventhough a growth requirement for Na+ was not evident. The efflux of 22Na' from 22Na+-loaded vesicles wasmore rapid at pH 9.5 than at pH 7 in the presence of an electron donor. On the other hand, vesicles at pH 7showed more rapid efflux than at pH 9.5 when the antiporter was energized by a valinomycin-mediated K+diffusion potential (inside negative).

Most alkalophilic bacteria isolated to date in alkalinemedia containing 1% Na2CO3 require Na+ for growth, andamino acid uptake by these cells was stimulated by theaddition of Na+ (9, 14, 15, 17). We have reported that Na+was required for o-aminoisobutylic acid uptake by the cellsof some alkalophiles that were isolated in alkaline mediacontaining K2CO3 instead of Na2CO3 (16).

It is clear that alkalophiles must maintain a cytoplasmicpH that is more acidic than the external pH. For example,cells of Bacillus firmus RAB exhibited an immediate rise incytoplasmic pH in the absence of Na+ and lost viability veryrapidly at alkaline pHs (13). Then, at least some alkalophileshave special functions for pH homeostasis which are relatedto Na+ and H+ movement. From extensive studies on thebioenergetics of alkalophiles, Krulwich and co-workers (2,5, 7, 20, 21, 24) indicated that an electrogenic Na+/H+antiporter was required for pH homeostasis in obligatealkalophiles. An Na+/H+ antiporter is defined as a carrierthat translocates Na+ and H+ in opposite directions across amembrane. This antiporter was very active and acidified thecytoplasm in the alkaline range ofpH (18, 20, 21). In additionto the regulation of internal pH, the antiporter converted aproton motive force to an electrochemical gradient of Na+which could energize solute transport. Thus, Na+ wasreentered into the cells as a coupling ion for solute uptake.This Na+ circulation through the antiporter and thesymporter is a most important physiological function inalkalophiles. These facts clearly underlie the expectationthat alkalophiles will require Na+ for growth and solutetransport.

Recently, we have found that alkalophilic Bacillus sp.strain C-59, which was isolated in Na2CO3-containing me-dium (11), can grow well in K2CO3 medium and cannot growin K+-depleted alkaline medium, i.e., this strain exhibited norequirement for added Na+ for growth. In the current study,we have examined the effect of Na+ on the bioenergeticproperties of Bacillus sp. strain C-59 at alkaline pH.

* Corresponding author.

MATERIALS AND METHODS

Organisms and growth conditions. Alkalophilic Bacillus sp.strains C-59 and 2b-2 were used throughout this experiment.Taxonomical studies on these strains have been reported(11, 22). Bacillus sp. strain C-59 was grown at 37°C withshaking at pH 10 in an alkaline medium (starch, 1%; yeastextract, 0.5%; polypeptone, 0.5%; K2HPO4, 0.1%;MgSO4 * 7H20, 0.02%; Na2CO3 or K2CO3, 1%). Growth wasmeasured by optical density at 660 nm with a Hitachispectrophotometer (type 124).A* and ApH measurements. The value for membrane

potential (A1) was determined by a filtration assay (30) forwhole cells by the accumulation of [3H]tetraphenyl-phosphonium, which was added to a vigorously shaking cellsuspension (0.3 mg of protein per ml) at a concentration of 20,uM. Counts retained by the filters in the presence of 20 ,uMgramicidin were subtracted. A* was calculated from theNemst equation (13).ApH was measured by the distribution of a weak base,

