JOURNAL OF BACTERIOLOGY, Oct. 1971, p. 334-342Copyright 0 1971 American Society for Microbiology

Vol. 108, No. IPrinted in U.S.A.

Heterotrophic Metabolism of the ChemolithotrophThiobacillus ferrooxidans

ROBERT TABITA AND D. G. LUNDGRENDepartment of Biology, Syracuse University, Syracuse, New York 13210

Received for publication 18 June 1971

Glucose-6-phosphate dehydrogenase and the enzymes of the Entner-Doudoroffpathway, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconatealdolase (assayed together), are induced during heterotrophic growth of Thioba-cillus ferrooxidans on an iron-glucose-supplemented medium or on glucose alone.By contrast, autotrophic cells (iron-grown) contain low levels of these enzymes.Fructose 1,6-diphosphate aldolase, an enzyme of the Embden-Meyerhof pathway,is present at low levels irrespective of the growth medium, suggesting that this en-zyme is not involved in energy-yielding reactions but merely provides intermediatesfor biosynthesis. The Entner-Doudoroff and pentose-phosphate pathways are theprinciple means through which glucose is dissimilated and is presumed to be con-cerned with energy production. Isotopic studies showed that a high rate of CO2formation from specifically labeled glucose came from carbon atoms I and 4. Anunexpectedly high rate of evolution of CO2 also came from carbon 6, suggestingthat the triose phosphate formed during glucose breakdown and specifically as aresult of 2-keto-3-deoxy-6-phosphogluconate aldolase activity, was metabolized viasome unorthodox metabolic route. Cells grown in the iron-supplemented and glu-cose-salts media have a complete tricarboxylic acid cycle, whereas autotrophicallygrown T. ferrooxidans lacked both a-ketoglutarate dehydrogenase and reducednicotinamide adenine dinucleotide oxidase. Two isocitrate dehydrogenases [nicotin-amide adenine dinucleotide (NAD) and NAD phosphate (NADP) specific] werepresent. NAD-linked enzyme was constitutive, whereas the NADP-linked enzymewas induced upon adaptation of autotrophic cells to heterotrophic growth.

The iron-oxidizing bacterium Thiobacillus fer-rooxidans grows on simple sugars such as glu-cose as a sole source of energy (14, 19, 25).When grown on glucose, the iron oxidationmechanism of the cell is repressed (25). Growthon glucose results in poly-,8-hydroxybutyrate(PHB) accumulation (28) and an increase in theactivity of the enzyme glucose-6-phosphate dehy-drogenase (G6PD, reference 25). These resultssuggested that to metabolize glucose, the orga-nism drastically alters its metabolic profile. Astudy was made to see which of the enzymes ofthe cell change when T. ferrooxidans switchesfrom an autotrophic existence to a heterotrophicone. Radiorespirometric experiments with 14C-specifically labeled glucose were performed toconfirm the pathway(s) through which glucose ismetabolized.

MATERIALS AND METHODST. ferrooxidans was grown autotrophically on fer-

rous iron as an energy source in the 9K medium andheterotrophically on glucose (25); glucose replaced theiron in the 9K medium as previously described (25).

Preparation of cell-free extracts. Cell-free extractswere prepared by using cells harvested at 4 C by cen-trifugation in a Sorvall RC2-B centrifuge, washed twicewith 50 mm tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.9) buffer, and then suspended inthis buffer overnight prior to breakage. This procedureis known to facilitate cell breakage of autotrophicallygrown T. ferrooxidans (20); storage of whole cells at 4C did not affect subsequent enzyme determinations. A20% (w/v) cell suspension in the Tris-hydrochloridebuffer was used as a source of cell extract, and treat-ment for 15 min in a water-cooled (10 kc/sec) Ray-theon sonic oscillator was required for cell breakage.After sonic treatment, unbroken cells and debris wereremoved by centrifugation at 35,000 x g for 20 min at4 C, and the supernatant fluid was collected and usedas the crude cell-free extract for most of the enzymeassays. Both dialyzed and undialyzed extracts wereroutinely used with no differences noted.

Krebs cycle enzymes were assa3 -d by using cell-freeextracts from cells previously harvested at 4 C by cen-trifugation and immediately suspended in a potassiumphosphate (50 mm)-glutathione (1 mm) buffer at pH7.4. Cell pellets were washed twice with this buffer. Thepresence of glutathione or dithiothreitol (I mM) in thebuffer was essential for maximum enzymatic activity.

