Plant Physiol. (1987) 83, 535-5400032-0889/87/83/0535/06/01.00/0

Uptake and Metabolism of Carbohydrates byBradyrhizobium japonicum Bacteroids1

Received for publication August 5, 1986 and in revised form October 7, 1986

SEPPO 0. SALMINEN AND JOHN G. STREETER*Department ofAgronomy, The Ohio State University and Ohio Agricultural Research and DevelopmentCenter, Wooster, Ohio 44691

ABSTRACI

Bradyrhizobiumjaponicum bacteroids were isolated anaerobically andwere supplied with '4C-labeled trehalose, sucrose, UDP-glucose, glucose,or fructose under low 02 (2% in the gas phase). Uptake and conversionof '4C to CO2 were measured at intervals up to 90 minutes. Of the fivecompounds studied, UDP-glucose was most rapidly absorbed but it wasvery slowly metabolized. Trehalose was the sugar most rapidly convertedto C02, and fructose was respired at a rate at least double that of glucose.Sucrose and glucose were converted to CO2 at a very low but measurablerate (<0.1 nanomoles per milligram protein per hour). Carbon Number1 of glucose appeared in CO2 at a rate 30 times greater than theconversion of carbon Number 6 to C02, indicating high activity of thepentose phosphate pathway. Enzymes of the Entner-Doudoroff pathwaywere not detected in bacteroids, but very low activities ofsucrose synthaseand phosphofructokinase were demonstrated. Although metabolism ofsugars by B. japonicum bacteroids was clearly demonstrated, the rate ofsugar uptake was only l/30 to l/5o the rate of succinate uptake. The overallresults support the view that, although bacteroids metabolize sugars, therates are very low and are inadequate to support nitrogenase.

When '4C02 was supplied to soybean plants and the distribu-tion of "'C in nodules was determined, the most highly labeledfraction in bacteroids was clearly the neutral sugars (24). Sucrosecontained the bulk of the label and, between 2 and 5 h afterlabeling, 14C in sucrose declined as "1C in trehalose increased.14C in glucose and fructose in bacteroids also declined with time(24).The results indicated some metabolism of sugars by Brady-

rhizobium japonicum bacteroids, although we could not detectphosphofructokinase, Entner-Doudoroff enzymes, invertase, orNADP-dependent 6-P-gluconate dehydrogenase in bacteroids(24, 34). Later studies indicated that B. japonicum bacteroids doabsorb carbohydrates but that the absorption is by diffusion andis slow relative to the uptake of organic acids (25).The overall results for B. japonicum indicate that sugars prob-

ably are not the principal source of energy and reducing equiv-alents which support N2 fixation in the nodule. However, whiledirect uptake and metabolism of sugars may not be required forbacteroid functioning (6, 18, 30), some metabolism of sugars bybacteroids seems likely. The need for sugar metabolism would

'Supported in part by the United States Department of Agricultureunder agreement No. 83-CRCR-1-1306. Salaries and research supportprovided by State and Federal Funds appropriated to the Ohio Agricul-tural Research and Development Center, The Ohio State University.Journal Article No. 99-86.

include the synthesis of cell walls, glycogen, and trehalose inbacteroids (24, 29).We report here experiments in which 14C-labeled sugars were

supplied to anaerobically isolated bacteroids using incubationperiods long enough (60-90 min) to permit accurate estimatesof the rates of metabolism of various compounds. We foundslow but measurable respiration ofall sugars studied. In addition,we re-examined the complement of enzymes of carbohydratemetabolism in B. japonicum bacteroids and found levels ofactivity which were low but which were consistent with theobserved metabolism of labeled sugars.