[14C]methylamine, in a flow dialysis apparatus (26). Wholecells (0.9 mg of protein) were suspended with 200 p.l of 0.05M Na2CO3 or K2CO3 buffer (pH 10) in the upper chamber.Buffer was pumped through the lower chamber (spiral chan-nel) at a rate of 1 ml/min. Fractions (1 ml) were collected andassayed for radioactivity by liquid scintillation spectrome-try. The upper and lower chamber were separated by dialy-sis tubing. The upper chamber was stirred with a magneticstirring bar under a stream of oxygen. [14C]methylamine wasadded to a final concentration of 55 ,uM. [14C]methylamineuptake by membrane vesicles was also measured by a flowdialysis method. The vesicles were energized by the additionof Tris-ascorbate (20 mM) and TMPD (tetramethyl-p-phenylenediamine, 0.2 mM). Vesicle concentrations of 1.1mg of protein per ml were used. The ApH was calculated bythe formula of Waddel and Butler (29). The internal volumesof Bacillus sp. strain C-59 cells and vesicles were found to be6.0 and 1.1 p.l/mg of protein, respectively.

22Na' efflux experiments. Na+ efflux experiments wereperformed by following the time-dependent decrease in

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5762 KITADA AND HORIKOSHI

intravesicular 22Na' content of 22Na+-loaded membranevesicles as described by Bassilana et al. (1). Right-side-outmembrane vesicles in 50 mM K2CO3 buffer (pH 9.5) weresuspended in the same buffer containing 10 mM MgSO4 and10 mM NaCl at a final concentration of 20 mg/ml. Then, asmall portion of carrier-free 22NaCl was added to the vesiclesuspension (3,500 cpm/nmol), and the suspension was incu-bated on ice for 2 to 3 h. When 22Na efflux was conducted byK+ diffusion potential, valinomycin was added to give a finalconcentration of 20 ,uM. Na+ efflux determinations wereinitiated by diluting 2.5 ,ul of membrane vesicle solution into400 ,ul of 50 mM K2CO3 or K2 P04 buffer containing 10 mMMgSO4 and electron donors (Tris-ascorbate, 20 mM, plusTMPD, 0.15 mM) and quickly agitated with a Vortex mixerat room temperature. When a K+ diffusion potential was

produced, concentrated vesicles that had been equilibratedwith 22Na' in the presence of valinomycin were diluted800-fold into 100 mM choline chloride buffered with 10 mMTris chloride (pH 7 and 9.5) or 100 mM K2CO3 as a control.At given times, the Na+ efflux reaction was terminated byfiltering the diluted sample. The filter (0.45 ,um pore size;Millipore Corp.) was washed once with 2 ml of equilibriumsolution. Nonspecific binding of 22Na on the filters was

estimated from the amount of radioactivity retained on thefilter following filtration of diluted sample that had stood for2 h at room temperature and was subtracted from the Na+efflux data.

Transport of [14C]serine by cells or membrane vesicles.Preparation of right-side-out membrane vesicles for trans-port and antiporter studies was performed by the osmoticshock procedure (12). Protein was determined by the methodof Lowry et al. (23) with bovine serum albumin as astandard. The uptake of [14C]serine was determined by a

filtration assay described previously (15). The final concen-trations of protein and serine were 0.2 mg/ml and 300 ,uM forcells, or 1 mglml and 15 ,uM for membrane vesicles, respec-tively.

Materials. 22NaCl (carrier free), [14C]serine (173 mCi/mmol), [14C]methylamine (46 mCi/mmol), and [3H]tetraphe-nylphosphonium (35.5 Ci/mmol) were all obtained from NewEngland Nuclear Corp. Valinomycin, gramicidin, and CCCP(carbonyl cyanide chlorophenylhydrazine) were from SigmaChemical Co. Ascorbic acid and TMPD were purchasedfrom Tokyo Kasei Co., Ltd. All other chemicals wereobtained commercially at the highest purity available.

