JOURNAL OF BACTERIOLOGY, Nov. 1973, p. 1019-1028Copyright © 1973 American Society for Microbiology

Vol. 116, No. 2Printed in U.S.A.

Characterization of the Membranes ofThermoplasma acidophilum

PAUL F. SMITH, THOMAS A. LANGWORTHY, W. R. MAYBERRY, AND A. E. HOUGLAND

Department of Microbiology, University of South Dakota, Vermillion, South Dakota 57069

Received for publication 25 July 1973

Thermoplasma acidophilum grows optimally under aeration at 59 C and pH 2.Both intact cells and membranes disaggregate below pH 1 and above pH 5,producing no sedimentable particles. Increase in ionic strength at pH 5 or belowresults in cellular lysis and membrane disaggregation. Membranous componentsproduced by lysis at alkaline pH reaggregate upon reduction of both pH and ionicstrength. Osmotic environment plays little role in cellular stability.Membranes prepared by sonic lysis at pH 5 exhibit vesicular structures and are

composed of multiple proteins. Although the amino acid composition of themembrane proteins is similar to other mycoplasmal membranes, the number offree amino and carboxyl groups is less than half of those in Acholeplasma.Reduction of the number of free carboxyl groups results in membrane stabiliza-tion over a wide range of pH. Increase in the number of free amino groups reverses

the stability of membranes relative to pH. Acidophily in Thermoplasma can berelated to a significant reduction in repulsing negative charges on the membraneproteins.

The obligatory acidophily and thermophily ofthe wall-less organism. Thermoplasma acido-philum (5), provide for a unique model to studymembrane stability in a harsh environment.Belly and Brock (2) have reported a sensitivityof this organism to lysis at alkaline pH and inthe presence of detergents, but insensitivityto osmotic changes, various enzymes, primaryalcohols, digitonin, and heat. We have under-taken a study of various properties of the intactorganisms and isolated cytoplasmic membranesto assess the parameters associated with thisrequirement for a hot acidic environment. Aprevious report (10) has shown the existenceof very long-chained isopranol ether lipids whichcould impart stability under the acidic growthconditions and the proper liquid crystallinestate of the lipid bilayer at the abnormally hightemperature required for growth.

MATERIALS AND METHODSOrganisms (isolate 122-1B2; ATCC 25905) were

grown at 59 C (pH 2.0) and harvested as previouslydescribed (10). Aeration was achieved by shaking orby continuous sparging with the desired gas. Volumelosses of the culture medium were avoided by employ-ing an environment saturated with water vapor.Growth was assayed by measurement of the opticaldensity at 540 nm in a Beckman DU or Bausch &Lomb Spectronic 20 spectrophotometer. The method

of most probable number using serial 10-fold dilutionsin culture medium also was used for assessment ofviable organisms. It was found necessary to use a freshpipette for each dilution since carryover of organismsfrom lower to higher dilutions resulted in spuriousresults.

Organisms used to determine the lytic effects of avariety of conditions were washed twice by resuspen-sion in deionized water followed by centrifugation for15 min at 20,000 x g and 0 C in a Sorvall RC-2Bcentrifuge. A concentrated suspension of organisms indeionized water served for addition to 5-ml volumes ofdesired suspending medium. Lysis of intact organismswas determined by measurement, within 30 min, ofthe decrease in optical density at 540 nm, decrease inthe number of viable organisms, and the liberation ofprotein and 260-nm absorbing substances in thesupernatant medium. Sonic lysis was carried out bysubjecting suspensions of the organisms at 4 C to 10 kcoscillations in a Raytheon sonic oscillator and to 20 kcoscillations in a Branson Sonifier for varying timeperiods.