MATERIALS AND METHODSBacteroid Isolation. Soybeans, Glycine max [L.] Merr, cv

Beeson 80, were inoculated with Bradyrhizobium japonicumstrain 61A76 and grown in the greenhouse as described previ-ously (29). Nodules were harvested from 37- to 47-d-old plantsand ground under anaerobic conditions (AtmosBag flushed withN2) in a mortar with a pestle in 2 ml of 0.15 M Na phosphatebuffer (pH 7.5) per 1 g nodules. The brei was centrifuged incapped tubes (anaerobic) at 6000g for 10 min, and the bacteroidpellet resuspended in the same buffer (2 ml/g nodules) under N2flow.

"C-feeding. D_[U-'4C]fructose (274 mCi/mmol), D_[U-_4C]glu-cose (3 mCi/mmol), D_[1-'4C]glucose (55 mCi/mmol), D-[6-'4C]glucose (58 mCi/mmol), and [U-'4C]sucrose (552 mCi/mmol)were purchased from Amersham (Arlington Heights, IL), anduridine diphosphate D_[U-'4C glucose] (205 mCi/mmol) waspurchased from ICN (Irvine, CA).The reaction mixture consisted of 1 ml ofbacteroid suspension

in a final volume of 2 ml of 0.15 M Na phosphate buffer (pH7.5) with 0.04 mm myoglobin in 15 ml Corex tubes sealed witha serum stopper with an attached cup containing a fluted filterpaper impregnated with 10% KOH. The 02 in the gas phase wasadjusted to 2% by removing 1.4 ml of N2 and injecting 1.4 mlof Ar.02 (80:20). Reactions were started by injecting 1 umol at1 ;tCi/Mmol of the '4C-sugar into the tubes shaken at 250 rpm.At 0, 30, 60, and 90 min duplicate reaction mixtures weresampled. The filter papers were placed in counting vials, thetubes were centrifuged at 2°C at 25,000g, and the pellets wereextracted twice with 80% ethanol. The samples were evaporatedto dryness and taken up in 2 ml of water. The aqueous sampleswere added on 0.7 x 2.0 cm Dowex 50-H+ and Dowex 1-formatecolumns in tandem. The neutral fraction was eluted with H20,after which the columns were separated and amino acids wereeluted from the Dowex 50-H+ column with 2 N HCI, and theorganic acids from the Dowex 1-formate column with 4 NHCOOH. The eluates were evaporated to dryness and taken upin 80% ethanol.

Counting. The cocktail used with the KOH-impregnated filterpaper consisted of 6 g PPO/L of 2 parts toluene and one part

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SALMINEN AND STREETER

methoxyethanol. The cocktail for the ethanolic samples consistedof 24 g PPO, 1 g POPOP in 2 L toluene + 2 L Triton X-100.

Synthesis of U-'4C-Trehalose. Nineteen ml of Manhart andWong medium (15) containing 60 mg of glutamate as the sourceof C and N and 250 ,uCi at 280 ,uCi/,umol of [U-'4C]glutamatewas inoculated with 1 ml of B. japonicum 61A76 culturedbacteria and the flask shaken at 27°C. The growth of bacteriawas followed in a separate flask lacking the radioisotope bydetermining the A at 600 nm. At the end of the exponentialgrowth phase the cells were harvested and trehalose was extractedand purified as described before (35).Enzyme Assays. B. japonicum 6 1A76, R. phaseoli, 127K14,

and R. leguminosarum 128C53 were obtained from the NitraginCo., Milwaukee, WI. The other strains of B. japonicum wereobtained from the Nitrogen Fixation and Soybean GeneticsLaboratory, USDA/ARS, Beltsville Agricultural Research Cen-ter, Beltsville, MD. Preparation of soluble protein from bacter-oids and cultured bacteria was the same as described recently(29) except that bacteroids were isolated by centrifugation at4300g for 15 min. For the experiments with bacteroids purifiedon Percoll gradients, the procedure was as described previously(24).