RESULTS

Growth characteristics. Bacillus sp. strain C-59 was orig-inally isolated as a xylanase-producing bacterium (11) onNa2CO3-containing medium. Investigation of the effect ofNa+ or K+ on the growth of Bacillus sp. strain C-59 showedthat it could also grow well on K2CO3 medium. Indeed, thespecific growth rate in K2CO3 medium was slightly higherthan in Na2CO3 (0.57 and 0.43 h-', respectively). This straincould not grow on K+-depleted medium (data not shown).As the external pH was kept at more than 9.5 during the first6 h, Bacillus sp. strain C-59 can grow in very alkalineconditions without added Na+.Ap analysis. To analyze the bioenergetic properties of

Bacillus sp. strain C-59, ApH and were measured, whichtogether constituted the Ap (25). The flow dialysis pattern ofmethylamine uptake by cells of Bacillus sp. strains C-59 and2b-2 showed that cells of C-59 actively took up methylaminein the presence of K2CO3 as well as Na2CO3. This was

consistent with the generation of ApH, inside acid, without

TABLE 1. Ap patterns of alkalophilic Bacillus species as afunction of Na+ and K+ content of the medium

Ap patterns in medium (pH 10.0):

Strain Na2CO3 (50 mM) K2CO3 (50 mM)58.8 ApH A4i 58.8 ApH Ai&i(mV) (mV) (mV) (mV)

Bacillus sp. strain 2b-2 + 84 -142 0 -105Bacillus sp. strain C-59 +90 -131 +82 -126B.firmus RABa +65 -162 0 -152

a Data from Kitada et al. (13).

added Na+. On the other hand, the cells of 2b-2 failed to takeup methylamine in the absence of Na+. Methylamine uptakeby the cells of C-59 was inhibited by the inclusion ofgramicidin (20 ,uM) in both Na2CO3 and K2CO3 media. Themagnitude and orientation of the ApH and A,d in cells of C-59and 2b-2 are shown in Table 1. In the absence of added Na+,the cells of C-59 generated a substantial ApH, acid in, inalkaline conditions, while the internal pHs of the cells of 2b-2and Bacillus firmus RAB, which require added Na+ forgrowth and amino acid transport, were 10.0, i.e., ApH = 0.Cells incubated with either Na+ or K+ exhibited appreciableAi\ values, and there was essentially no difference betweenNa+ and K+ medium. These results indicate that primaryproton pumping allowed the formation of A4j, and a second-ary Na+/H+ antiporter plays a vital role in pH homeostasisin the alkaline region. The question then arises how Bacillussp. strain C-59 could keep the cytoplasmic pH lower than inthe external milieu without added Na+. Does C-59 possess aspecial function for the regulation of internal pH other thanan Na+/H+antiporter, or does this antiporter have a specialhigh affinity for Na+ like that of Bacillus alcalophilus (20)?To examine this question in more detail, methylamine up-take by membrane vesicles was measured by a flow dialysisassay. Membrane vesicles energized in the absence of Na+could not take up methylamine actively (Fig. 1). By contrast,the vesicles equilibrated with 10 mM NaCl actively took upmethylamine. This was consistent with generation of a ApH,inside acid. This indicates that an Na+/H+ antiporter isinvolved in pH homeostasis in C-59 which requires relativelylower concentration of Na+. In the presence of 50 mMK2CO3, some indication of intravesicular acidification wasalso observed (see Discussion).

Transport system. The investigation was then extended to['4C]serine uptake by cells or membrane vesicles from C-59and 2b-2. Serine uptake by cells of C-59 was stimulated bythe addition ofNa+ to a maximal level at around 10 mM Na+(Fig. 2A). Further addition of Na+ up to 100 mM producedno increased stimulation. Conversely, cells of 2b-2 werestimulated for serine uptake at higher levels than for C-59;2b-2 also demonstrated a requirement for higher concentra-tions of Na+, attaining maximal uptake at Na+ concentra-tions of 100 mM or greater. Figure 2B shows the effect ofNa2CO3 and K2CO3 concentration on serine uptake by cellsof C-59. The profile of serine uptake in the presence ofNa2CO3 was similar to that in the presence of NaCl. Serineuptake was also stimulated by the addition of K2CO3. In thiscase, serine uptake was higher than for Na2CO3 at concen-trations higher than 20 mM, reaching a maximum at approx-imately 50 mM.The different Na+ requirements for serine uptake were

notable in membrane vesicles. At concentrations of addedNa+ less than 10 mM, the vesicles from 2b-2 exhibited littleserine uptake, whereas the vesicles from C-59 showed