Preparation of membranes proved to be difficult.As will be noted later, lysis at alkaline pH resulted inapparent dissolution of the membranes since nosediment was observable even after centrifugation for1 h at 100,000 x g in a Spinco model L ultracentrifuge.Suitable preparations exhibiting vesicular structureswith amorphous debris by electron microscopy wereobtained by sonic oscillation at 10 kc for 30 min oforganisms suspended in 0.05 M acetate buffer (pH5.0) or by 20 min of sonic oscillation at 20 kc of

1(19

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SMITH ET AL.

organisms suspended in deionized water (pH 5.5).The pellet resulting from centrifugation for 30 min at24,000 x g appeared as three layers. The top layer wasclear and amber colored; the middle layer was opaqueand grayish brown in color similar to pellets of intactorganisms; and the bottom layer was dark brown,opaque, and of minimal amount. The top layer wascarefully resuspended and washed several times indeionized water. After several washings this materialbecame fluffier and more easily resuspendable. Thismaterial was employed for all of the experimentsinvolving membranes. Acholeplasma laidlawii Bmembranes prepared as previously described (16)were used for comparative purposes.

Freeze-dried membranes were employed for analy-sis of the crude chemical composition and for totalamino acid content. The total lipid content wasdetermined by weighing the dried residue containingthe material extractable with chloroform-methanol(2:1, vol/vol) made 0.1 N with HCl. Protein wasdetermined by the method of Lowry et al. (11).Polysaccharide was assayed as glucose by the an-throne and phenol-sulfuric acid (1) methods appliedto the aqueous phenol extract, prepared as describedby Westphal and Jann (18), or to the lipid-freemembrane. Solution of the lipid-free membrane waseasily accomplished by suspension in 0.01 M phos-phate buffer, pH 8.0. Membrane preparations used foramino acid analysis were treated by the method ofWeinstein et al. (17) for removal of lipids. Three-mil-ligram portions of freeze-dried lipid-free membraneswere hydrolyzed in constant-boiling HCl for 25 h at100 C under nitrogen in Teflon-lined screw-cappedtubes. After removal of the acid in vacuo, the hydroly-sates were analyzed on a Beckman amino acid ana-lyzer, model 116, by the method of Spackman et al.(15).

Free amino groups on the membrane proteins weredetermined by the ninhydrin method as modified byRosen (14). Free carboxylic acid groups were meas-ured by the method of Hoare and Koshland (8)wherein the increase in glycine residues is measuredafter reaction of the membrane proteins with glycinemethyl ester in the presence of urea and 1-cyclo-hexyl-3-(2-morpholinoethyl)-carbodiimide methol-p-toluenesulfonate (Aldrich Chemical Co., Milwaukee,Wis.). Quantitation of glycine in untreated andtreated membranes was performed after hydrolysis in6 N HCl by chromatography of the trifluoroacetyln-butyl ester derivatives (7) on a biomedical gaschromatograph (Hewlett Packard, F and M model402) equipped with a flame ionization detector and a3370A digital electronic integrator. Analyses wereperformed on a glass column (1.8 m by 0.6 cm) packedwith TABSORB (0.325% ethlene glycol adipate onhigh-performance Chromosorb G; Regis ChemicalCo., Morton Grove, Ill.) at a temperaiure of 100 C for4 min followed by a programmed temperature (5 C permin) up to 250 C with helium as carrier gas at a flowrate of 60 ml/min.

Disk electrophoresis was carried out by the method(gel system la) described by Maurer (12), i.e., 7%polyacrylamide at pH 8.9 with the Canalco apparatus(5 mA per gel). Wet membrane pellets were solubi-lized in Tris(hydroxymethyl)aminomethane-glycine

buffer, pH 8.3, used as the electrode buffer forpolyacrylamide electrophoresis. Samples to be elec-trophoresed were incorporated into spacer gel. Gelswere stained with 1% amido black 10B in 7% aceticacid and destained electrophoretically.

