Planit Physiol. ( 1(90) 41, 1330-1336

Enzymes of the Glyoxylate Cycle in Rhizobiaand Nodules of Legumes1

Gordon V. Johnson', Harold J. Evans, and TeMay ChingDepartments of Botany and Farm Crops, Oregon State University, Corvallis. Oregon

Receixed April 25, 19(16.

Sitinn;wry. The relatively high level of fatty aci(ds in soybl)eaI no(duiles and(I rhizobiafrom soybean noduiles suiggested that the glyoxylate cycle might have a role in no(ldllemetabolism. Several species of rhizobia in pulre cul]ture w\ere found( to have malatesynthetase activity when grown on a ntimber of different carbon sources. Significanitisocitrate lyase activity was induced when oleate, which presuimably may act as an acetylCoA precursor, was utilized as the principle carbon souirce. AMalate synthetase wvas

active in extracts of rhizobia from nodules of bush bean (Plioscoluts zulllgoitris L.), cow-pea ( Vfig o(i sincusis L.), lupinc (Lu pinus (ontgu(stifoliius L.) and(I soybean (Glycilne ;111.-L. MIerr.). Activity of malate synthetase was, however, barely detectable in rhizobiafrom alfalfa (l[edicoq7go satiz7a L.), red clover (Trifoliu;n proltense L.) antd pea (Pistionsctizumn L.) nodllles. Appreciable isocitrate lyase activ-it\x was not (letecte(l in rhizobiafrom nodules nor was it indutce(d by depletion of enidogeniouis substrates hy incuibationof excisedl btish beanl nodules. Althouigh rhizobia has the potential for the formatiolnof the key enzymes of the glyoxylate cycle, the absence of isocitrate lyase activity in

bacteria isolated from noduiles indicated that the glyoxylate cycle (loes not operatein the symbiotic growtlh of rhizobia ancd that the observed high content of fatty .cid(!in nodlulles and no(llile bacteria probably is related to a struletu1ral role.

MIarked metabolic differenices exist between rhi-zol)ia collected from legtume niodules ain(I those clil-tulrel on synthetic media. Nitrogen fixation byrhizobia occtirs only tindler coin(litions of svlmbioticgrowth. If more information on the activity ofvariotus metabolic pathways coulldI be procuired fromthe symliotic and cuiltuire(d rhizobia, a letter nider-standing of symbiosis possibly couild be achieved.Evidence for the function of the tricarboxylic acidcycle, the oxidative pentose phosphate pathway andthe Embden-Meyerhof glycolytic pathway in rhi-zobia has been provided (13), but the glyoxvlatecycle in rhizobia has not been investigate(l.

The key, enzymes of the glyoxylate cycle are:isocitrate lyase (isocitratase) which catalyzes thecleavage of isocitrate to glyoxylate aind suiccinate,antI( malate synthetase catalyzing the formation ofmalate from glyoxylate anId acetvl CoA. Isocitratelyase anid malate synthetase prov-ide a mechanism forsyinthesis of C-4 intermedliates of the tricarboxylicacidl cYcle from acetYl CoA derived from acetate,ethanol or fatty aci(ds. By operatioln of the glyoxy-

Techllical paper No. 2099 Oregoni Agricultural Ex-perimnent Station. This research was supported in partby granlts from the Hill Foundationi anid the NationalScience Foundation (GB2518).

2 Present address: Departmeint of Biologxy 'Universityof New- Mexico, Albuquierqute, NexN- Mexico.

late cycle and(l reversal of glycolysis all cellullar conl-stituienits mav be derived from C-2 preculrsors.

Examinatioin of rhizobia for the key enzvrme: ofthe glvoxylate cycle was fuirther encoulrage(d by theobserVation that soybcan inodulles aind bacteria ex-tractedl from soybeani nodules contained relativelvlarge amouints of fatty acids. Polv---hvdroxvbu1tyrate has been stuggested as an important energystorage form in leguiminouis noduile bacteria ('12).Utilizationi of this polymer woulld be expecte(l torestult in formatioln of acetoacetate andl then acetvlCoA (9).

Isocitrate Ivase and( malate sy nthetase occurw-idely in bacteria, yeast, fulngi and(l algae ( 10, 16).The formatioln of these enzymes in many Imlicroor-ganisms is known to be greatly infltuelnce(d b)y thenatuire of the carbon source utilize(d for grow th (16,20). In higher plaints appreciable isocitrate Ivaseantl malate synthetase activities have been (letecte(lonly in tissuies in wihich fats are b)eingiutilizId (3,22).