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~~~~BIOENERGETICS OF AN ALKALOPHILIC BACILLUS SP. 5763

1

6-4 -

~~ H O- ------ A~~~~~~~4: .7

24 L.,

-r7-2-77-p =22

~ ~ ~ =-7

20 2!~~~iiiII~~~~I-,PH'K-77~Toi1u~~2

5

FIG. 1. Proton movements in right-side-out membrane vesicles

of Bacillus sp. strain C-59 energized at pH 9.5. Flow dialysis

experiments' were conducted as described in Materials and Meth-

ods. The counits of ['4C]methylamine flowing out in the dialysate

were monitored during experiments. The energy source, ascorbate

(Tris salt)-TMPD, was added at the points indicated by the arrows.

The cation used was (A) 100 mM KCl in 50 mM Tris buffer, (B) 10

MM NaCl in 50 mM Tris buffer, (C) 10 mM Na2CO3 buffer, or (D) 50

mM K2C03 buffer.

maximum accumulation (Fig. 3). Thus, Na'/serine symport

in vesicles from C-59 showed a requirement for a lower

concentration of added Na'. Since there was almost no

serine upitake in the absence of added Na', it is likely that

serine was taken up by the electrochemical sodium gradient

which was formed through an Na'/H' antiporter. The result

in Fig. 4 showed that this was the case. The membrane

vesicles were washed thoroughly with 50 mM Tris hydro-

chloride (pH 8) and suspended in the same buffer containing

10 MM MgSO4 and [14C]serine. A sodium ion gradient across

0

L.

FEC;10

0

C

0

2.0

1.5

1.0 [

0

0i

0 10 20 30 40 50

NaCL (mM)

FIG. 3. Effect of Na' concentration on ['4C]serine uptake by

membrane vesicles of Bacillus sp. strains C-59 (0) and 2b-2 (M). The

reactions were stopped after 5 min.

the membrane was imposed by the addition of NaCi (50 mm)

into the above solution. The addition of Na' caused a

transient uptake of serine (Fig. 4). When K' was substituted

for Na', no stimulation of serine uptake was observed.

Efflux of2Na' from membrane vesicles of Bacillus sp.

strain C-59. The results in Fig. 1 and 4 suggest that the

membrane from C-59 possesses an Na'/H' antiporter and

that this antiporter plays a crucial role in the maintenance of

relatively acidified cytoplasm and in generating a sodium

gradient. This antiporter activity was examined by measur-

ing the efflux of 22Na froM 22Na-loaded vesicles that were

energized either by the addition of an electron donor,

ascorbate-TMPD, or by the establishment of a valinomycin-

mediated potassium diffusion potential. In the experiment in

Fig. 5, vesicles equilibrated with 10 mM radioactive Na'

inside at pH 9.5 were diluted 100-fold into the' buffer con-

taining 10 mM NaCI at pH 7 or 9.5. In the absence of an

electron donor, there was almrost no 22Na effiux at either pH.

In the presence of the electron donor, rapid efflux of 22Na

was observed at both pHs 22Na efflux at pH 9.5 was more

rapid than that at pH 7. The addition of CCCP (20 p.M)

.0

I

0

0-1

60302000

CL

A3

(A) (B)

--U-

I

) 0 40 6 8 0NoI. rKL M

NcO2CO3 or K2C03 (mM)

FIG. 2. Effect of NaCi and Na2CO3 concentration on the uptake of [14C]serine by the cells of Bacillus sp. strains C-59 and 2b-2.

Experimental conditions were described in Materials and Methods. (A) Serine uptake by C-59 in the presence of NaCl (0) or KCI (A). The

dotted line shows the serine uptake by 2b-2 in the presence of NaCI (double scale). (B) Serine uptake by C-59 in the presence of Na2CO3 (0)

or K2C03

uP7-

V--

VOL. 169, 1987

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5764 KITADA AND HORIKOSHI

JG0-4-

0L.06

E0

4-

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0E£

0

0.