Solubilization of both treated and untreated mem-branes was measured as the decrease in opticaldensity at 540 nm. Membranes were treated both inthe presence and absence of urea to alter the ioniccharges by reaction of the carboxylic acid groups withglycine methyl ester (removal of negative charges)and with ethylene diamine (removal of one negativecharge and addition of one positive charge for eachreactive carboxylic acid group). No obvious solubili-zation of membranes occurred during these reactionswhen conducted in the absence of urea. After thereaction, the membranes were washed repeatedlywith water to remove reagents and finally resus-pended in deionized water. Reaction in the presenceof 7.5 M urea resulted in solubilization of the mem-branes which formed reaggregates upon dialysisagainst 0.001 N HCl. These aggregates also were usedfor experiments on membrane solubilization. No at-tempt was made to block free amino groups since thealkaline reaction conditions result in solubilization ofthe membranes.Two types of membrane preparations were ex-

amined by electron microscopy. One consisted of thesonically prepared membranes; the other was a reag-gregated preparation produced by lysis of' the intactorganisms in 0.01 M phosphate buffer (pH 8.0)followed by dialysis against water. Water washedpellets were fixed by suspension in unbuffered 5%glutaraldehyde for 3 h. After postfixation in 1% OS04and dehydration in a graded ethanol series andacetone, the pellets were embedded in Epon resin.After being stained with lead citrate and uranylacetate, thin sections were cut on an LKB Ultratomeand viewed under an RCA electron microscope (modelU3g) at 50 kV.

RESULTS

Definition of the optimal conditions forgrowth was necessary to ensure the use oforganisms in the exponential phase. The opti-mal temperature appeared to be 59 C althoughequivalent yield could be obtained at 45 C if theincubation time was extended (Fig. 1). Theoptimal pH was shown to be pH 2 as reported byDarland et al. (5) (Fig. 2). No change in pHoccurred during growth. The effect of the size ofinoculum from a 24-h culture is seen in Fig. 3.No explanation is available for the decreasedtotal yield with inoculum sizes of less than 5%.This phenomenon was noted repeatedly. Aninoculum of greater than 5% of a 24-h cultureresulted in elimination or shortening of the lagphase of growth. As the cultures age autolysisand/or loss of viability usually occurs. Therelation of optical density to the most probablenumbers of viable microorganisms is shown inTable 1. Although the accuracy of this method

1020 J. BACTERIOL.

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

VOL. 116, 1973

EE0.5

0

ct).-04-a

0.30

_0.2

0_

MEMBRANES OF THERMOPLASMA

Time (hr)FIG. 1. Effect of temperature of incubation on the growth of Thermoplasma acidophilum.

Ec 0.50

0.410LO

a)C* 0.3C:

0

-5 0.2

CL0 0.1

pH4

pH 3

pH 2,pHS

20 60 80Time (hr)

FIG. 2. Effect of pH on the growth of Thermoplasma acidophilum.

140

is limited, in general, the numbers of viableorganisms matched reasonably well with theoptical density through the exponential phase ofgrowth. The reduction in optical density as theculture ages is seen as a drastic loss of viability.The technique of dilution and plating could notbe used since the organisms fail to grow ade-quately on solid culture media. Aeration byshaking or sparging increases the growth rateand improves the overall yield of organisms(Table 2). Continuous gassing with nitrogenlowered the yield to slightly less than obtainedwith stationary-phase cultures. No significantgrowth occurred in an atmosphere of carbon

dioxide. This gas actually lowered the yieldwhen mixed with an atmosphere of nitrogen.These results led to the use of organisms grownwith shaking in a medium of pH 2 at 59 C. A 5%inoculum was added, and incubation was car-

ried out for 40 h so as to provide a maximal yieldof organisms in the late exponential phase.

Suspension of the organisms in deionizedwater (pH 5.5) did not result in any reduction ofoptical density or the appearance of any 260-nmabsorbing material and protein in the superna-tant water. However, viability was reduced atleast 100-fold below the viability determined on

suspension in fresh culture medium. Both pH

1021

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SMITH ET AL.

