JOURNAL OF BACTERIOLOGY, Oct. 1976, p. 248-256Copyright X 1976 American Society for Microbiology

Vol. 128, No. 1Printed in U.S.A.

Properties of Escherichia coli Mutants with Alterations inMg2+-Adenosine TriphosphataseLAWRENCE W. ADLER AND BARRY P. ROSEN*

Department ofBiological Chemistry, University ofMaryland School ofMedicine, Baltimore, Maryland 21201

Received for publication 7 June 1976

A mutant Escherichia coli, selected for resistance to the antibiotic neomycin,was unable to utilize nonfermentable carbon sources for growth. Two strainswere selected from this mutant on the basis of their ability to grow utilizingsuccinate as a carbon source. All three strains had approximately equal amountsof the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) protein, but theactivity of the enzyme differed in each strain. The Mg2+-ATPase from each of thethree strains lost activity upon solubilization and appeared to undergo rapiddissociation once solubilized. This dissociation is similar to that described for thewild type after cold exposure.

Various laboratories have isolated mutantsof Escherichia coli that are defective in theMg2+-adenosine triphosphatase (ATPase) (BF1)(1, 6, 11-15, 18, 20). Some of these strains wereselected for inability to utilize nonfermentablecarbon sources for growth (1, 13-15), whereasothers were selected for resistance to antibioticssuch as neomycin and streptomycin (6, 11, 12,14, 18, 20).Many of the strains selected for antibiotic

resistance have been found to be defective intheir ability to couple oxidative energy to activetransport of small molecules (11, 14, 19, 20).The question arises as to whether loss of enzy-matic activity of the BF1 is directly related tothe inability to concentrate solutes andwhether resistance to antibiotics in BF1 mu-tants can be correlated with either BF1 activityor ability to couple oxidation to transport.This paper reports some physiological and

biochemical properties of a neomycin-resistantmutant that has previously been shown to bedefective in the BF1 and to be unable to couplerespiratory energy to active transport (12). Theproperties of two partial revertants of this mu-tant isolated by ability to utilize succinate forgrowth (12) are also explored. The data suggestthat a correlation can be made between sensi-tivity to neomycin and ability to catalyze activetransport, but that a similar correlation be-tween Mg2+-ATPase activity and active trans-port activity may not exist.

MATERIALS AND METHODSBacteria and media. Cultures were grown in a

basal salts medium (16) supplemented with variouscarbon sources as noted. Solid media consisted of

nutrient agar, neomycin agar (basal salts mediumcontaining 11 mM glucose and neomycin sulfatesupplemented with 2% agar), and succinate agar(basal salts medium containing 85 mM sodium suc-cinate supplemented with 2% agar).The strains used were E. coli K-12 strain 7 (5) and

the BF,-deficient derivatives NR70 (11) and NR76(12). The isolation of the spontaneous partial revert-ants NR76A and NR76B has been described previ-ously (12).

Preparation of membrane-bound and solubleBF,. Membrane vesicles with bound BF, were pre-pared by lysis of intact cells with a French pressurecell as previously described (17). Cytoplasmic frac-tions refer to the supernatant solutions obtainedafter centrifugation of the lysed cells at 105,000 x gfor 1 h. Ethylenediaminetetraacetic acid (EDTA)-treated membranes (stripped membranes) andEDTA extract (crude soluble BFI) were preparedfrom washed membrane vesicles as described previ-ously (17).ATPase assays. Three procedures were utilized

for the assay of ATPase activity, as described previ-ously (17). The first assay, performed on white-spotplates, gave a qualitative estimate of the ATPaseactivity of column fractions. The second assay wasused for a rough quantitation of ATPase-containingsolutions. The third assay was used for the moreaccurate determination of activities and kinetic con-stants. It consisted of the same assay mixture asused in the other two assays with the addition of anATP-regenerating system. The reaction was linearwith both time and protein concentration.

Immunological assays. Antiserum was preparedagainst a purified preparation of BF, from strain 7as described previously (17). Specificity of the anti-sera toward the BF, was verified by the presence ofasingle precipitin arc on crossed immunoelectropho-resis of crude soluble BF, against the antiserumaccording to the method of Laurell (8). The localiza-tion of the precipitin arc corresponded to the locali-

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VOL. 128, 1976

zation of azide-inhibitable ATP-hydrolyzing activitydetermined by histochemical staining of the slidesfor inorganic phosphate (7).