Materials and Methods

(Cultural Methods. Pure culltuires of effectivestrainis of Rhli-obuimn incliloti F29, R. lcynminhIOS01rootC56, R. plhascoli Kll, R. trifolii P28 and(l R. japoi-clort 61A76 were investigated. These species ofrhizobia are the endophytes of alfalfa, pea, bean,

1330

JOHNSON ET AL.-GLYOXYLATE CYCLE ENZYMNES IN RHIZOBIA

clover and sovlbean associations respectively. Thecultures were grown in 400 ml of medium in 1 literflasks with cointinuous shaking at 25 to 30°. Thebasic cultture medium for all species of rhizobia conl-tained the following components in g/liter: K2,HPO4,1.00; KHPC)4, 1.00; MgSO4*7H2O, 0.36; CaSO4,0.13; FeCl236H^O, 0.004 and yeast extract (DifcoBacto), 1.00. Carbon sources were added to thebasic medium as indicated in the tables. For theculttures of R. mieliloti 60 lug/liter Co as CoCL wasadded to the basic medium and the pH was adjustedto 6.5 to 7.0. For culture of R. japonicumn, R. leg-imtintosarumtSl, R. trifolii, and R. phaseoli 0.7 g/literKNO. was added to the basic medium and the pHwas adjusted to 6.3. Cultures were inoculated with1 % by volume of log phase cultures of R. mzelilotisupplied with mannitol or the other species of rhi-zobia supplied with arabinose and glycerol.

Alfalfa (M1edicago sativa L., var. Duipre), bushbean (Phlaseolius vulgaris L., var. Top Crop), redclover (Trifoliumt pratentse L., Common var.). cow-pea (Vigna sinzentsis L., var. Iron Clay), lupine(Lutpinuis angustifolius L., var. Borre Blue), pea(Pisutm satizvuin L., var. Little Mlarvel) and soybean(Glycine inax L. MIerr., var. Chippewa) seeds wereinoculated with commercial inoculum and planted inpots containing Perlite. The legumes, except soy-beans, were supplied throughout their growth witha nutrient solution lacking nitrogen and containingthe following macronutrient salts in g/liter: K2 SO4,0.28; MgSO4/7H.O, 0.49; KH.P04, 0.02; K.HPO4,0.15; CaSO4*2H2O, 1.03; and CaCl, 2H.,O, 0.06.The nuttrient solution supplied to soybeans containedin g/liter:K.SO4, 0.34; KH.P0,, 0.12; and K2HP04,0.01 and the other macronuttrient salts at the con-centrations previously indicated. Micronutrientswere supplied at the following coIncentratioins inmg/liter: Fe, 1.0 as FeEDDHA; B, 0.25 as H3B03;Mn, 0.25 as MInSO4.*/H,O; Zn, 0.05 as ZnS,O4I7H.O;Cu, 0.02 as CuSO,-5H20; Mo, 0.01 as Na2MoO42H.O and Co, 0.05 as CoCI2*6H. O. After emer-gence the cultuires were irrigated with nutrient so-lution daily for 2 days followed by tap water everythird day. The legumes, except soybeans whichwere cultured in a growth chamber, were grown ina greenhouse with sunlight supplemented by fluo-rescent lights during the winter months.

Chemtical Constituients of Nodule.s and Bacteria.The procedure for lipid extraction and analysis wasthat of Ching (5). Five g of soybean nodules wereground in 75 ml of an ether:ethanol solution (2:1,v/v) for 2 mintites in an Omnimixer. Homogenizednodule material or bacteria from 5 g of nodules wasthen extracted 3 times with 75 ml of ether:ethanolat room temperature for a total of 18 hours. Theresidue was washed with the solvent, dried in avacuum oven overnight at 500 and then weighed.The filtered crude lipid extracts were separatedfrom aqueous material by 3 extractions with 2 %K,CO3. The washed lipids were dried in a vacuutmevaporator and fLurther dried overnight in a vacutum

oven at 50°. Dried lipids were saponified by 10 %NaOH in 75 % ethanol for 20 hours. After saponi-fication, the aqueous phase was acidified and fattyacids were extracted with petroleum ether :ether(1:1, v/v). The fatty acids were methylated withdiazomethane in ether and concentrated under N2.Fatty acids were determined using a 12 foot columncontaining 14 % diethylene glycol succinate onChromosorb NV, 80/100 mesh, with a F & M gaschromatograph (thermal conductivity detector). Theunsaponifiable fraction was dried and weighed.

Lipids were extracted from 2.9 g samples ofbush bean nodules with 3 different 20 ml volumesof chloroform:methanol (2:1, v/v) for 3 houirs atroom temperature. The lipids were washed withdistilled water and dried in a vacuum oven at 60°for 24 hours prior to weighing (11). Soltuble sugarswere determined in washings of the lipid extractand the water soltuble portion of the 80 % ethanolextract by the anthrone method (23). Starch wasdetermined as sugar by the anthrone method afterhydrolysis of the sugar-free residue with perchloricacid ( 14).