5.0 %1.0 %

20 40 60 80 100 120 140 160

Time (hr)FIG. 3. Effect of inoculum size on growth of Thermoplasma acidophilum.

TABLE 1. Relation of optical density (OD,4,) to mostprobable number of Thermoplasma acidophilum

Highest 10-foldTime (h) OD540 dilution

showing growth

0 0.008 64 0.015 68 0.035 712 0.075 823 0.420 928 0.460 932 0.610 1049 0.725 1072 0.690 796 0.560 2

and ionic strength of the suspending mediumproduced effects on the stability and viability ofthe organisms. The results of suspending theorganisms in 0.1 M buffers of varying pH areshown in Fig. 4. Surprisingly, maximal stabilitybased upon viability measurements was seen atpH values of 3 to 4 in spite of the optimal pH forgrowth being 2. Apparent lysis occurred belowpH 2 and at pH 6 and above. Based uponviability measurements, a small portion of thepopulation was capable of recovery from theeffects of pH values from 6 through 8. Above pH8, lysis essentially was complete. Neither the

TABLE 2. Effect of gaseous environment on thegrowth of Thermoplasma acidophilum

Optical density at 540 nmGas

6 h 24 h 48 h

Stationary 0.023 0.220 0.260Air 0.024 0.390 0.600N2 0.026 0.150 0.235CO2 0.025 0.043 0.04595% N2 + 5% CO2 0.023 0.067 0.140

cellular concentration nor the temperature hasany noticeable effect on these results.

Increasing the ionic strength of the suspend-ing medium brought about lysis of the orga-nisms at pH values of normally maximal stabil-ity (Fig. 5). On the contrary, a slight increase instability was effected at pH values of 6 to 8 byincreasing ionic strength. Viability paralleledthe optical density values. Similar results wereobtained using NaCl, KCI, MgCl2, and MnCl2for varying ionic strength. Sucrose in concentra-tions up to 20% had no protective effect. Ly-sates resulting from treatments at pH 6 orabove, or at high ionic strength, exhibited littleor no sediment upon centrifugation at forcesas high as 100,000 x g. Apparently the envelop-ing membranes were disaggregated under theseconditions. A gelatinous clump of unidentified

0.6

0.5

0.4

0.3

0.2

EC0

to

-0

(.

0

1022 J. BACTERIOL.

"I".%

I& 3.0 %

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MEMBRANES OF THERMOPLASMA

material consistently appeared at pH 7. Thestringy nature of this material suggested nucleicacids.

Addition of the chelating agent(ethylenediaminetetraacetate, disodium salt) inconcentrations up to 5 mM had no effect on thestability of the organisms suspended in deion-ized water. No change in optical density and noleakage of protein or nucleic acid into thesupernatant fluid were detected. The poly-amine, spermine, had no protective effect at pH5 or 7 in concentrations varying from 10-5 to10-2 M. At pH 5, increased lysis occurred whichcould be accounted for by the increase in ionic

CP

0

14

20 0

0.6

_, ao_

1.4-

300 1.2-

1.0-

200 0.8 </ 1"

100 0.4- ',

strength brought about by the concentration ofadded spermine. The anionic detergent, sodiumdodecyl sulfate, was a more effective lytic agentthan was the cationic detergent, cetyl trimethylammonium bromide (Fig. 6).The reduction in optical density and viability

of organisms suspended in the culture mediumas a result of sonic oscillations at 10 and 20 kc isshown in Table 3. The viable count was a morereliable measure of cellular destruction. Theoptical density increased with 20-kc oscilla-tions. Reliable viability data could not be de-rived from water suspensions due to the loss ofviability in deionized water. Similar optical

pHFIG. 4. Effect of pH on the stability of intact cells of Thermoplasma acidophilum: pH I to 3, KCI-HCI

buffers; pH 4 to 5, acetate buffers; pH 6 to 8, phosphate buffers; pH 9 to 12, borate buffers, all at 0.1 Mconcentration.