Immunodiffusion and immunoelectrophoresis as-

says were performed using microscope slides coveredwith 3 ml of 1% agarose in 0.05 M barbital-hydro-chloride buffer, pH 8.6. In the immunoelectropho-resis assay, the proteins were allowed to migratetoward the cathode for 4 h at 100 V. In both assays,diffusion was allowed to proceed for 12 to 16 h at23°C, followed by two washes with 0.9% NaCl. Theslides were stained for 15 min at 23°C in 0.1% naph-thol blue black in 7.5% acetic acid, followed by de-staining in 7.5% acetic acid. For the localization ofcross-reacting material in column fractions, thedensity of the precipitant arcs was estimated by eye

without washing or staining.Ammonium sulfate fractionation and agarose

column chromatography of the BF,. Solid ammo-nium sulfate (430 g/liter) was added to crude solublefractions containing the BF, with stirring over aperiod of 15 min. The suspension was stirred for anadditional 15 min, followed by centrifugation at12,000 x g for 10 min. The pellet was resuspended in1 ml of a buffer consisting of 0.05 M tris(hydroxy-methyl)aminomethane - hydrochloride, 0.5 mMEDTA, 1 mM j3-mercaptoethanol, and 10% glycerol(vol/vol). The solution was clarified by centrifuga-tion and applied to 1.6-cm-diameter column filled to90 cm with BioGel A-1.5M. The column was elutedat 12 ml/h with the same buffer. Fractions of 2 mlwere collected. All steps of the procedure were per-formed at 23°C.

Transduction. The generalized transducing bac-teriophage 363 was used for transduction experi-ments (4). The bacteriophage was grown on strainMA96 (ilvA, obtained from W. Maas) and used toinfect strains unable to utilize succinate for growth.Dilutions were spread onto succinate plates contain-ing isoleucine and valine. Colonies were then scoredfor the ability to grow in the absence of isoleucineand valine.

Protein assays. The protein content of columnfractions was estimated by using a nomogram (2)based on the absorbances at 280 and 260 nm. Otherprotein determinations were performed according toa modification of the method of Lowry et al. (9).

Chemicals. Neomycin sulfate, rabbit skeletalmuscle pyruvate kinase (type II), and naphthol blueblack were obtained from Sigma Chemical Co.BioGel A-1.5M was purchased from BioRad Labora-tories, special enzyme grade ammonium sulfatefrom Schwarz/Mann, and dicyclohexylcarbodiimide(DCCD) from Eastman Kodak Co. All other chemi-cals were reagent grade.

RESULTS

Growth properties of the mutant and itsrevertants. The mutant NR76, selected forspontaneous resistance to 50 ,g of neomycinsulfate per ml, is unable to grow in basal saltsmedium using succinate, D-lactate, acetate, a-

ketoglutarate, malate, or pyruvate as carbon

Mg-ATPase MUTANTS OF E. COLI 249

source. By selection for spontaneous growth onsuccinate agar plates, it was possible to isolatestrains derived from NR76 that had regainedthe ability to utilize those carbon sources. Re-vertant strains with wild-type characteristics,as well as partial revertants, were obtained.Neither of the two strains chosen for furtherstudy completely regained the wild-type char-acter. NR76B showed visible colonies on succi-nate agar plates after 2 days, whereas NR76Arequired 3 to 4 days. The parent, strain 7, ex-hibited visible growth overnight on such plates.The ability of these strains to utilize fermenta-tive and aerobic carbon sources in liquid me-dium (Table 1) was similar to the results foundon agar plates. Strain 7 showed the fastestgrowth rate on each carbon source, followedclosely by NR76B. NR76A grew less well, butbetter than NR76.Each strain was examined for sensitivity to

neomycin (Table 2). The cultures were streakedonto agar plates supplemented with varyingconcentrations of neomycin. After 40 h, eachstrain showed varying amounts of growth onplates with no neomycin, and the size of thecolonies was roughly proportional to the gener-ation time on basal salts medium supplementedwith glucose. Strain NR70 (11) was used as acontrol-resistant strain. NR76B showed nogrowth on plates containing as little as 5 ug ofneomycin per ml, and the colonies of strain 7were greatly reduced in size. On the otherhand, no reduction in colony size was noted forthe other three strains. Increasing concentra-tions of neomycin caused reductions in colony