L-

LI)

6I

4[3

2

1

0.0 1 2 3 4 5

Time (min)FIG. 4. Transient uptake of f14C]serine by the addition of Na+.

Membrane vesicles of Bacillus sp. strain C-59 were preincubatedwith ['4C]serine for 3 min at 37°C, and then NaCl (0) or KC1 (A) wasadded to a final concentration of 50 mM at the time indicated by thearrow.

inhibited 22Na efflux in the presence of the electron donor.Although the data are not shown, the presence of 10 to 50mM nonradioactive Na+ in the dilution medium had no effecton the patterns of 22Na efflux. When the antiporter wasenergized by a valinomycin-mediated potassium diffusionpotential (inside negative), there was also rapid 22Na efflux atboth pHs. The vesicles showed more rapid efflux at pH 7than at pH 9.5.

DISCUSSION

The growth data showed that Bacillus sp. strain C-59could grow in alkaline conditions without added Na+. Flowdialysis analysis indicated that methylamine was taken up in50 mM K2C03 buffer, reflecting a ApH, acid in. Since thealkaline medium used here contained a fermentable sugar,starch, acidic metabolites might be produced to lower theinternal pH. To avoid this possibility, methylamine uptakewas examined by flow dialysis with right-side-out membranevesicles. There was no appreciable uptake of methylamine inthe absence of Na+. Thus, it is possible that C-59 reallyrequired Na+ for growth but that the alkaline medium mightcontain considerable amounts of Na+ contamination. Theresults in Fig. 1 indicated that Na+ was necessary tomaintain lower cytoplasmic pH than external pH in thealkaline region and that an electrogenic Na+/H+ antiportermay be involved in this reaction.Brey et al. (3) had proposed that the Na+/H+ antiporter

was not needed for pH homeostasis in Escherichia colibecause there was no evidence that Na+ was required for itsgrowth. They concluded that an K+/H+ antiporter played avital role for pH homeostasis in E. coli because K+ wasabsolutely required for growth and was continually accumu-lated to high concentration in the cells. In the case of C-59,however, K+ did not appreciably stimulate methylamineuptake by membrane vesicles (Fig. 1). It was unlikely that aK+/H+ antiporter regulated the internal pH in the alkalineregion.

It has been reported that Bacillus alcalophilus did notexhibit any requirement for added Na+ for growth but

possessed the Na+/H+ antiporter, having a higher affinity forNa+, which served the vital physiological function of main-taining lower cytoplasmic pH (20).

Observations of respiration-dependent and valinomycin-induced 22Na efflux supported the idea that 22Na efflux frommembrane vesicles from C-59 would represent the activity ofthe electrogenic Na+/H' antiporter. Na+-dependent H+influx could not be determined, but this 22Na+ efflux was notdue to a respiration-dependent sodium pump (27, 28) be-cause the efflux was inhibited by the addition of CCCP (20IsM).At an internal pH of 9.5, respiration-dependent 22Na'

efflux was more rapid at an external pH of 9.5 than at a pHof 7.0. Thus, high external H+ concentration (pH 7) had nostimulatory effect on 22Na efflux. This result was in goodagreement with the fact that the antiporter must normallyfunction at an alkaline pH. On the contrary, 22Na efflux wasmore rapid at pH 7 when the antiporter used a valinomycin-mediated K+ diffusion potential. This means that the exter-nal H+ concentration stimulated 22Na" efflux from thevesicles. Similar results have been obtained in the experi-ments on ATP synthesis in starved cells of Bacillus firmusRAB (8), but the antiporter in that strain is not active atneutral pH (4).