VOL. 116, 1973 1023

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SMITH ET AL.

1.2

10Ec0

Inc

0

0.cL0

0.8

0.6

0.4

0.2

pH 2

pH 3

pH 5pH 6

0 0.2 04 06 0.8 1.0 1.2r/2

FIG. 5. Effect of ionic strength on stability of Thermoplasma acidophilum at acid pH, [r/2 adjusted withNaCl.

0.5 W ; . EDTAEC

°0 0.40\In

0.3

CTAB'0.2

0.0.0

0 ~~SDS0 I 2 3 4 5

mMFIG. 6. Effect of a chelating agent and detergents on the stability of intact cells of Thermoplasma

acidophilum. EDTA, Ethylenediaminetetraacetate; CTAB, cetyl trimethylammonium bromide; SDS, sodiumdodecyl sulfate. Organisms suspended in 0.05 M KCI-HCI buffer, pH 2.

1024 J. BACTERIOL.

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MEMBRANES OF THERMOPLASMA1

density readings were obtained with water sus-pensions of organisms. No change in pH wasobserved after sonic treatments.The combined use of a suspending medium of

moderate ionic strength (about 0.05) at a pH(pH 5) near the value producing total lysis andsonic oscillation permitted the recovery ofmembranous material. In no preparation didbreakage near completion. Rather, the maximaldegree approached an estimated 50%.

Figure 7 is an electron micrograph of amembrane preparation produced by sonic lysis.

TABLE 3. Effect of sonic treatment on Thermoplasmaacidophiluma

10 kc 20 kcTime (min)

OD540 MPN OD540 MPN

0 1.343 1010 1.343 10100.5 1.343 1010 1.462 10101 1.303 107 1.462 1072 1.169 105 1.422 1073 1.122 104 1.485 1055 0.924 104 1.485 10210 0.624 101 1.485 101

aODD540, optical density atprobable number.

540 nm; MPN, most

It exhibits trilaminar vesicular elements andamorphous material. The dark granules areconsidered to be artifacts resulting from thestaining process; if not, no explanation can bemade for their appearance. The nature of theamorphous material is unknown at present. It isunlikely to be ribosomes as these should sedi-ment in the preparation lysed at alkaline pHbefore reaggregation.

Polyacrylamide gel electrophoresis of soni-cally prepared membranes produced a multi-plicity of protein bands. All protein appeared toenter the separation gel (Fig. 8). As with mem-brane proteins in general, those of Thermo-plasma are a heterogeneous mixture. Grosschemical analysis revealed a composition ofapproximately 60% protein, 25% lipid, and 10%carbohydrate. The nature of the lipid compo-nent has been reported. The amino acid compo-sition of the membrane protein (Table 4) didnot reveal any major differences from mem-brane proteins of other mycoplasmas (4, 6, 13).A difference in the membrane protein compo-nent when compared with A. laidlawii B be-came apparent upon determination of the freeamino and carboxyl groups. Although the ratiosof free carboxyl to free amino groups weresimilar in each of the organisms, the total

9

C~~~~~l.

* .. };

V, ..1.

-tei, . 4.FIG. 7. Electron micrograph of a

acidophilum.thin section of sonically prepared membranes from Thermoplasma

l--

a IL0.

1025VOL. 116, 1973

If

-,b-'t -b

.., .,%,IL16

.0.1

Ws-Ir

I

le

"I--'Wi.-A

4h.-3.u I.I.

I

.. 1.

.."- , t..

% IV*_

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SMITH ET AL.

remove one potential negative charge. Table 6shows the results of suspending untreated andtreated membranes in buffers of varying pH andmolarity. The untreated membranes behavedsimilarly to the intact organisms, i.e., stabilitydecreased as pH increased. Membranes treatedwith glycine methyl ester appeared relativelystable at all pH values tested. Membranestreated with ethylene diamine exhibited a be-havior opposite to the untreated membranes,i.e., stability increased as the pH increased.Similar results were obtained with membranesreacted with glycine methyl ester in the pres-

ence of urea followed by apparent reaggregationupon dialysis to remove reactants.