TABLE 1. Growth of strains 7, NR76, NR76A, andNR76B on various carbon sourcesa

Doublings/hStrain 100 mM 11 mM 55 mM

succinate D glucose glycerol

7 0.75 1.67 1.25NR76B 0.40 1.43 0.67NR76A 0.00C 1.11 0.56NR76 0.00d 0.71 0.42

a Overnight cultures of each strain were washedunder sterile conditions and inoculated into flaskscontaining basal salts medium supplemented with100 mM succinate, 11 mM glucose, or 55 mM glyc-erol. Growth was estimated by turbidity as mea-sured with a Klett-Summerson colorimeter equippedwith a no. 66 filter.

b Carbon source.c Growth was detectable after 3 days on succinate

agar plates.d No growth was detectable after 7 days on succi-

nate agar plates.

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250 ADLER AND ROSEN

TABLE 2. Sensitivity to neomycin sulfateRelative growth on minimal-glucose agar

Strain0a,b 5a.b 10a.b 25a.c 50.c

7 ++++ + _ _ _NR76B +++ - - - -NR76A ++ ++ + ± -NR76 ++ ++ ++ + -NR70 + + + + + +

a Neomycin sulfate (micrograms per milliliter).b Scored after 40 h at 37°C.Scored after 60 h at 37°C.

sizes for those strains as well, in the orderNR76A, NR76, and NR70.

Similar results were obtained in liquid cul-tures. Strains NR76 and NR76A were unaf-fected by 15 ,ug of neomycin sulfate per ml inbasal salts medium supplemented with 0.2%glucose. Strains 7 and NR76B ceased growthwithin one doubling after the addition of neo-mycin. Thus, in these strains an inverse rela-tionship exists between the ability to utilizeaerobic carbon sources and resistance to neomy-cin. The strains that were resistant to highconcentrations of neomycin grew poorly or notat all using succinate, whereas the strains thatgrew well on succinate were sensitive to lowconcentrations of neomycin. Thus selection forsuccinate-utilizing strains from a neomycin-re-sistant strain resulted in the selection of neo-mycin-sensitive strains.

Genetics of the mutants. A number of mu-tants defective in aerobic metabolism havebeen reported to be located at 73.5 min on theE.coli chromosome (1, 6, 11, 13, 14, 18, 20). Theability to utilize nonfermentable carbon sourceswas co-transducible with ilv with frequencies of25 to 30% in strains NR70, NR76, and NR76A.Co-transducibility was not measured withstrain NR76B because of its ability to utilizesuccinate. These results together with thoseshowing a defective BF1 (see below) and a nor-mal membrane site or BFo (12) suggest that thelesion in these strains are in the uncA region ofthe chromosome.

Activity of the membrane-bound and solu-ble BF. Each of the strains was examined forATPase activity in membranes prepared bylysis with a French pressure cell. The partialrevertant NR76B was found to have about 8% ofthe membrane-bound BF1 activity of the origi-nal wild type, strain 7. The slower-growingpartial revertant, NR76A, and the mutantNR76 each showed about 1% of the activity ofstrain 7.The diminished membrane-bound Mg2+-

ATPase activity found in the mutants and re-

vertants could result from one of a number ofpossible sources: (i) the enzyme might bindpoorly to the membrane and exist predomi-nately in the cytoplasm; (ii) the Km of the en-zyme might be increased; (iii) there might be adecrease in the amount of enzyme; or (iv) theVmax might be reduced.

It can be readily seen from the data in Table 3that the activity is not localized in the cyto-plasm of the mutant or revertants, since thecytoplasms contained only small amounts ofATPase activity when compared with the totalamount of activity found in the wild type.The BF1 could be solubilized by treatment of

the membrane with a buffer of low ionicstrength containing EDTA (12). Approximately50% ofthe activity ofthe wild type was found inthe supernatant solution after centrifugation,with about 20o remaining on the strippedmembranes (Table 3). In other experiments, upto 70% of the activity was solubilized, with aslittle as 5% remaining on the stripped mem-branes. On the other hand, only about 10 to 15%ofthe activity ofthe mutant and the two revert-ants could be found in the supernatant solution(Table 3), with negligible activity remaining onthe stripped membranes (data not shown).Thus, BF1 from each ofthe three strains lost 80to 90% of its activity when removed from themembrane.