All reagents were of the highest purity available from SigmaChemical Co. Purified enzymes were also purchased from Sigmaas follows: aldolase, type X; triose-P-isomerase, type III; a-glycerophosphate dehydrogenase, type I; UDP-glucose dehydro-genase, type III; lactate dehydrogenase, type II; phosphogluco-mutase; glucose-6-P dehydrogenase, type XI.

Sucrose synthase (EC 2.4.1.13) was assayed by three methods:(a) measurement of UDPGlc formation using a continuouscoupled assay to UDP-glucose dehydrogenase (21), (b) quanti-tative analysis of UDPGlc after stopping the sucrose synthasereaction with heat (36), and (c) formation of ['4C]sucrose from[UDP-'4C]glucose (28). Sucrose phosphorylase (EC 2.4.1.7) wasmeasured with a spectrophotometric assay for the formation ofD-glucose- 1-P from sucrose (19).The Entner-Doudoroff enzymes 6-P-gluconate dehydratase

(EC 4.1.2.2) and 2-keto-3-deoxy-6-P-gluconate aldolase (EC4.1.2.14) were assayed by NADH oxidation, coupling pyruvateformation to lactate dehydrogenase (31). The slightly modifiedreaction mixture consisted of 63 mm Tris * HCI (pH 8.2), 1.1mM DTE, 0.37 mM MnCl2, 0.26 mM NADH, 3 units of lactatedehydrogenase, 3.7 mM 6-P-gluconate, and extracted protein(0.2-0.5 mg) in a total volume of 2.7 ml. The controls lackedsubstrate, and AA340 was a linear function of time and proteinconcentration.

Phosphofructokinase (EC 2.7.1.11) was assayed in a reactionmixture containing 63 mm Hepes-NaOH buffer (pH 7.8), 3.6mm fructose 6-P, 1.8 mM MgCl2, 1 mM DTE, 0.26 mM NADH,1.5 mM ATP, 2 units of aldolase, 2 units of glycerophosphatedehydrogenase, 12 units of triose-P isomerase, and extractedprotein (0.2-0.6 mg) in a total volume of 2.7 ml. PPi: fructose-6-P 1-phosphotransferase (EC 2.7.1.90) was assayed in a reactionmixture which was the same as that just described except thatDTE was omitted, PPi (2.2 mM) was used in place of ATP, andthe total volume was 2.5 ml (1). The control for these assays wasminus ATP or PPi, and for both assays NAD formation was alinear function of time and protein concentration.

Protein concentration in extracts was determined by themethod of Lowry et al. ( 14).

RESULTSMetabolism of '4C-Labeled Sugars. The possibility that the

bacteroid preparations were contaminated with mitochondriawas checked using two assays: MDH2 activity was tested with

2 Abbreviation: MDH, malate dehydrogenase.

two inhibitors of mitochondrial MDH (37). A rate of 2.24 ±0.13 (mean ± SE) ,umol * mg-' protein * min-' was measuredfor controls, whereas 70 min preincubation with 1 mm iodoace-tate or 1 mM phenylmethanesulfonyl fluoride gave rates of 1.95± 0.05 and 2.06 ± 0.06, respectively. Using a Cyt oxidase assay(3) we obtained identical rates, 77 ± 1.5 and 78 ± 0.5 nmol mg-'protein min-' in the absence or presence ofCO, which is knownto inhibit mitochondrial Cyt oxidase but not Cyt oxidase inbacteroids (2). These results indicated that the bacteroid prepa-rations were not contaminated with mitochondria.The reproducibility between different bacteroid preparations

was good; e.g. the total uptake rates of glucose in Tables I andII, with different bacteroid preparations, are not statisticallydifferent. In still another experiment with [U-'4C]glucose theuptake was 599 (60) (mean [SE]) picomol * (mg-' bacteroidprotein) * 90 min-'.The uptake ofsugars was approximately linear for the duration