DISCUSSIONSome of our results are at variance with those

of previous reports (2, 5), notably the stability

TABLE 4. Amino acid composition of the membranesof Thermoplasma acidophilum

Amino acid MoV/100 mg"

Lysine 6.05Histidine 1.30Arginine 3.39Aspartic 8.61Threonine 4.30Serine 4.52Glutamic 7.14Proline 3.73Glycine 7.51Alanine 6.70Half Cystine None detectedValine 6.70Methionine 2.49Isoleucine 6.96bTyrosine 7.31Phenylalanine 4.27

a Average of three determinations.bIsoleucine value contains the small value found

for alloisoleucine.

FIG. 8. Polyacrylamide gel pattern of membraneproteins from Thermoplasma acidophilum, pH 8.9.

numbers of these groups per unit protein in T.acidophilum were less than half those found inA. laidlawii (Table 5).

Alterations of the free carboxyl groups weremade to determine whether an imbalance ofelectrical charges would alter the solubility char-acteristics of the membrane. Glycine methylester attached to the free carboxyl groups wouldremove one potential negative charge; ethylenediamine would add one positive charge and

TABLE 5. Free amino and carboxyl groups inmembrane proteins of Thermoplasma acidophilum

and Acholeplasma laidlawii

Free amino Free carboxylOrganism groups" groupsb -COOH/(lmol/mg (AmolImg -NH2

of protein) of protein)

T. acidophilum ... 0.47 4 0.03 1.76 ± 0.10 3.8A. laidlawii ...... 1.05 ± 0.11 4.27 ± 0.33 4.0T. acidophilumiA. Iaidlawii .... 0.45 0.41t

a Average of three preparations.Average of two preparations.Ratio.

1026 J. BACTERIOL.

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MEMBRANES OF THERMOPLASMA

TABLE 6. Solubility of membranes from T.acidophilum treated to alter the charges on

membrane proteins

Optical density, 540 nm

Suspending Reacted Reactedmedium Untreated with with

glycine ethylenemethylester diamine

0.01 M, pH 5 0.750 0.660 0.2600.1 M, pH 5 0.600 0.640 0.2850.5 M, pH 5 0.300 0.580 0.280

0.01 M, pH 6 0.150 0.660 0.2950.1 M, pH 6 0.150 0.530 0.3500.5 M, pH 6 0.190 0.560 0.350

0.01 M, pH 7 0.070 0.680 0.3300.1 M, pH 7 0.140 0.620 0.3300.5 M, pH 7 0.135 0.560 0.330

0.1 M, pH 8 0.5900.5 M, pH 8 0.640

Deionized water 0.760 0.640 0.300

of the organisms at various pH and ionicstrength. The data of Belly and Brock (2)indicate a stability, based upon optical densitychange and protein liberation, from pH 1through 8, and no significant optical densitychanges occur in NaCl solutions of concentra-tions to 1.0 M. Our data with the same organismindicate a reasonable stability from pH 1through 5 and instability at all pH values toincreasing ionic strength. No explanation forthese discrepancies can be given. Our resultsrelative to the optimal pH, time, and tempera-ture of growth conform with the results ofDarland et al. (5). The organism is a facultativeaerobe, but both growth yield and rate of growthimprove greatly upon aeration. The existence ofnaphthoquinones in the organism (10) suggestthe presence of an oxidative respiratory chain.