Preparationt of Cell-free Extracts. Culttures ofrhizobia were harvested in the log phase of growth,at the times indicated in the tables. Cells were col-lected by centrifulgation at 10,500 X g for 10 min-utes. Subsequent operations were carried ouit at0 to 40. After harvesting, the cells were washedtwice by suspension in cold 0.01 M potassium phos-phate buffer at pH 6.8 followed by centrifugationat 35,000 X g for 10 minutes. The yield of thecultures was estimated from the fresh weight of thewashed cells.

Noduiles were removed from the roots of activelygrowing leguimes and washed to remove Perlite.Subseqtuent operations were carried out at 0 to 40.Bacteria were isolated from the nodules by crushingthe nodules with a cold mortar and pestle in 3 vol-uimes of potassium phosphate buffer (0.01 M at apH of 6.8) and then squeezing the crushed nodulesthrough 4 layers of cheese cloth (4). The bacteriawere separated from the hemoglobin and othersoluble components by centrifugation at 35,000 X gfor 10 minutes. The bacteria from nodules werewashed and weighed in the same mannier as werethe pure culture bacteria.

Washed bacterial cells from either pure cultuiresor nodules were suspended in 4 volumes of 0.02 Mpotassium phosphate buffer at pH 7.4 containing0.01 M 2-mercaptoethanol and 10-4 NI NaXEDTA.This suspension, surrounded by an ice bath, "was soni-cated in an MSE sonicator after addition of 0.2 gof cold levigated alumina for each g of cells. Inorder to minimize heating during sonication, thecell suspensions were sonicated for 2 minutes, theincooled to 1° and sonicated for an additional 2 min-utes. Cell-free extracts were obtainedI by centrifu-gation at 35,000 X g for 20 minutes.

Extracts of rhizobia were stirred with 5 mg/mlof activated charcoal (Darco, 5-60 mesh) for 5

1331

PLANT PHYSIOLOGY

m-inuites in order to remove pyridine nlcleotides thatmay hav-e been present in the cruide extract (3).Charcoal was removed by centrifuigation.

For the characterization of isocitrate lyase andmalate synthetase from R. m,ieliloti grown on oleate,ice cold satuirated (NH)2,SO4 at pl4 7.4 and con-tainling 0.01 M\ 2-mercaptoethanol was ad(le(d dropwise w%ith stirring to the charcoal treatecl cell-freeextract to prec pitate the enzymes. Precipitatedprotein was recovered hy centrifugation at 35,000X q for 10 minuttes, and resuispended in 0.02 Mapotassiuim phosphate buiffer at pH 7.4 containing10- -INa.EDTA and 0.5 mg/ml GSH.

The roots of soybeans anid cowpeas after removalof noduiles were homogenized for 1 minutte in an icecol(d Omnim xer in 1.4 voltumes of the buffer utsedin preparation of cell-free extracts of rhizobia. Thehomogenate was strained through 4 layers of cheesecloth followed by centrifuigation at 35,000 X'g for20 minuites. Only malate synthetase activity wasdetermined in root extracts.

Enz-vnie Assays. Isocitrate ly-ase and malatesynthetase activities were determinedl uising charcoaltreated crtude extracts of bacteria from pure ctultturesan(l nodules. Isocitrate lyase activity wvas (leter-mined by the proceduire of Daron aind Gunsalus (6).The complete reaction mixtture consisted of: 100MAmoles TrisCl pH 7.9, 3.0 ,umoles MgI2, 2.0,mmoles cysteine-HCl, 7.5 )Umoles Na isocitrate, cell-free extract cointaining 0.25 to 10.0 mg of protein,and water to a final volume of 1.5 ml. Soditum iso-citrate was prepared from DL-isocitric acid lactone(Sigma, type II). After 10 minuttes incuibatioinwith the suibstrate at 300 the reactioni Was terminatedby the additioin of 0.1 ml of 80 % trichloroaceticacid. After centriftugatioin to remove precipitatedprotein, glyoxylate was determine(d colorimetricallyas the 2,4ldinitrophenylhydrazone. The optical den-sity of the complete reaction mixtuire was correctedfor the optical density of the reaction mixtuire lack-ing suibstrate. Glyoxylate formation was fouind tobe proportional to enzyme conicentration uip to 0.9,umole glyoxylate in the reactioin mixtuire an l iso-citrate lyase activity was measturedI within the pro-portional ranige.

Malate synthetase activity was estimated fromthe decrease in optical density at 240 m,u upon cleav-age of the thioester bond of acetyl CoA in thepresence of glyoxylate uising a Cary model 11 spec-trophotometer (8). The complete reaction mixtureconsistecl of: 273 ,umoles TrisCl pH 8.0, 10 /molesMgCl.,, 0.14 ,mole acetyl CoA, 1.4 ,umoles Na gly-oxylate and cell-free extract containing 0.1 to 0.6mg of protein in a final volume of 3.0 ml. AcetylCoA was prepared daily from Coenzyme A (Sigma)ancl redistilled acetic anhydride (MNIerck) as (lescribedby Stadtman (21). A weak deacylase system, asindicatedI by a graduial (lecrease in optical densityprior to the addition of glyoxylate, -was detectedin only a few extracts. \Vhen deacylase activitywas present, the rate of dlecrease in optical density-

after the additioln of glxoxylate wcas correctedI foithe rate of decrease prior to addition of this sub-strate. Althouigh malate sy-nthetase activity wasestimate(I from the change ini optical density (itiringthe first 1.0 or 2.0 minuttes of the reactioni, theseresuilts have been expressed on the basis of 10 min-ultes in ordler to ftacilitate comparison with thevalues for isocitralte lvase activity.