It was not possible to determine accuratelythe kinetic parameters of the BF1 of NR76 andNR76A because both the membrane-bound andsoluble fractions contain a large amount ofATP-hydrolyzing activity not due to the BF1, asdemonstrated by the lack of inhibition by thespecific antiserum or by azide.To eliminate the possibility that there were

different amounts of BF, protein in the mutantor revertants, those amounts were quanti-tated. Varying amounts of membranes fromstrains 7, NR76, and NR76A were incubatedwith a constant amount of an antiserum thathad been prepared against the purified BF1 ofstrain 7. After centrifugation, the supernatantsolutions were assayed for the ability to inhibitMg2+-ATPase activity in membranes of strain7 (Fig. 1). The results indicate that there areroughly equal quantities of cross-reacting ma-terial on the membrane of the three strains,suggesting that there is an intrinsic differencein the proteins themselves. In support of thispossibility, it was found that the cross-reactingmaterial from NR76 and NR76A exhibited areduced electrophoretic mobility in immunoe-lectrophoresis assays. This observation will bediscussed in greater detail in a later section.Thus, it appears that the most likely expla-

nation for the reduced Mg2+-ATP activities is

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Mg-ATPase MUTANTS OF E. COLI 251

TABLE 3. Mg2t-ATPase activities of cytoplasmic, membrane, and crude soluble BF1 fractions

ATP-hydrolyz- Mg2+-ATPase ac- % of wild-type % of mem-Strain Enzyme source ing activitya tivityb membrane branebouddd7 Cytoplasm 1260 320 21 21

Membranes 3130 1510 100 100Soluble BF, 970 970 52 52

NR76B Cytoplasm 960 30 2 24Membranes 168 126 8 100Soluble BF, 13 11 1 9

NR76A Cytoplasm 910 10 0.7 59Membranes 60 17 1 100Soluble BF1 6 2.4 0 14

NR76 Cytoplasm 490 0 0 0Membranes 8 12 1 100Soluble BF, 4 1.8 0 15

a Values are given as nanomoles of inorganic phosphate per minute per milligram of membrane protein.Since the volume of the crude soluble BF, and cytoplasmic fractions were the same as the membranes, thevalues were normalized by calculating specific activities using the protein values of the membranes. Sincethe protein concentrations of the vesicle preparations of different strains varied, this treatment allowscomparisons to be made between the various strains.

b Mg2+-ATPase activity was determined by assaying the membranes and cytoplasms in the presence ofBF1 antiserum and the crude soluble BF, in the presence of 10 mM NaN3. The amount of ATP-hydrolyzingactivity inhibitable by NaN3 or antiserum represents Mg2+-ATPase activity. Values are given as nanomolesof inorganic phosphate per minute per milligram ofmembrane protein. Normalization was as described in a.

e Calculated as [(activity of fraction)/(activity of strain 7 membranes)] x 100.d Calculated as [(activity of fraction)/(activity of membranes)] x 100.

an alteration in the protein resulting in a de-creased Vma., although an effect on the Kmcannot be discounted.Binding of the soluble BF1 to stripped mem-

branes. As shown above, the BF1 ofthe mutantand the two partial revertants lost most of itsactivity upon solubilization. When the solubi-lized BF, from NR76B was added to strippedmembranes from NR76B in the presence ofMg2+, it bound to the membrane and exhibitedan activity near that of the original NR76Bmembrane-bound BF1 (Table 4). Since it is pos-sible that only a portion of the protein wasremoved by solubilization, several variations ofthe reconstitution experiment were performed.It was found that BF, from strain 7 boundnormally to the membrane of NR76B, and thatthe BF1 from NR76B bound to stripped mem-branes of strain 7 with a reversal of the inacti-vation. The same experiments were performedusing stripped membranes from strain NR70(Table 4). Membranes from NR70 were usedbecause of the lack of any residual Mg2+-ATP-ase activity. These experiments suggest that itis unlikely that a subunit of the BF1 of NR76Bremains on the stripped membranes since thesoluble fraction from that strain is able to bindto stripped membranes from strains 7 or NR70with a restoration of activity. Moreover, there

is no impediment to the binding ofthe BF, fromstrain 7 to stripped membranes of strainNR76B. It also appears that the defect in strainNR76B resides in the soluble BF1 and not inBFo.