Difficulty was experienced in preparing mem-branes of Thermoplasma. Under acidic condi-tions the organisms are quite resistant to physi-cal breakage, particularly at low ionic strength.At pH greater than 6, the membrane disaggre-gates into non-sedimentable fragments. A com-promising condition of pH and ionic strengthpermitted breakage of a portion of the orga-nisms by sonic treatment. The membranes thusprepared were free of intact cells but containedamorphous material, the nature of which is notknown. Gross chemical analysis showed a com-

position similar to other mycoplasma mem-

branes. Multiple protein bands were seen in

polyacrylamide gel electrophoresis, indicating amultiplicity of protein species.A previous report (10) described the existence

in T. acidophilum of long-chained isopranolether lipids which could be related to thethermophilic and acidophilic nature of the orga-nism. The results of this preliminary examina-tion of the membrane proteins also can becorrelated with this requirement of a harshenvironment. The behavior of intact organismstoward pH and ionic strength of the suspendingmedium suggested that ionic interactions weremajor factors in maintenance of the structuralintegrity of the cytoplasmic membrane. Al-though the amino acid composition is similar tothe amino acid composition of membranes frommesophilic mycoplasma, the number of freeamino and carboxyl groups is less than half thenumber in mesophilic organisms. This reducedquantity of chargeable groups indicates agreater degree of hydrophobicity for the Ther-moplasma. The relative hydrophobic nature ofthis organism is characterized by settling out ofsuspension and tight packing of the cellularpellet upon centrifugation. Other mycoplasmasbehave in opposite fashion. The chargeablegroups would be further reduced at the lowoptimal pH for growth and stability because of areduction in the ionization of the free carboxylgroups. Increasing the pH would result in agreater ionization of these carboxyl groups andconceivably upset the normal balance of posi-tive and negative electrical charges in the mem-brane. The resulting repulsion of negativecharges could effect a disruption of the mem-brane proteins causing cellular lysis. Such apossibility is supported by the effects of altera-tions on the free carboxyl groups. Removal ofpotential negative charges by addition of gly-cine methyl ester stabilizes the membranes atpH values near neutrality. Removal of a poten-tial negative charge and the addition of anotherpositive charge by addition of an ethylenediamine radical reverse the pH effect of normalmembranes, i.e., instability occurs at acid pHbecause of an increase in the number of repuls-ing positive charges. A similar, but not identi-cal, phenomenon occurs with halophilic bacte-ria and with bacterial membranes treated bysuccinylation to increase the number of ioniza-ble carboxyl groups on the membrane proteins(3). In these situations, disaggregation of mem-branes is hypothesized as due to repulsion ofnegative charges which can be reduced byincreasing the ionic strength of the suspendingmedium.The lytic effect of increased ionic strength is

compatible with a role for ionic interactions in

1027VOL. 116, 1973

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SMITH ET AL.

membrane stabilization. The ions of the neutralsalt first interact electrostatically with thecharged groups on the membrane protein, re-sulting in a typical salting in process. Althoughnot shown in Fig. 5, further increase of the ionicstrength to 2 or above results in an increase inoptical density, typical of the salting out proc-ess of proteins. These data support a mecha-nism of balanced ionic interactions for stabiliza-tion of membrane proteins in Thermoplasma.This balance is near the optimum in the culturemedium which has a calculated ionic strengthof about 0.25 and a pH of 2.0.The stability of Thermoplasma has a corol-

lary in the extreme halophilic bacteria. The H+concentration has a stabilizing effect onThermoplasma analogous to the high salt con-centration on the extreme halophiles (9). Rais-ing the pH for Thermoplasma and lowering thesalt concentration for the halophiles results indisintegration of the membranes. Osmotic envi-ronment is not the sole basis for protection ineither type of organism since sucrose does notstabilize them. In contrast to the extreme halo-philes, Mg2+ does not protect the Thermo-plasma membrane from disintegration. Nor dothe proteins in the Thermoplasma membranecontain an overabundance of acidic amino acids.Rather, these proteins appear to be more hy-drophobic based upon the number of accessiblecharge groups.Our preliminary examination of the mem-

branes of T. acidophilum suggests that ther-mophily can be related to the long isopranolchains of the lipids and that acidophily can berelated to the sole presence of ether lipids and toa drastic reduction in chargeable groups on themembrane proteins. The obligatory require-ment for this harsh environment probably re-sults from the invariable nature of the proteinsand lipids comprising the major segment of thelimiting membrane.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Servicegrant Al-04410-11 from the National Institute of Allergy andInfectious Diseases.