The protein conteint of cell-free extracts wasletermine(d 1w the method of Loxvrv et al. ( 17

Identification of Pr-oduicts of En,,Vma(tit cCRacctionIs.The fractioni obtained by 50 to 75 % ( NH,)),Sosatuiratioii of a cell-free extract of R. iic.Ziloti grouwnon oleate wvas uised for the idenitificationi of theproduicts of the isocitrate Ivase and(I malate synthe-tase reactionis. The 2,4 dinitrophenylhydrazones ofketo-aci(ds from the complete reactioni mixtuire, frointhe reactioni mixtuire without substrate, and fromll astandlardl sanmple of Na glyoxylate were forme(d anidextractedl as (lescribedl by Ranisol (19, metho(d 1).The hydrazonies of glyoxylate -xvere idlentifie(d b1paper chromatography wvith 3 solveent systems: Amethanol :beuzene :z-butaLnoI :H..O ( 4 :2:2 :2 ) B)water satlirate(l nt-buitanol C ) n-lbutalol : N.5xNH,OH:ethanol (7:2:1):

The (leproteiniizecl suiperniatant soluition fr-omli acomplete malate synthetase reaction mixtuire anld areactioin mixtulre lacking acetyl CoA were passedthrough a column of 50 to 100 mesh Dowex S(O\V-X4. The efflueint and washings w\ere colncenitrate(dusing a flash evajpwrator. The reaction pro.luict wasidlentified by paper chromatographN with malic acidtusing isoamyl alcohol saturatedl wsith 4 N formic acidas a solvent system (7).

Results

FPatty' Acid CUoiipositiont of Soybcan Vodulcs awdSy'Imbiotic Bactcria. In table I the composition ofesterifieed fatty acids from a sample of intact soy-hean nodulles andl the bacteria isolated from sovLeaniiodu1les is shlowx-n. In the 'whole nodufles, palmitic,oleic, linoleic anid linolenic are the pre(dominantfattv acids. OnlI small amouints of other fattyacitls are present. Oleic acid makes uip 42 % bywxeight of the esterified fatty acids in the nodutilebacteria. Palmitic andl linoleic acids also are majorconstituielnts. A smaller weight percent of linoleniicacidI is fouind( ii the lbacteria than in whole no(duiles.On a fresh weight basis the lipid content of nodutlesand bacteria from the nodules was 20 % and(I 17 %respectively.

Envmzycs of the Glyoxylatc CYcic inz Rhiozobia inPntrc C lttnrc. The influtence of the carbon souirceon the activity of the key enzymes of the glyoxylatecy-cle in cell-free extracts of R. njlcliloti growvn inpuire cuiltulre is shoxx-n in table II. In experimeintI it is apparent that while appreciable malate svn-thetase activitv was detectable in all extracts, isoci-trate lyase activitv was virtuallv absent exceptwvhen oleate was suippliedI as a carboon soturce.

1332

JOHNSON ET AL.-GLYOXYLATE CYCLE ENZYM-.\E;S IN RHIZOBIA

Table I. Fatt! Acid C(omposition of SaponifiablcMatcrial fromit Intact Soybean Nodules and Bacteria

frtonm Soy bean Nodules

Five g of fresh nodules from approximately 7-week-old soybean plants contained 1.00 g of lipids of which-0.57 was saponifiable. The bacteria from 5.0 g ofnodules containied 0.16 g of lipids of which 0.12 g wassaponifiable.

Percentage by weightof total fatty acids in:

Fatty acid Nodules Bacteria

Butyric, valeric, caproic, C4,5,6 2.7 38Heptoic, caprylic, C7,8 0.4 0.4Capric, C10 0.6 0.2Lauric, C12 1.0 0.3Myristic, C14 1.4 0.6Palmitic, C16 15.3 17.9Pa'imitoleic, C16=1 1.0 1.0Stearic, C18 2.7 1.9Oleic, C181 23.3 42.2Linoleic, C18 2 26.2 19.7Linolenic, C183 17.3 7.6Arachidic, C22 0.4 0.0Eicosadienoic, C20=2 4.1 02Behenic, C22 1.7 0.0Unidentified fatty acid 2.3 4.3

Since the basic medium incluides 0.1 % (w/v)yeast extract, experiment II was conducted todetermine the influence of yeast extract alone on

the activity of glyoxylate cycle enzymes. \Whenyeast extract was suipplied at a level of 0.3 %(w/v) isocitrate lyase activity was negligible. Mal-ate synthetase activity increased 2 to 3-fold witholeate as the carbon source compared to the activitywith other carbon sources which failed to induceisocitrate lyase.