It is so far not clear why the external pH had differenteffects on the 22Na efflux rate between respiration-dependentand valinomycin-induced energization. Since the actualmembrane potential could not be determined, it is unclearwhether the value of the diffusion potential at pH 9.5 and atpH 7 is the same. The hypothesis by Haines (10) about alocalized pathway, however, is suggestive of one model, i.e.,some of the protons pumped out by respiration are madeavailable to the proton-utilizing antiporter in some localizedway, as observed in ATP synthesis by B. firmus RAB (6, 8,19).

Finally, we have to refer to the effects of K2CO3 onmethylamine and serine uptake. As shown in Fig. 1 and 2B,methylamine and serine were taken up actively in thepresence of K2CO3. The potassium carbonate used was thehighest grade, but 50 mM K2CO3 buffer (pH 10) contained 12ppm (0.5 mM) of Na+ from the atomic absorption analysis.

-(A) (B)

Time (sec)FIG. 5. 22Na' efflux from 22Na-loaded membrane vesicles of

Bacillus sp. strain C-59 at pH 7.0 (U) and 9.0 (0). Efflux of 22Na'was energized by either respiration (A) or K+ diffusion potential (B).Control experiments (A) were carried out in the presence of eitherCCCP (10 ,uM) (A) or K2CO3 (50 mM) (B).

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BIOENERGETICS OF AN ALKALOPHILIC BACILLUS SP.

Therefore, methylamine uptake by cells might be due to thecontaminating Na+ in K2CO3 buffer, because membranevesicles required considerable amounts of Na+ for its up-take. It is unlikely, however, that the stimulation of serineuptake was due to the contaminating Na+, because serineuptake was higher in K2CO3 over 50 mM concentration (Fig.2B). It will be of interest to study the influence of K2CO3 andC032- on physiological properties in alkalophiles, especiallythose able to grow on K2CO3 medium.

LITERATURE CITED1. Bassilana, M., E. Damiano, and G. Leblanc. 1984. Kinetic

properties of Na+-H+ antiport in Escherichia coli membranevesicles: effect of imposed electrical potential, proton gradientand internal pH. Biochemistry 23:5288-5294.

2. Bonner, S., M. J. Mann, A. A. Guffanti, and T. A. Krulwich.1982. Na+/solute symport in membrane vesicles from Bacillusalcalophilus. Biochim. Biophys. Acta 679:315-322.

3. Brey, R. N., B. P. Rosen, and E. Sorensen. 1980. Cation/protonantiport systems in Escherichia coli. J. Biol. Chem. 255:39-44.

4. Garcia, M. L., A. A. Guffanti, and T. A. Krulwich. 1983.Characterization of the Na+/H+ antiporter of alkalophilic bacilliin vivo: &qt-dependent 22Na' efflux from whole cells. J. Bacte-riol. 156:1151-1157.

5. Guffanti, A. A., R. Blanco, R. A. Benenson, and T. A. Krulwich.1980. Bioenergetic properties of alkaline-tolerant and alkalo-philic strains of Bacillus firmus. J. Gen. Microbiol. 119:79-86.

6. Guffanti, A. A., R. F. Bornstein, and T. A. Krulwich. 1981.Oxidative phosphorylation by membrane vesicles from Bacillusalcalophilus. Biochim. Biophys. Acta 635:619-630.

7. Guffanti, A. A., D. E. Cohn, H. R. Kaback, and T. A. Krulwich.1981. Relationship between the Na+/H+ antiporter and Na+/substrate symport in Bacillus alcalophilus. Proc. Natl. Acad.Sci. USA 78:1481-1484.

8. Guffanti, A. A., R. T. Fuchs, M. Schneier, E. Chiu, and T. A.Krulwich. 1984. A transmembrane electrical potential generatedby respiration is not equivalent to a diffusion potential of thesame magnitude for ATP synthesis by B. firmus RAB. J. Biol.Chem. 259:2971-2975.

9. Guffanti, A. A., P. Susman, R. Blanco, and T. A. Krulwich.1978. The proton motive force and a-aminoisobutyric acidtransport in an obligately alkalophilic bacterium. J. Biol. Chem.253:708-715.