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The cells were suspended to a 20% (w/v) suspension inthe aforementioned buffer and placed in the standardRaytheon cup, and N, gas was bubbled through thecell suspension for 10 min. The cells were broken by anexposure for 15 min. Residual whole cells and debriswere removed by centrifugation at 35,000 x g for 20min at 4 C. The supernatant fluid was separated andused for Krebs cycle enzyme assays as well as for re-duced nicotinamide adenine dinucleotide (NADH) oxi-dase assays.

Enzyme assays. G6PD [D-glucose-6-phosphate,nicotinomide adenine dinucleotide phosphate (NADP)oxidoreductase, EC 1.1. 1.49] was assayed spectro-photometrically at 340 nm by observing the rate of ap-pearance of either reduced NADP (NADPH) orNADH. The reaction mixture contained: 15 mM Tris-hydrochloride (pH 7.9); 6.67 mM MgCI2; 0.83 mm glu-cose-6-phosphate; 0.33 mm NADP or nicotinamideadenine dinucleotide (NAD, pH 7.0), cell-free extract,suitably diluted in 50 mm Tris (pH 7.9), and water to3.0 ml. These concentrations of substrates and coen-zymes were found to be saturating. The reaction wasinitiated by the addition of either NADP or NAD.

6-Phosphogluconate dehydrogenase [6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating),EC 1. 1. 1.44] was assayed by using the same assay asfor G6PD except that 2.67 mm 6-phosphogluconate wasused in the place of glucose-6-phosphate. The potas-sium salt of 6-phosphogluconate was prepared from thebarium salt by the method of Horecker and Smyrniotis(9).

Fructose- 1, 6-diphosphate aldolase (fructose- 1, 6-diphosphate D-glyceraldehyde-3-phosphate lyase, EC4.1.2.13) was measured spectrophotometrically byusing a coupled assay containing triose-phosphateisomerase and a-glycerophosphate dehydrogenase; therate of NADH oxidation was followed at 340 nm, andthe activity of the enzyme was calculated from the ini-tial rate of NADH oxidation. The reaction mixturecontained: 0.1 M Tris-hydrochloride (pH 7.5), 5 mMfructose-1 , 6-diphosphate, 0.02 ml of a-glycerophos-phate dehydrogenase-triosephosphate isomerase (10mg/ml), 0.25 mm NADH (pH 7.0), and cell extractand water to 3.0 ml. The reaction was initiated byadding cell extract.The Entner-Doudoroff (ED) enzymes, 6-phospho-

gluconate dehydrase (6-phosphogluconate dehydratase,EC 4.2. 1. 12) and 2-keto-3-deoxy-6-phosphogluconatealdolase (EC 4. 1.2. 14), were assayed together by de-termining the amount of pyruvate formed from 6-phosphogluconate by the method of Keele, Hamilton,and Elkan (10). The reaction mixture contained 0.1 MTris-hydrochloride buffer (pH 7.6), 8 mm gluconate-6-phosphate, 6 mm FeSO4 7H2O (freshly prepared), 3mM glutathione (freshly prepared), and extract andwater to 1.0 ml. FeSO4 was required for maximum ac-tivity. Pyruvate was determined as the 2, 4-dinitro-phenylhydrazone (7). The enzymatic formation of pyr-uvate was confirmed by comparing the absorptionspectra of the dinitrophenylhydrazone of a pyruvatestandard to the test compound, as well as by com-paring the optical density (OD) ratio (in nanomoles),of both products OD49,-OD.4. (22).

Aconitase [citrate (isocitrate) hydrolyase, EC4.2.1.2] was assayed by the method of Anfinsen (2)

and was based on the spectrophotometric determina-tion at 240 nm of cisaconitic acid. The reaction mix-ture contained 9.7 mM DL-isocitrate, 48.3 mm phos-phate buffer (pH 7.4), and extract and water to 3.0 ml.

Fumerase (fumarate hydratase, EC 4.2.1 .2) wasassayed spectrophotometrically at 240 nm by the pro-cedure of Racker (18). The reaction mixture contained0.05 M sodium L-malate (pH 7.4), 0.05 M phosphatebuffer (pH 7.4), and extract and water to 3.0 ml.

Isocitrate dehydrogenase [threo-D.-isocitrate: NAD(or NADP) oxidoreductase (decarboxylating) EC1.1.1.41 and EC 1.1.1.42] was assayed spectropho-tometrically at 340 nm after the rate of appearance ofNADH or NADPH, or both. The reaction mixturecontained 10 mm Tris-hydrochloride (pH 7.9), 0.33 mMMnCI2, 0.17 mM DL-isocitrate, 0.33 mM NADP or 3.33mM NAD, and extract and water to 3.0 ml.