Effect of ethanol and DCCD on the mem-brane-bound BF1. Another property of themembrane-bound BF1 ofE. coli is its sensitivityto DCCD (10). It was of interest to determinewhether the sensitivity to DCCD differedamong the various strains. Since DCCD is onlysparingly soluble in aqueous solutions, it isusually added in ethanol. Ethanol itself wasfound to have a stimulatory effect on the BF1 atconcentrations below 0.06 M (0.01 ml of ethanolin an assay mixture of 0.3 ml) and an inhibitoryeffect at higher concentrations. To prevent anycomplicating effects of ethanol on interpreta-tion of the results with DCCD, all assay mix-tures in which DCCD was used contained aconstant amount of ethanol (0.01 ml per assay).The activity of the membrane-bound BF1

from strain 7 was found to have a differentsensitivity to DCCD than that from strainNR76B. At neutral pH the inhibition caused byDCCD became constant at concentrations ofDCCD greater than 50 ,M (Fig. 2A and B).However, treatment and assay at basic pH re-sulted in a reversal of DCCD inhibition at con-

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252 ADLER AND ROSEN

pg MEMBRANE MTEINFIG. 1. Estimation of the relative concentrations

of BF, on the membranes of strains 7, NR76, andNR76A. Various amounts of control membranes, as

indicated, from strains 7 (0), NR76 (a), andNR76A (0) were mixed with 5 p1 ofanti-BF, serum,

adjusted to a total volume of 0.1 ml with TKMGbuffer, and incubated for 15 min at 23°C. The sus-

pensions were centrifuged at 105,000 x g for 1 h at4°C. The supernatant solutions were carefully re-

moved, and 10 p1 ofcontrol membranes from strain 7(3.5 U/ml) was added to each supernatant solution.After 15 min at 23°C, duplicate 30-pl aliquots wereremoved and assayed for ATPase activity by assay

three, as described in the text.

centrations greater than 50 ,M in the case ofstrain 7 (Fig. 2A), whereas no such reversalwas found for the membrane-bound BF1 ofstrain NR76B (Fig. 2B). When the solubilizedBF1 from strains 7 and NR76B, respectively,were bound to the membrane of strain NR70,identical results were obtained, showing thatthe difference in DCCD sensitivity resides inthe BF1 and not in the BFo. It is clear, then,that there are significant differences betweenthe BF1 proteins of these strains. The signifi-cance of the effect of DCCD is not clear, espe-cially since the reversal of the inhibition byhigh concentrations of DCCD has not been ob-served by other investigators. It does not ap-pear that DCCD at high concentrations solubi-lizes the protein, since centrifugation of theDCCD-treated membranes removes all ATPaseactivity from the supernatant. A loosening ofthe bond between the BF1 of the wild type andthe membrane cannot be ruled out.Immunological characterization of the sol-

uble BF1 from strains 7, NR76, NR76A, andNR76B. On immunoelectrophoresis, solubleBF1 from the mutant NR76 and the revertant

NR76A invariably yielded a precipitin arc thatcorresponded to an antigen with reduced elec-trophoretic mobility when compared withstrain 7 (Fig. 3). On the other hand, the precipi-tin arc observed for the partial revertantNR76B corresponded to a protein with an elec-trophoretic mobility equal to that of the wildtype. When immunoelectrophoresis slides werestained for ATPase activity, all four strainsyielded a band of activity that corresponded tothe location of the precipitin arc of the wildtype, although the activity bands for the threemutants were much fainter than that of strain7. Moreover, no activity was observed at a posi-tion corresponding to the more slowly migrat-ing antigen. These results suggest that the BF1proteins ofNR76 and NR76A dissociate rapidlyupon removal from the membrane into a formretaining immunological but not catalytic ac-

TABLz 4. Effect ofsolubilization and reconstitutionaon Mg2+-ATPase activity of strains 7 and NR76B