10. Haines, T. H. 1983. Anionic lipid headgroup as a proton-conducting pathway along the surface of membrane: a hypoth-esis. Proc. Natl. Acad. Sci. USA 80:160-164.

11. Horikoshi, K., and Y. Atsukawa. 1973. Xylanase produced byalkalophilic Bacillus no. C-59-2. Agric. Biol. Chem. 37:2097-2103.

12. Kaback, H. R. 1971. Bacterial membranes. Methods Enzymol.22:99-120.

13. Kitada, M., A. A. Guffanti, and T. A. Krulwich. 1982.Bioenergetic properties and viability of alkalophilic Bacillus

firmus RAB as a function of pH and Na+ contents of theincubation medium. J. Bacteriol. 152:1096-1104.

14. Kitada, M., and K. Horikoshi. 1977. Sodium ion-stimulateda-[1-14C]aminoisobutyric acid uptake in alkalophilic Bacillusspecies. J. Bacteriol. 131:784-788.

15. Kitada, M., and K. Horikoshi. 1980. Sodium ion-stimulatedamino acid uptake in membrane vesicles of alkalophilic Bacillusno. 8-1. J. Biochem. (Tokyo) 88:1757-1764.

16. Kitada, M., and K. Horikoshi. 1980. Further properties ofsodium ion-stimulated a-(1-14C)aminoisobutyric acid uptake inalkalophilic Bacillus species. J. Biochem. (Tokyo) 87:1279-1284.

17. Koyama, N., A. Kiyomiya, and Y. Nosoh. 1976. Na+-dependentuptake of amino acids by an alkalophilic Bacillus. FEBS Lett.72:77-78.

18. Krulwich, T. A. 1983. Na+/H+ antiporters. Biochim. Biophys.Acta 726:245-264.

19. Krulwich, T. A. 1986. Bioenergetics of alkalophilic bacteria. J.Membrane Biol. 89:113-125.

20. Krulwich, T. A., A. A. Guffanti, R. F. Bornstein, and J.Hoffstein. 1982. A sodium requirement for growth, solute trans-port, and pH homeostasis in Bacillus firmus RAB. J. Biol.Chem. 257:1885-1889.

21. Krulwich, T. A., K. G. Mandel, R. F. Bornstein, and A. A.Guffanti. 1979. A non-alkalophilic mutant of Bacillus alca-lophilus lacks the Na+/H+ antiporter. Biochem. Biophys. Res.Commun. 91:58-62.

22. Kudo, T., and K. Horikoshi. 1979. The environmental factersaffecting sporulation of an alkalophilic Bacillus species. Agric.Biol. Chem. 43:2613-2614.

23. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

24. Mandel, K. G., A. A. Guffanti, and T. A. Krulwich. 1980.Monovalent cation/proton antiporters in membrane vesiclesfrom Bacillus alcalophilus. J. Biol. Chem. 255:7391-7396.

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26. Ramos, S. S., S. Schuldiner, and H. R. Kaback. 1979. The use offlow dialysis for determinations of ApH and active transport.Methods Enzymol. 55:680-688.

27. Tokuda, H., and T. Unemoto. 1981. A respiration-dependentprimary sodium extrusion system functioning at alkaline pH inthe marine bacterium Vibrio arginolyticus. Biochem. Biophys.Res. Commun. 102:265-271.

28. Tokuda, H., and T. Unemoto. 1982. Characterization of therespiration-dependent Na' pump in the marine bacteriumVibrio alginolyticus. J. Biol. Chem. 257:10007-10014.

29. Waddel, W. J., and T. C. Butler. 1959. Calculation ofintracellular pH from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO). Application to skeletal muscle of thedog. J. Clin. Invest. 38:720-729.

30. Zilberstein, D., V. Agmon, S. Schuldiner, and E. Padan. 1982.The sodium/proton antiporter is part of the pH homeostasismechanism in Escherichia coli. J. Biol. Chem. 257:3687-3691.

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