The authors acknowledge the assistance of M. Houglandfor the electron microscopy.

LITERATURE CITED

1. Ashwell, G. 1969. New colorimetric methods ot sugar

analysis, p. 85-95. In E. F. Neuteld and V. Ginsburg(ed.), Methods in enzymology, vol. 8. Academic PressInc., New York.

2. Belly, R. r., and T. D. Brock. 1972. Cellular stability ot athermophilic, acidophilic mycoplasma. J. Gen. Micro-biol. 73:465-469.

3. Brown, A. D. 1964. The development ot halophilic proper-ties in bacterial membranes by acylation. Biochim.Biophys. Acta 93:136-142.

4. Choules, G. L., and R. F. Bjorklund. 1970. Evidence otstructure in Mycoplasma membranes. Circular dichro-ism, optical rotatory dispersion, and infrared studies.Biochemistry 9:4759-4767.

5. Darland, G., T. D. Brock, W. Samsonoff, and S. F. Conti.1970. A thermophilic, acidophilic mycoplasma isolatedfrom a coal refuse pile. Science 170:1416-1418.

6. Engelman, D. M., and H. J. Morowitz. 1968. Characteri-zation of the plasma membrane of Mycoplasma laid-lawii. IV. Structure and composition of membrane andaggregated components. Biochem. Biophys. Acta150:385-.396.

7. Gehrke, C. W., D. Roach, R. W. Zumwalt, D. L. Stalling,and L. L. Wall. 1968. Quantitative gas-liquid chroma-tography of amino acids in proteins and biologicalsubstances. Analytical BioChemistry Laboratories,Inc., Columbia, Mo.

8. Hoare, D. G., and D. E. Koshland, Jr. 1967. A method forthe quantitative modification and estimation of car-boxylic acid groups in proteins. J. Biol. Chem.242:2447-2453.

9. Kushner, D. J. 1968. Halophilic bacteria. Advan. Appl.Microbiol. 10:73-99.

10. Langworthy, T. A., P. F. Smith, and W. R. Mayberry.1972. Lipids of Thermoplasma acidophilum. J. Bacte-riol. 112:1193-1200.

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

12. Maurer, H. R. 1971. Disc electrophoresis and relatedtechniques of polyacrylamide gel electrophoresis.Walter de Gruyter, Berlin.

13. Morowitz, H. J., M. E. Tourtellotte, W. R. Guild. E.Castro, and C. Woese. 1962. The chemical compositionand submicroscopic morphology ot Mycoplasma gal-lisepticum, avian PPL05969. J. Mol. Biol. 4:93-103.

14. Rosen, H. 1957. A moditied ninhydrin colorimetric analy-sis for amino acids. Arch. Biochem. Biophys. 67:10-15.

15. Spackman, D. H., W. H. Stein, and S. Moore. 1958.Automatic recording apparatus tor use in the chroma-tography of amino acids. Anal. Chem. 30:1190-1206.

16. Smith, P. F., W. L. Koostra, and W. R. Mayberry. 1969.Observations on membranes ot Mycoplasma laidlawiistrain B. J. Bacteriol. 100:1166-1174.

17. Weinstein, D. B., J. B. Marsh, M. C. Glick, and L.Warren. 1969. Membranes ot animal cells. IV. Lipids ofthe L cell and its surtace membrane. J. Biol. Chem.244:4130-4111.

18. Westphal, O., and K. Jann. 1965. Bacterial lipopolysac-charides, p. 83-91. In R. L. Whistler (ed.), Methods incarbohydrate chemistry, vol. 5. Academic Press Inc.,New York.

1028 J. BACTERIOL.

on February 22, 2020 by guest

http://jb.asm.org/

Dow

nloaded from