Ammonium sulfate fractionation of cell-freeextracts of R. mneliloti cultuired with oleate as thecarbon source resulted in a concentration of isoci-

trate lyase in the 50 to /75 % (NH4)2S.D4 satutrationfraction. This fraction was used for identificationof the product, glyoxylate, and characterization ofisocitrate lyase. That glyoxylate was formed en-

zymatically from isocitrate was confirmed by paperchromatography. The major hydrazones of authen-tic glyoxylate and complete reaction mixtures hadcomparable RF values in solvent systems A, B andC, while hydrazones were not observed in reactionmixtuires without isocitrate.

AMalate synthetase activity was distributed inseveral of the (NH) 2SO precipitated fractions;however, the enzyme was characterized in the 50to 75 % (\H4)2SO satulration fraction preparedfrom oleate culltured R. mteliloti. Malate was idlen-tified as the product of the reaction of glyoxylateand acetyl CoA by chromatographic comparisoni ofthe acids separated from the complete reaction mix-tulre with a malic acid standard. When acetyl CoAwas omitted from the reaction mixttlre the spotcorresponding to malate did not appear.

Isocitrate lyase from oleate cultured R. mtelilotiwas found to require isocitrate, magnesium, andcysteine-HCl for maximulm activity and to exhibita pH optimtlm between pH 8.0 and pH 8.5. Ac-tivity of malate synthetase from R. mneliloti was

foulnd to be dependent on glyoxylate, acetyl CoA,and magnesium with an optimum pH range betweenpH 8.0 and pH 9.0. The requirements and pl1optimums for isocitrate lyase and malate synthetasefrom R. ineliloti are similar to those reported forother species of microorganisms (6,8) and higherplants (3, 22).

The influence of the carbon source in the growthmediuim on the specific activity of the key enzymes

of the glyoxylate cycle in several other species ofrhizobia is shown in table IlI. When R. legumiiito-sarum711, R. phaseoli, and R. trifolii were cultulredwith oleate as the principle carbon source, activeisocitrate lyase systems were detected while isoci-trate lyase activity was negligible when these species

Table II. Infliience of Carbon Source on Activity of Key Enzymecs of the Glyoxrylate Cycle in R. melilotiCultures -%vere harvested after 18 to 20 hours of growth.

Fr wt of cells Specific activity*Carbon source*" (g/800 ml) Isocitrate lase Malate synthetase

Experiment ID-Mannitol 2.22 0.01 0.31Succinate 2.23 0.00 0.26Glycerol 2.39 0.00 0.37L-Gluitamate 2.07 0.02 0.37O'eate 1.47 2.17 0.93

Experiment IIYeast extract 2.64 0.02 0.50D-M---initol + oleate 2.01 0.01 0.75O!eate 1.39 2.40 1.28* Specific activities of isocitrate lyase and malate sy nthetase are expressed as zmoles of glyoxylate formed/10

min/mg protein and umoles acetyl CoA utilized/10 min/mg protein respectively.** All carbon sources were supp'ied at a level of 0.3 % (w/v) in medium.

1333

Table ILL. Key Enfyincs of t11 Gl/,vx-0a/h Ccle in R. leguminosaruim, R. pliaseoli, R. trifolli, and R. jal)onicumR. lcguminosarmni, R. phao/i, ailI R. trifoli were hiarveste(d after 24 lhoturs of grrow th. R. japonicumi wN-as lidr-

vested after the follow-ing grow th periocls oni the various carbon sources: L-arabinose + glycerol, 34 hiouirs; L-glu-tanmate, 49 lhours; succinate, 39 hours.

Species

RI. /cgl ainosarion

R. phascol/i

R. trifo/li

R, japonicmao

Carbon source; *

L.-arabinose + glycerolOleateL-arabinose + glycerolOleateL--tralinose + gl! cerolOleateL-arabinose + gly-cerolL- lutamnateSuicciinateOleate***

Specific activ ity3Isocitra,tc la-seeMalate sx lthetase

(.()03.110.011.580.022.010.010.010.02

1.432.161.771.661.372.410.700.450.99

Specific activities of isocitrate l-ase anid malate synthetxtse are expressed as jimoles gixo\x\ late formed/10 mllitl,nig protein and gnmoles acetyl CoA utilized/10 min/mg protein respectively.Carbon sources -were added to the basic medium at the following levels (w A ): L-arabinose, 0.1 %, glycerol,0.5 % oleate acid, 0.3 %; L-glutamiate, 0.3 %; succinate, 0.3 %.R. japon1icman1w failed to grow wlheni oleate Nas supplieed.

wvere cutltured oni the mediumiii conltaininig aralbilloseand gly,cerol. Malate synthetase was active in allextracts and was influience(d only slightly by thenature of the carbon souirce. R. japonllicilii failed togrow with oleate as the priniciple carboni souirce.Appreciable malate synthetase activity was fotund inR. japonicum ctultture(d on gluitamate, sticcinate oraral)inose anid glycerol; however, the isocitrate lyaseactivity of these extracts was negligible.