of the experiments (Fig. 1). The rates, however, were quite lowin comparison to those measured with succinate. For instance,the rate of uptake of succinate was 26.5 nanomol * mg-' bacte-roid protein . h-', and the absorbed succinate was almost com-pletely (97%) metabolized. The corresponding uptake rates forUDPGlc, an immediate precursor oftrehalose (29), and trehalosewere 2.7 and 0.9 nanomol.The respiration ofthe sugars taken up was calculated assuming,

e.g. 1 trehalose -* 12 CO2 and a constant specific activity of 1MCi/Amol in the sugar supplied (Fig. 2). Trehalose was the bestrespiratory substrate, whereas the sugar taken up most readily,UDPGlc, had the lowest rate. Fructose was respired much morerapidly than glucose, which was indistinguishable from sucrose.A further comparison between fructose and glucose (Table I)

with the same bacteroid preparation confirmed the results seenin Figures 1 and 2. The uptake of fructose was 31% greater andconversion to CO2 2.6 times faster than the corresponding ratesfor glucose. In 90 min, 41% of the fructose taken up wasconverted to C02, whereas only 21% ofthe glucose was respired.Most of the 14C was in the neutral fraction after feeding glucoseor fructose. The amount of '4C in the amino acid fraction wasslightly higher from fructose than from glucose on a relative basis(Table I). In another experiment (data not shown), fructosefeeding resulted in six times more label in the amino acid fractionthan glucose feeding. The distribution of radioactivity in theneutral, amino acid, and organic acid fractions followed thepattern seen with glucose. No labeling ofthe amino acid fractionwas seen with sucrose feeding (data not shown).The recoveries from the ion-exchange columns were in the

range of 80 to 97% and usually within 10% among samples ineach individual experiment. The notable exception was UDPGlc,where recovery from the Dowex 1 -formate column was only20%. Evidently the binding ofthe phosphate group to the columnwas too strong to be eluted with 4 N HCOOH. This was verifiedby applying standard ['4C]UDPGlc solution directly onto thecolumn. The recovery was only 2.5%. The fact that recoveries ofboth the sample and the standard were low is consistent with theassumption that most of the '4C in the bacteroids fed with ['4CJUDPGlc (Fig. 1) was present as UDPGlc. The UDPGlc in thebacteroids was, thus, very slowly converted to '4CO2 (Fig. 2).A comparison between [1-'4C]glucose and [6-'4C]glucose (Ta-

ble II) showed that 32% of the label taken up was released fromC-l in 90 min, whereas only 1% of the label was released fromC-6.Enzyme Activities. Three assays for sucrose synthase were

used. A problem with the assay (A) continuously coupled toUDP-glucose dehydrogenase is that the pH optima of the dehy-drogenase and of sucrose cleavage are several pH units different(21). A pH of 7.5 was used as a compromise (21) but, at this pH,we found that dehydrogenase activity limited the apparent reac-

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UPTAKE AND METABOLISM OF CARBOHYDRATES

Table I. Uptake and Metabolism of[U-'"C]Fructose and [U-'4C]GlucoseResults are obtained using the same bacteroid preparation. Mean (SE) of duplicate samples for 90 min is

given.

Radioactive Substrate FractionsSubstrate Converted Total Conversion

Applied to C02 Uptake Neutral Amino acid Organic toC02

pmol-mg' protein %[U-'4C]fructose 298 (18) 730 (29) 267 (17) 138 (6) 25 (21) 41[U-'4C]glucose 116 (13) 556 (27) 205 (29) 89 (3) 23 (5) 21

Table II. Uptake and Metabolism of[J-'4CJGlucose and[6-'4CJGlucoseMean (SE) of duplicate samples is given.

Radioactive , Percent ofGlucose Min 4CO2 Total the TotalSupplied Uptake

pmol -mg-' protein1-'4C 45 87 (27) 288 (63) 30

90 193 (7) 597 (31) 326-'14C 45 4 (0) 293 (17) 1

90 6 (0) 604 (14) 1

3

-

.