Succinic dehydrogenase [succinate: (acceptor) oxi-doreductase, EC 1.3.99.1] was assayed spectropho-tometrically at 600 nm after the reduction of 2,6-di-chlorophenolindophenol (DCPIP), mediated by phena-zine methosulfate (PMS), by the method of Arrigoniand Singer (3). The reaction mixture contained 20 mMsuccinate, I mm potassium cyanide, 56 mm phosphatebuffer (pH 7.6), and cell-free extract and water to 3.0ml. The reaction mixture was incubated for 3 min, andthe reaction was initiated with 0.08 mM DCPIP and 1.1mM PMS.

a-Ketoglutarate dehydrogenase was assayed by spec-trophotometrically observing (600 nm) DCPIP reduc-tion as described by Watson and Dworkin (30). Thereaction mixture contained 33 mm phosphate buffer(pH 7.0), 6.67 mM MgCI2, 0.067 mm thiamine pyro-phosphate (TPP), 6.67 mm a-ketoglutarate (pH 7.0),0.033 mm DCPIP, and extract and water to 3.0 ml.NADH oxidase [NADH: (acceptor) oxidoreductase,

EC 1.6.99.1 ] was assayed by spectrophotometrically ob-serving (340 nm) the oxidation of NADH. The reac-tion mixture contained 0.25 mm NADH, 66.7 mmphosphate buffer (pH 7.4), and cell-free extract andwater to 3.0 ml.

For all assays, controls were routinely performedand consisted of the reaction mixture minus the specificsubstrate or minus the cell-free extract. Substrate con-centrations were at saturating levels in all cases. Allenzymes were inactivated by boiling for 5 min.

In all cases, except where noted, enzyme activity wasexpressed as units per milligram of protein. One unit isthe amount of enzyme needed to oxidize or reduce IjAmole of substrate per min. Assays were always re-peated three times, and results were reproducible. Sep-arate batches of cell-free extracts were routinelychecked to confirm enzyme activity.

Piotein was determined spectrophotometrically bythe method of Warburg and Christian as described byLayne (I 3).

Radiorespirometric studies. The radiorespirometricexperiments were performed as described by Wang etal. (29), modified by Perry and Evans (17). Cells weregrown in an 9KG medium as previously reported (25),or in the glucose-salts medium (25), harvested after 60hr of growth, and resuspended in the salts solution usedin the 9K medium; the final pH was 2.5. Cells wereplaced in the main compartment of a reaction vesselcontaining 20 ml of the salts of the growth medium

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with 20 Mmoles of carrier glucose in the side arm. Thefollowing counts per min of appropriately labeled glu-cose were added to the reaction vessel: glucose-1-"4C,517,520; glucose-2-14C, 461,580; glucose-3-'4C, 66,123;glucose-3,4-'4C, 490,010; glucose-6-'4C, 558,208.

The CO2 produced was trapped in 10 ml of mono-ethanolamine; the gas collection column was previouslywashed twice with 5 ml of absolute ethyl alcohol. Sam-ples (I ml) were collected and the radioactivity was

measured in a Mark I Analyzer (Nuclear-ChicagoCorp.). The counting vial contained I ml of the trapsolution plus 14 ml of a counting mixture consisting of0.4% 2,5-diphenyloxazole (PPO) and 0.01% 1,4 bis-2-(5-phenyloxazolyl)-benzene (POPOP) in toluene.

For the estimation of participation of the ED andpentose-phosphate (PP) pathways, the following for-mulas were used (29): Gpp = G,-G4, where Gppequals the percentage of glucose oxidized by the PPpathway and G1 and G4 equal the total activity re-covered in metabolic CO, from cells utilizing glucoselabeled in carbon atoms I and 4, respectively. G4 iscalculated by the following equation.

G4 = 2G3, 4- Gs,

where G..4 and G. equal the total activity recovered inmetabolic CO2 from cells utilizing glucose labeled incarbon atoms 3, 4, and 6, respectively. The percentageof glucose dissimilated by the ED pathway was calcu-lated from the relationship: GED = 1 -G,.

Isotopes were purchased from New England NuclearCorporation Boston, Mass. PPO and POPOP wereproducts of the Packard Instrument Co., LaGrange,Ill. All other chemicals were obtained from SigmaChemical Co., St. Louis. Mo.