Source of ATPase ac-Membrane Conditions added solu- tivityb

ble BF,Strain 7 Control - 3.16Strain 7 Stripped - 0.14

- - Strain 7 2.32Strain 7 Stripped Strain 7 2.04NR76B Stripped Strain 7 2.07

NR76B Control - 0.231NR76B Stripped - 0.014

- - NR76B 0.059NR76B Stripped NR76B 0.185Strain 7 Stripped NR76B 0.295

Strain 7 Control - 2.93- - Strain 7 2.94

NR70 Stripped Strain 7 2.26

NR76B Control - 0.196- - NR76B 0.018

NR70 Stripped NR76B 0.175

a Stripped membranes and soluble fractions weremixed in a volume ratio of 1:3, and MgCl2 was addedto 10 mM. The suspensions were centrifuged for 1 hat 105,000 x g at 40C, washed once with buffer, andresuspended to 3 to 5 mg of protein per ml.

b Activity is expressed as nanomoles of inorganicphosphate per minute per milligram of membraneprotein, using the protein value of the appropriatecontrol membrane suspension. Since the strippingand reconstitution procedures cause significant al-terations in the protein content of the vesicles, thisnormalization treatment allows for comparison ofthe various membrane preparations with eachother. By normalizing the soluble BF, solutions inthe same way, it is possible to compare the amountof soluble BF, added to the amount bound bystripped membranes.

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Mg-ATPase MUTANTS OF E. COLI 253

I I I I, I munological identity with the other mutantsA: Strain 7 B: Strain NR76B became correspondingly stronger (Fig. 4B and

4C).=°75 1 e 9 0 g

50~~~~~~~:

IV 75 X

,--4;1,'7- ;,. -'9U

7.0 9.0+125 ~~~~~~~~~~~~~8.0-

DCCD CONC. (n*)

FIG. 2. Effect of DCCD on Mgou-ATPase activity . ..in strains 7 and NR76B. Membranes were diluted .into assay buffer adjusted to various pH values andcontaining an ATP-regenerating system. Incubation -,was carried out for 15 mn in the presence of varyingconcentrations ofDCCD, as indicated, before initia- ..- .tion of the reaction by addition ofATP. Assays wereperformed at pH 7.0 (x), 7.5 (A), 8.0 (0), and 9.0 -. ........9-.

tivity. However, a small amount of the activeprotein remained, although there was too littleto be seen as a precipitin arc.

Since it was possible that the BFt proteins ofstrains NR76 and NR76A dissociated duringthe process of preparation, two variations ofthe fromimmunoelectrophoresis were performed. In thefirst instance, membranes of the four strainswere resuspended in the stripping buffer andimmediately were subjected to immunoelectro-phoresis without first centrifuging to removethe stripped membranes. In the second in- si w n t bstance, membranes of the four strains were .solubilized by incubation with 10%1 Triton X-_~P.100 for 15 min at 37C, and the resulting solu- fr-4 .tion was subjected to immunoelectrophoresis. 4/'9In both cases, the precipitin arcs were at the ''jsame location as found for inmmunoelectropho- ~-resis of the soluble BF, described above. ~/The results of immunodiffusion ofthe soluble -

BF, of the four strains against the BF, antise- '4rum are shown in Fig. 4. Immunodiffusions offr-eshly prepared soluble BF, from NR76 andNR76A displayed precipitin arcs that showedpartial identity with that of the wild-type,whereas NR76B displayed two precipitin arcs,

FG .Imneetohrsso rd oulone showed identity with that of the wild type, FG .Imneetohrsso rd ouland the other showed identity with the arcs of BF, from strains 7 and NR76. Approximately 5 pg ofthe other strains (Fig. 4A). Immnunodiffusion of crude BF, from the two strains was subjected tothe soluble BF, proteins 48 and 96 h after prepa- electrophoresis as described in the text, after whichratin,espetivly, howd tht te ar of50 pl of anti-BF, serum was added to the centralratin,rspecivel,shwed hat he ac oftrough. Diffusion was allowed to proceed overnight.

NR76B that showed immunological identity The slide was then incubated with several changes ofwith that of the wild type became fainter and 0.9% NaCl to remove unreacted protein, dried, anddisappeared by 96 h, whereas the arc with im- stained with naphthol blue black.

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254 ADLER AND ROSEN

..