-U06

x

0

0

a.

c

-J

w0-

W0 30 60 90TIME (minutes )

FIG. 1. Total uptake of radioactive substrates by B. japonicum bac-teroids. The values represent the sum of radioactivity found in thebacteroids and the substrate equivalent of '4CO2, assuming constantspecific activity of I gCi/Mmol of the sugar supplied. The data are

expressed on a per mg bacteroid protein basis. The bars represent 2 SE ofduplicate samples. (0), Glucose; (A), fructose; (A), sucrose; (0), trehalose;(0), UDPGlc.

tion rate and that apparent sucrose cleavage was inversely relatedto protein concentration. The assay (B) where pH was changedafter stopping sucrose cleavage with heat (36) appeared to givereasonable results. However, we were concerned that losses ofUDPGlc upon heating (29) would render this assay unsatisfactoryfor the very low activity we expected to find in bacteroids.

In contrast to these problems, the radioactive assay (28) forsucrose synthesis gave good results: low control values and rates

Ch.CP~~~~~~~~~~~~~~~~~~~~~~~~~C

E

0 30609

0c0

z

0wi

0

00 30 60 90TIME (minutes)

FIG. 2. CO2 evolution from labeled substrates by B. japonicum bac-teroids. The calculations assumed a constant specific activity of 1 ACiljumol of the sugar supplied and, e.g. I glucose -* 6CO2. Other details asin Figure 1.

which were linear with protein concentration and time. Usingthis assay we found much higher activity in cytosol than inbacteroids (Table III) and this is in agreement with earlier results(21). Although activity in bacteroids was quite low, the radioac-tivity in sucrose in complete mixtures was at least 10 times thatin control mixtures, and activity was still present after purifica-tion of bacteroids on Percoll gradients.

In agreement with the previous report (24), we were unable todetect pyruvate formation from 6-P-gluconate in B. japonicumbacteroids (Table III). This result was obtained using an assaywhich was sensitive to G0.5 nmol pyruvate/min and whichindicated levels of enzyme activity in R. phaseoli and R. legum-inosarum similar to those reported by others (16). In optimizingthis assay it was found that DTE was not required but thatactivity was about 70% lower when Mn2+ was omitted (data notshown).The absence of phosphofructokinase was previously reported

in B. japonicum bacteroids (24). However, we discovered subse-quently that an inhibitory (high) concentration ofATP had beenused in those assays. Using a correct ATP concentration (1.5mM) we measured phosphofructokinase activity in cytosol whichwas 6-fold higher than that previously reported (24); we werealso able to detect low activity in bacteroids (Table III). Particu-

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SALMINEN AND STREETER

Table III. Activity ofEnzymes of Carbohydrate MetabolismValues represent results from one or, generally, two experiments (multiple assays/experiment). Results for

two experiments were usually within 10% and were averaged to give the result shown.

Enzyme Source of Protein and Conditions Enzyme Activity

Sucrose synthase

Entner-Doudoroff enzymes'

Phosphofructokinase

PPi-fructose-6-P l-phospho-transferase

B. japonicum 6 1A76 bacteroids; assay CSame as above but bacteroids purifiedon Percoll gradient

Soybean nodule cytosol; assay CR. leguminosarum 128C53, cultured

bacteriaR. phaseoli 127K14, cultured bacteriaR. phaseoli 127K14, bacteroidsB. japonicum USDA 6, 31, 76, 94, 110,

122, 138, cultured bacteriaB. japonicum 6 1A76, USDA 110 bacter-

oidsSoybean nodule cytosolB. japonicum 61 A76, USDA 110 bacter-

oidsB. japonicum 61A76 bacteroids purifiedon Percoll gradient

Soybean nodule cytosolB. japonicum 6 1A76, USDA 110 bacter-

oids

nmol mg-'protein * min-'