Enzynies of the Glyoxy-l(ate Cycle i11,tSymbioticRhizobia. The resuilts of enizymatic assays on ex-tracts from symbiotic rhizobia from inoduiles ofseveral species of leguimes are reported in table IN.An active malate synithetase system was presenit inextracts of bacteria from butsh bean, cowpea, lutpineand( soybeain noduiles buit bacteria from alfalfa,clover and pea nodluies exhibited very weak malatesvnthetase activity. Siginificanit isocitrate 1vase ac-

Table IV. Kcy Enzsivics of t1/c (I/voxNlatc Cycle uiBacteria fromi Aoditl/s oj Lcgumainous Plan/s

Species

AlfalfaBtushl bean

CloverCoN-peaCowpeaLupinePeaSoybean

Age(dax s)

4027623362333243

Specific activity-Isocitrate Malate

Ivase Sx lutlhetase

0.010.00

0.(10.010.010O.()(0.000.01

0.060.42(1.02

0.520.340.410.060.57

* Specific activities of isocitrate lI-ase and malate sx-ii-

thetase are expressed a,s /.Lmoles glyoxylate formed/10ulin/nig1 protein andI ,umoles acetx-I CoA uitilized/10miim/ng protein) respectivel.

tivitv was lnot foundl(l in any of the symbioticallycultulre(d bacteria investigatedl. i\Ilalate syinthetaseactivity in bacteria from cowpea lno(Ililes decrease(lbetween 33 and 62 days of gro-wth as the ino(dtulesaili host planits neare(I senlescenice. Little or noisocitrate lyase or malate synthetase activity coIldbe (letectedl in several stuperinatanits of no(duile ho-mogenates teste(l. Extracts prepare(l from soybeanalndl co-wpea roots after the removal of niodules hadnegligible malate synthetase activity.

An experimenllt was colu(Iticted to (leterminie if,

uinder conditionis of anl inadequate energy, supply toio(luiles from the host plaint, symbiotic rhizobia.woul(l utilize fatty a<ci ls presenit in the nlo(duiles viathe glyoxylate pathway. When excised bush beanno(lliles were inicubate(d aerol)icallv in a bti-ffer solui-tioin for 0, 10, 20, 36 alndI 60 houirs, appreciableinduction of isocitrate Ivase (ii(l niot ocCIr. Little ifany change in the activity of nialate t.vntlictab.e was(letectalble in these bacterial extracts even after 60holurs of incubation. In table V the analysis of thex-hole butsh bean niodulles initially and after 60) hoursof incubation showvs that -while the lipi(d contentchanige(d negligibly, the silgar and starch conitelnt(of the nodules dclcreased( lby 70 %, and(I 40 % respec-tivel v. Semiqllaintitative analysis of the compoIlentllSof the lipi(d fractioni failed to reveal apprecilablechaniges in the rclative amouniits of variouls clalssesof lipids.

Discussion

The data in table II indclicatedl that thc alfalfaendophyte, R. meliloti, hadl malate synthetase re-gar(lless of the grow th sulbstrate suipplie(d to piirecuiltuires. Appreciable isocitrate Ivase was iin(lulce(lonly when oleate was suipplie(l as the princil)al car-bon souirce. With this medlituml- the specific activities

1334 PLANST PHYSIOLOGY

JOHNSON ET AL.-GLYOXYLATE CYCLE ENZYMES IN RHIZOBIA

Table V. Inflzence of 7'irne of Incuibation of ExciscdBit,h Bcan N odulcs on Somiie Nodui(c ConstitzuentsTenl g samiples of nodules excised from 31 day old

bush bean plants were suspended in 200 ml of 1 fifth-strength nitrogen-free nutrient solution buffered with0.008 Ni KH,PO4-KHP04 at a final pH of 6.7. Thesuspended nodules were incubated on a rotary shaker at260 for times up to 60 hours.

Time of incubation (hrs)Conistituient 0 60

% of dry wtLipids* 9.9 10.7Residue** 53.2 42.1Free sugars 4.07 1.31Starch 2.13 1.40

* Lipids were separated inlto the followuii- classes bythin layer chromatography: pigments and sterolesters, trigly cerides, free fatty acids, sterols, digly-cerides and gl-colipids, and phospholipids and amino-lipids (18). The relative amounts of the variousclasses of lipids as indicated b) densitometry did notchange appreciably between 0 and 60 hrs of incui-bation.