FIG. 4. Time-dependent dissociation of solubleBF,. Immunodiffusion was performed as describedin the text. After diffusion, the slides were treated as

given in the legend to Fig. 3. The center well in eachslide contained 5 p ofanti-BF, serum. In each slidewells 2, 4, and 6 contained 25 pg ofBF, from strainNR76, NR76A, and NR76B, respectively, whilewells 1, 3, and 5 contained 25 pg ofBF, from strain7. (A) BF, fractions were used immediately afterextraction from the membrane. (B) Immunodiffusionwas performed 48 h after isolation of the proteinfractions. The proteins were stored at 23°C for thatperiod. (C) The protein fractions were left at 23°C for96 h before immunodiffusion. (D) Soluble BF, fromstrains NR76, NR76A , and NR76B were left at 23°Cfor 96 h. BF, from strain 7 was frozen in liquidnitrogen immediately after isolation, followed byslow thawing at 23°C.

These results suggest that the mutation inNR76 and NR76A results in BF, proteinswhich, when removed from the membrane, are

prone to very rapid dissociation, and that thereversion in NR76B results in a BF, proteinmore stable than that of the other two but lessstable than that of the wild type. Since the BF,of the wild type has been shown to dissociateupon freeze-thaw treatment, it was of interestto determine whether the apparent dissociationof NR76, NR76A, and NR76B corresponded tothe dissociation of the freeze-thawed wild-typeBF,. When the soluble BF, of strain 7 wassubjected to freeze-thaw treatment, the result-ing precipitin arc showed identity with those ofNR76 and NR76A and with the arc of NR76Bafter 96 h (Fig. 4D). These results are consis-tent with the hypothesis that the mutation inNR76 and NR76A results in a BF, protein thatdissociates very readily when removed from themembrane and is indistinguishable from thedissociated BF, of the wild type produced byfreeze-thaw by the criterion of immunological

cross-reactivity. The reversion in the case ofNR76B results in a BF1 that is more stable thanthat of strains NR76 and NR76A, yet less stablethan that of the wild type.

Partial purification of the BF1 from strains7, NR76, NR76A, and NR76B. An attempt wasmade to purify the BF, of each of the fourstrains in order to characterize the differencesin these proteins. The crude soluble BF, from 8g ofeach strain was precipitated by the additionof solid ammonium sulfate to a final 65% ofsaturation. The pellets were dissolved and ap-plied to BioGel A-1.5M columns, as describedunder Materials and Methods. The fractionswere assayed for protein, ATPase activity, andprecipitation by the BF, antiserum (Fig. 5).The elution profile for the wild-type enzymeshowed a sharp peak of antibody-precipitablematerial that coincided with the ATPase activ-ity at a position corresponding to a molecularweight of approximately 350,000. No enzymaticactivity was found in the NR76 and NR76Acolumn fractions, but a peak ofantibody-precip-itable material was found at a position corre-sponding to a lower molecular weight. Theeluate from the agarose column containing theNR76B soluble fraction contained two peaks ofantibody-precipitable material. The first peakcoincided with the ATPase activity and ap-peared at the same position as that of the wild-type enzyme. The smaller second peak was de-void of enzymatic activity and appeared at thesame position as those of NR76 and NR76A.Immunodiffiusion assays of the antibody-pre-

cipitable material from each column (Fig. 6)yielded a single arc for the peak fractions of theNR76 and NR76A material. Both peaks fromgel filtration of the NR76B material gave linesof identity with each other and with the arcs ofthe NR76 and NR76A fractions. The peak tubefrom the separation of the strain 7 BF, yieldedtwo arcs, a heavy outside line and a fainterinner arc. The latter showed a line of identitywith the arcs from the other three strains. Im-munoelectrophoresis of the material from eachpeak ofantibody-precipitable material revealedthat the antigens from NR76 and NR76A andboth antigens from NR76B migrated at a slowerrate than the major antigen from strain 7. Theimmunoelectrophoretic patterns obtained wereidentical with those shown in Fig. 3 for thecrude soluble BF, except that a second fainterarc in the material from the wild type appearedat the same mobility as the antigen from themutants.Thus it appears that the BF1 protein disso-

ciates upon solubilization from the membrane.The dissociation is relatively slow in the case ofthe wild-type protein, more rapid in the case of

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Mg-ATPase MUTANTS OF E. COLI 255

N116B

oZ ;~~~~o

S".~~~~~~UCa~~~~~5

m ~ ~ ~ ~RCIN17 N1UMBER

anienct as descie inteet=0. __ 11166~~~~~~~~I"1UL4rI."~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~~~2 6 04C -~~~~~~~FATONNME