5.30.75

32512

50<5b

<0.5c

<0.5c

28010

1.6

1 3d<0.5c

a Assay for the formation of pyruvate from 6-P-gluconate. b AA30 of controls approximately 90% ofcomplete mixtures so that actual rate is uncertain. c No activity was detected. This value is an estimate ofthe minimum rate measurable with the methods employed. d Rate in the presence of 5 to 10 gM fructose-2,6-P.

late phosphofructokinase (13) could not be detected, and thePPi-dependent phosphotransferase was also not detectable inbacteroids with or without catalytic amounts of fructose-2,6-P.The PPi-dependent activity was found in cytosol (Table III), butthe activity was <10% of the ATP-dependent phosphofructoki-nase activity in cytosol.

DISCUSSION

Studies on the uptake of sugars in bacteroids have yieldedconflicting reports. Glenn and Dilworth (5) demonstrated activedisaccharide uptake systems for fast-growing species of culturedrhizobia, whereas little or no uptake was seen in slow-growingspecies. They found no uptake ofsucrose or glucose by bacteroidsof either slow- or fast-growing species. However, evidence forglucose utilization by R. lupini bacteroids was obtained withsimilar short (5 and 15 min) incubation periods (12, 26). Theuptake of glucose by B. japonicum is by passive diffusion (25).The rates reported here indicate there is a definite, albeit quiteslow, uptake ofsugars. Even UDPGlc, where a charged phosphategroup appeared to facilitate the uptake, had a rate of only aboutone-tenth of the rates obtained with succinate.We also found clear evidence ofthe metabolism ofthese sugars

in the bacteroids. This agrees with data of other workers (33),but it is in disagreement with a report indicating that in aerobi-cally isolated bacteroids from soybean nodules no oxidation ofglucose, sucrose, or trehalose occurred under any conditions(23). It is possible that maintenance of anaerobic conditions inour study would account for this difference.

Trehalose gave the highest rate of CO2 production (Fig. 2). Inlight of the high trehalase activity in the bacteroids (29) this isnot surprising. High uptake rate and trehalase activity wouldquickly lead to high glucose levels. The glucose formed wouldappear to be metabolized principally via the pentose phosphate

pathway. Because the phosphogluconate dehydrogenase is NAD-linked (17, 22, 24), this could contribute to the NADH supply.It could also, however, create a demand for NADPH needed forfatty acid synthesis beyond that provided by glucose 6-P dehy-drogenase alone.Although sucrose is the major carbohydrate transported to the

nodules (7, 10, 11), it is doubtful that sucrose plays a major rolein bacteroid metabolism. Its entry was the slowest measured (Fig.1), and its respiration rate was comparable to glucose (Fig. 2).Considering that fructose, which should be produced from su-crose breakdown, was more readily converted to C02, the lowrate ofCO2 evolution from sucrose could be indicative that it ishydrolyzed to hexose relatively slowly. This is in contrast totrehalose which may well serve as a ready reserve carbohydratein the bacteroids.

Fructose was converted to CO2 more rapidly than glucose (Fig.2; Table I). In light ofthe reported enzyme activities (24) showingthe presence of hexokinase and phosphoglucose isomerase seem-ingly allowing for a ready interconversion, this is somewhatsurprising. Fructokinase, forming fructose-l-P would, if coupledto fructoaldolase forming glyceraldehyde and dihydroxyacetonephosphate, bypass the phosphofructokinase step. However, noevidence for it was found in bacteroids (24). Thus, we have noexplanation for the difference in the utilization of fructose andglucose. However, all quantitative results agree with a more rapidturnover of fructose, i.e. nodules have much less fructose thanglucose.

Results from the feeding of labeled compounds to bacteroidsindicated that sugars are metabolized, although the rates werevery slow relative to succinate. Results obtained for enzymesappear to substantiate pathways for metabolism of all of thesugars which were respired by bacteroids.