** Residue remaining after extraction of lipids andmethatnol soluble materials.

of isocitrate ly-ase and malate synthetase were ofthe same order. Oleate would be expected to bedegraded b) ,-oxidation to acetyl CoA which mightthen be tutilized in the synthesis of carbon skeletonsof celltular constittuents via the glyoxylate cvcle andreversal of glycolysis. WVhen oleate and mannitolwere both stupplied, significant induction of isoci-trate lyase was not detectable indicating that manni-tol was tutilized in the formation of carbon skeletonsof cellutlar constitulents. In this meditum oleate mayhave been degraded to acetyl CoA by the 8-oxida-tion pathway: however, acetyl CoA apparently wasnot then tutilized via the glyoxylate cycle.

The role of isocitrate lyase in the growth ofR. lcguiinoso(r1ui1, R. phoiseoli, and R. trifolii on anacetyl CoA precursor seems to be very similar toits role in R. iieliloti (table III). Acetate has beenused as a growth substrate to indtuce isocitrate lyasein many microorganisms (16), however, the speciesof rhizobia in-estigated failed to make satisfactorygrowth oIn Na acetate in ouir laboratory.

The operation of the glvoxylate cycle in bac-teria has beein proposed to be controlled largelyby intracelltular reguilation of the synthesis andactivity of isocitrate lyase (16). Kornberg (15)has proposed that induiction of isocitrate lyase inEschiericli(i coli is controlled by an intracellularrepressor closely related metabolically to oxalacetateor malate. Acetyl CoA acts to reduice the level ofoxalacetate aind malate by formation of citrate thuispermitting the synthesis of isocitrate lvase. Phos-phoenolpyrutv-ate and isocitrate have been reportedas intracelluilar inhibitors of isocitrate lyase inE. coli B ( 1.

WN'hile anl appreciable level of isocitrate lyasehas been detected in se'veral species of bacteria

when not utilizing an acetyl CoA prectursor asthe carbon souirce, an investigation with mutantsof I,. coli lacking this enzyme indicates that isoci-trate lyase is not essential for the ultilization ofglultamate or glycolate (2). Thus isocitrate lyasemay not play an essential role in rhizobia exceptwhen an acetvl CoA precursor is the principalcarbon soturce. This is consistent with the virtualabsence of isocitrate lyase activity in extracts ofrhizobia from nodules (table IV) or bacteria cul-tuired on carbon soutrces other than oleate.

Rhizobia from nodtules of bush bean, cowpea,lupine and soybean were fotund to have active malatesynthetase systems, however, this enzyme was barelydetectable in bacteria from alfalfa, clover or peanodules. While malate synthetase has been consid-ered by some investigators to be a constitutive en-zyme, Reeves and Ajl (20) report that E. colictultulred anaerobically on a gltucose-citrate mediumexhibit neither isocitrate lyase nor malate synthe-tase activity and that both enzymes are adaptive.The detection of appreciable levels of malate syn-thetase in bacteria from noduiles is of interest inview of the partially anaerobic environment believedto be maintained in noduiles (13).

The occurrence of malate svnthetase in aero-bically cultuired bacteria suipplied with a varietyof carbon sources has led to the suggestion thatglyoxylate may arise metabolically from souircesother than isocitrate via isocitrate lyase. Whileglyoxylate may be derived from glycolate, glycineor oxalate (16) by known metabolic pathways, anactual pathway of formation of glyoxylate fromlonger chain carbon compouinds in bacteria is notknown. Translocation of glyoxylate from host tis-suie or formation of glyoxvlate from other sources,possibly glycolate, may be responsible for the appre-ciable activ,ity of malate svnthetase in bacteria fromnoduiles of certain species of leguimes.

The relatively h;igh content of lipids in soybeanand bush bean nodtules (tables I and V) apparentlydoes not uinder normal conditions provide raw ma-terial for growth or energy via the glyoxylate cvclesince negligible activity of isocitrate lyase was foundin noduiles. The differential composition of fattyacids in soybean noduiles and bacteria is of interestas it is the first attempt to characterize the fattyacids in these materials. A detailed study of thecomposition and function of these lipids wouldprobably provide an explanation of their high levelin noduiles.

WN hen excised buish bean nodules were incuibatedaerobically for as long as 60 hours, lipids fromnodules were neither ultilized for the synthesis ofcelluilar constituients nor oxidized for energy. Fail-tire of lipids to be utilized or isocitrate lyase tobe induiced in the excised noduiles may perhaps beattribuited to the presence of adequiate endogenoussuigar and starch. By 60 houirs of incubation thecondition of the nodules had beguin to deteriorateas evidenced by the disappearance of the hemo-globin.

1335

PLANT PHIYSIOLOGY

Acknowledgment

The authiors express their appreciatioin to Dr. George

Sorger for lils review, of this mianuscript.Literature Cited

1. AsHWORTH, J. M. AND 1963.

Fine control of the glyoxyLete cycle by allosteric

inhlibitioni of isocitrate Ivase. Biochim-. Biophy s.