FI. .Aaoecrmtgahofteslbefacinfrmsris7NR6NR6,adN7B

FG5.Agarose chromatographywapefrdof theamnu uft-raesoluble fractions from

ecstrains7NR6NRA,adR6B

as described in the text. The fractions were assayed for protein, relative ATPase activity, and relativeantigenicity as described in the te-xt.

the partial revertant NR76B, and even morerapid in the case of the protein from strainsNR76 and NR76A. The lack of activity on activ-ity stains of immunoelectrophoresis slides ofthe mutants and the lack of activity of thesmaller-molecular-weight peaks from BioGelchromatography suggest that the dissociationleads to a loss of catalytic activity. Thus theinactivation of the BF, proteins of the mutantsupon solubilization from the membrane is mostlikely a result of the dissociation of the pro-teins. This dissociation may be reversible, asindicated by the reactivation of the BF, ofstrain NR76B upon rebinding to the mem-brane.

DISCUSSIONTwo selection procedures have been reported

for the isolation of mutants of E. coli withdefects in aerobic metabolism. One is a directselection for strains that cannot utilize forgrowth substrates of the electron transportchain (1, 13, 15). The second method involvesthe selection of antibiotic-resistant strains (6,11, 12, 14, 18). Why the latter method yields

mutants with defects in energy metabolism isunclear. Although not all strains have beenexamined in detail, it appears that strains se-lected for antibiotic resistance show a decreasedability to accumulate small molecules (11, 14,19, 20). This suggests thatE. coli might becomeresistant to neomycin or streptomycin due to aloss of ability to concentrate these antibiotics.

Since many mutants with defects in aerobicmetabolism contain a defective BF, (1, 6, 11, 13,15, 18, 20), it was of interest to examine thatprotein in several strains with varying degreesof neomycin sensitivity and Mg2+-ATPase ac-tivity. Our results show that the two are notdirectly related. Strain NR76 exhibits about 1%of the wild-type Mg2+-ATPase activity and isresistant to high concentrations of neomycin.Yet strain NR76B, which has only about 8% ofthe wild-type Mg2+-ATPase activity, is as sensi-tive to neomycin as is the wild type. Althoughstrain NR76B was selected from strain NR76 byability to grow on succinate agar, it is stilldefective compared to strain 7. Thus, a defect inthe BF, may result in inability to grow onsuccinate, malate, and acetate but not in neo-

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256 ADLER AND ROSEN

FIG. 6. Immunodiffusion assays of the antigenicmaterial derived from strains, 7, NR76, NR76A,and NR76B. Immunodiffusion assays were per-formed as described in the text. The center well con-

tained 5 of anti-BF, serum. The outer wells con-

tained 5 1d from the tubes of the columns that were

identified as containing antigenic material, as de-scribed in the legend to Fig. 3. NR76B(1) andNR76B(2) refer to the high- and low-molecular-weight peaks, respecitvely.

mycin resistance. Ofinterest is the fact that theability of these strains to actively transportnutrients occurs in the following order: strain 7> NR76B > NR76A > NR76 > NR70 (12). Thesame order is found for the impermeability ofthe membrane to protons (12), and, as shownabove, sensitivity to neomycin. Again, thesefacts suggest that resistance to neomycin is a

result of increased proton perneability, whichcauses dissipation of the electrochemical gra-dient of protons, leading to decreased ability toaccumulate solutes including neomycin.The biochemical nature ofthe mutation lead-

ing to the NR76 phenotype and the nature ofthe subsequent reversions leading to theNR76A and NR76B phenotypes must still bedetermined. One of the original goals of thisinvestigation was the purification of the BF1from the strains described in this report. Ofinterest is the identity ofthe altered subunit(s).The apparent dissociation ofofthe protein fromstrains NR76, NR76A, and NR76B has ren-

dered this objective more difficult.ACKNOWLEDGMENTS

We thank A. Haing and I. Williams for technical assist-ance.

This investigation was supported by Public Health Serv-ice grant GM-21648 from the National Institute of GeneralMedical Sciences, grant BMS-75-15986 from the NationalScience Foundation, and a Basil O'Connor Grant from theNational Foundation-March of Dimes.

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