Morell and Copeland (20) previously reported low activity of

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UPTAKE AND METABOLISM OF CARBOHYDRATES

sucrose synthase and alkaline invertase in B. japonicum bacte-roids. Using a different assay and bacteroids purified on Percollgradients, we have confirmed the presence of low sucrose syn-thase activity in bacteroids (Table III). However, we have notpreviously detected invertase in Percoll-purified bacteroids (24,34), although we sometimes can measure very low activity inbacteroids isolated by differential centrifugation. We attributethe activity observed in this latter case to contamination fromcytosol and suggest that this may explain the result of Morelland Copeland (20). In any event, sucrose synthase may providea mechanism for sucrose hydrolysis in B. japonicum bacteroids.Sucrose phosphorylase was not detected in bacteroids.

Trehalase is probably the means by which trehalose is hydro-lyzed (29, 34). Trehalase activity in B. japonicum bacteroidsappears to be 5 or more times greater than the activity of sucrosesynthase, and this may explain why the respiration of trehalosewas much faster than the respiration of sucrose (Fig. 2). Thegreater rate of CO2 evolution from trehalose than from glucosewould appear to be due to the higher uptake rate of the former.Trehalase activity would lead to high glucose levels in the bac-teroids, whereas the slow uptake of glucose would result in lowendogenous glucose.

Glucose and fructose appear to be metabolized via the glycol-ytic or pentose phosphate pathways but not via the Entner-Doudoroff pathway. This conclusion puts us in conflict withElkan's group who have emphasized the importance of theEntner-Doudoroff pathway in Rhizobium (9, 33). Our results arein agreement with other reports which indicate that fast-growingRhizobium have much higher Entner-Doudoroff activity thanslow-growing bacteria and that, for the fast-growing species,bacteroids have much lower enzyme activity than cultured bac-teria (16, 30). We could not detect enzyme activity in eithercultured bacteria or bacteroids of B. japonicum and concludethat this pathway is not operative in soybean nodules.Glucose and fructose catabolism via the glycolytic pathway is

possible because phosphofructokinase (Table III) and other rel-evant enzymes (24) are present in B. japonicum bacteroids.However, the phosphofructokinase activity is quite low, and thePPi-dependent enzyme (4, 27, 38) could not be detected. Thus,flow of carbon via this route may be slow, and this would beconsistent with the much more rapid conversion of [1-'4C]glucose than [6-14C]glucose to '4CO2 (Table II). The relativelyrapid metabolism of glucose via pentose phosphates would, inturn, be consistent with the rela,tively high 6-P-gluconate dehy-drogenase in bacteroids; we previously reported enzyme activityof 40 nmol mg-' protein . min-' (24) and found similar rates instudies reported here (data not shown). It should be emphasized,however, that B. japonicum lacks the normal NADP-dependentenzyme (16, 22) and that there is still some doubt about theproduct of the NAD-dependent enzyme (22, 33). Ribulose-5-Pwas identified as the product of the partially purified enzymefrom B. japonicum (17), and similar evidence is available forNAD-6-P-gluconate dehydrogenase from other bacteria (32), buta different result was obtained by Mulongoy and Elkan (22). Ourresults with differentially labeled glucose indicate an early loss ofcarbon No. 1 from glucose and this would be consistent with theformation of ribulose-5-P via the NAD-dependent dehydrogen-ase.

There is increasing evidence that the enzymes of carbon me-tabolism in Rhizobium reflect the carbon compounds availableand, therefore, that enzymes active in bacteroids are one indi-cation of the carbon compounds supplied to bacteroids by thehost (30, 33). Our results support this hypothesis. That is, al-though enzyme activities which can account for the breakdownof sucrose, glucose, and trehalose found in B. japonicum bacter-oids were more than enough to account for the rates of CO2production (Fig. 2), these enzyme activities were very low relative

to activities of tricarboxylic acid-cycle enzymes (8). The lowactivity of enzymes for metabolism of sugars in bacteroids is inagreement with the low rate of respiration of sugars relative tothe rapid respiration of succinate.

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