Acta 73: 519-22.

2. AsHWvORTII, J. Mt. AND H. L. KORNBERG. 1964.

The role of isocitrate lyase in Eschcricliza coli.

Biochilm. Biophys. Acta 89: 383-84.

3. CARPENTER, \W. D. AND H. BEEVERS. 1959. Dis-

tribution and properties of isocitratase in plants.

Planit Phv siol. 34: 403-09.

4. CHIENIAE, G. AND H. J. EVANS. 1959. Propertiesof a particulate iiitrate reductase from the nodules

of the soybean pk.ent. Biochimii. Biophys. Acta 35:

140-53.

5).CHIN(;, TEMAYV. 1963. Fat uitilizationi in germinat-

in Douglas fir seeds. Plant Phvsiol. 38: 722-

28.

6. DARON, H. H. AND C. GU-Ns.Ai.Us;. 1962. Citra-

tase anid isocitratase. In MXethodls in Enzymology.

S. P. Colowick and( 'N. 0. Kaplan, eds. Academ-ic

Press, New York. Vol. 5: 622-33.

7. DEHERTOGHi, A. A., P. A. MAYE UX, A_ND H.

EVANS. 1964. TlI.e relationshipj of cobalt require-

mient to prop)iolnate metabolism in Rhiz-obiun. J.

Biol. Cbem. 239: 2446-53.

8. DiXON, G. H. AND H. L. KORNBERG. 1962. Malate

svnthetase fromi baker's veast. In Methiods in

Enzymology. S. P. ColoNvick andc N. 0. Kaplani,eds. Academic Press, 'New York. Vol. 5: 633-

37.

9. FRUTON, J. S. AND S. SI-M-MoNDS. 195-8. GeneralBiochemistry. Johnl Wiley and Sonis, New York.

10. HAIGH, W. G. AND H. BEEVERS. 1964. The occur-rence and assav of isocitrate lyase in alg,-,. Arch.

Biochem. Biophys. 107: 147-51.

11.HANAHAN, D. J. 10960. Lipid Chemistry. Jolmr\Wilev anid Sonis, New, York.

12. HAYWARD. A. C., W. G. C. FORSYTH, AND J. B.

ROBERTS. 19059. SN ithiesis and breakdowni of polyacidI by bacteria. J. Geni. M i-

crobiol. 20: 510-18.

13). JORDAN D. C. 1962. The bacteroids of the genus.-

Bacteriojl. Rcv. 26 119-41.

14. KAIA'N, J. 1953. Rapid phiotom--etric methiod for

the deterimintion of glycogen. Archi. Biocliemi.Biophy... 47: 408-18.

1 5. KoRN.\BER(, H. L. 1963. The role of acetate In iso-

citrate ase induitctioni. Biochimi. Biophyv-. _\'Cta

73: 517-19.

16. KoRNBERG, H. L. A\NDS. R. ELSDEN. 1961. The

metalbolism of 2-carbon complounds by, microorgani-isms,. Advain. Enzvmol. 23 401-70.

17. 0WRY, 0. H., N. J. RoSEBROUGH, A. L. FARR, A\ND

R. J. RA-NDALL. 1951. Protein measuremenit with

the Folini pheniol reagenit.. Biol. Chemi. 193:

265-75.

18. MANGOTLD. H. K. 1961. Thin-layer chromatog-ra-

phy of lipids. J. Aim. Oil Chiemiists' Soc. 37:

708-27.

19. RAN SON, S. 1L. 1955. Noui-x olatile miono-. dII-

anid tricarboxy lie acids ( Chromatogra'?phic and ioni

exchiange miethiods) Iii Modern Methiods of

Planit Anah sis. K. Paech and -M. V. Trace\-, edIs.Springer-Verlag,, Berlini. V'ol. 2: 539-82.

20. RiLEX,\ES, H. C. AND) S. Aju.. 1960. Occuirrenice and(

funictioni of isocitratase anid malate sviuthetas;e in

h:r-_teria. J. Bacter-iol. 79: 341-45.

21. STADT-MAN, E. R. 1957. Preparation and assay ot

acv]I coeizm me A anid othier thiol esters: use

h1N(lrox\ lamine. In: 'Methods in EnzVmnologv.. S. P.

ColoNN ick and 'N. 0. Kaplani, edis. Academilc Press.

_New York. Vol. 3: 931-41.

22. YA-MA-MoTo, Y. AND H. BEEVERS. 1960. MNalates\-ithetase in highier planits. Planit Phy siol. 35:

102-08.

23. YFEMM'\, E. W\. AND A. J. WILLIS. 1963. Tile esti-

miationi of c:~erbohxvdrates in planit extracts bxy ani

thironie. Biochiem. 1. 57 508-14.

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