RESULTSEnzymes of glucose metabolism. Iron-grown

cells, once adapted to glucose possessed highlevels of G6PD, the enzyme which catalyzes theinitial reaction for both the pentose-phosphateshunt and the ED pathways (25). Enzymes func-tioning in these pathways were assayed in crudecell-free extracts of T. ferrooxidans to determinethe pathway(s) through which glucose is dissimi-lated. In addition, fructose- 1, 6-diphosphate aldo-

lase, a glycolytic pathway enzyme, was assayedin extracts of cells grown either autotrophicallyor heterotrophically. The results (Table 1) showthat during autotrophic growth (iron-growncells), the levels of the glucose catabolic enzymeswere low, whereas, as previously shown (25), thecapacity for iron oxidation and CO2 fixation washigh. This low level of the glucose-associatedenzymes in autotrophically grown cells indicatesthat these enzymes function in biosynthesis anddo not appear to be directly involved in energymetabolism (15). However, when glucose servedas an energy source, as with 9KG medium or inthe glucose-salts medium, G6PD (both NADP-and NAD-linked) was about 50 times above theautotrophic level. The presence of an inhibitor toG6PD in crude cell extracts from iron-growncells was ruled out, for adding this extract tocell-free extracts of Escherichia coli K-12 had noeffect; E. coli is known to have a high level ofG6PD. Nor was inhibition observed when ex-tracts from iron-grown cells were added to par-tially purified G6PD obtained from glucose-grown cells (26). Either NADP or NAD servedas coenzyme for the G6PD from T. ferroxidans(26).The activity of 6-phosphogluconate dehydro-

genase was low or undetectable in all extractstested. However, the enzymes of the ED pathway(6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase) in cell ex-tracts prepared from iron-glucose-grown cellsshowed a 30- to 40-fold stimulation over extractsprepared from iron-grown cells (Table 1).

Figure 1 compares pyruvate formation withtime from 6-phosphogluconate as a substrate.Cell-free extracts of T. ferrooxidans grown on9K and on 9KG medium were compared to ex-tracts from Psuedomonas aeruginosa, an orga-nism known to degrade glucose by way of EDpathway (6).The high levels of G6PD and ED enzymes

were not observed when other sugars or carbon

TABLE 1. Effect ofgrowth substrate on enzymes involved in glucose metabolism by Thiobacillus ferrooxidansa

Activity (10-' units/mg)Enzyme Iron-grown Iron-glucose- Glucose-grown

cells cells cells

Glucose-6-phosphate dehydrogenase (NADP) ..... ........ 1.29 52.7 76.7Glucose-6-phosphate dehydrogenase (NAD) ..... ......... 1.55 62.0 90.26-phosphogluconate dehydrogenase (NADP) ..... ......... 3.70 <0.034 <0.086-phosphogluconate dehydrogenase (NAD) ..... .......... 1.23 1.02 1.596-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phos- 0.40 12.4 15.9

phogluconate aldolase.Fructose-1,6-diphosphate aldolase ....... ................ 1.29 0.47 0.88

a In each case, cells were taken from the late log phase of growth. Abbreviations: NAD, nicotinamide adeninedinucleotide; NADP, NAD phosphate.

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HETEROTROPHIC METABOLISM OF T. FERROOXIDANS

TIME (mn)FIG. 1. Pyruvate formation from 6-phosphoglu-

conate plotted against time. Pseudomonas aeruginosaserved as a positive enzyme control; extracts were pre-pared from cells grown in a glucose-casein hydrolysatemedium (6), harvested by centrifugation in the cold,washed twice with phosphate buffer, and then treatedas described for Thiobacillus ferrooxidans. Cell extractprotein (1.95 mg) was used in the assay. Protein (0.88)was present in the extract prepared from iron-glucose-grown cells, and 2.S2 mg of cell extract protein was

used in the assays for iron-grown cells.

sources were used for energy substrates (Table 2).Glucose-grown cells were readily transferred toseveral different organic substrates (19, 25)which supported good growth. However, thesecells showed altered activities for both G6PDand ED enzymes. Cells grown on sucrose or

fructose apparently do not use the ED pathway.Exactly how these various sugars are metabo-lized by T.ferrooxidans remains to be determined.The lower ED activity in gluconate-grown cellsprobably reflects the decreased rate of growthwhen gluconate is used as the carbon and energysource (25).Enzymes of the tricarboxylic acid cycle. Five

representative enzymes of the tricarboxylic acidcycle, plus NADH oxidase, were assayed in cellextracts prepared from iron-grown (autotrophic),iron-glucose-grown (mixotrophic), and glucose-

TABLE 2. Effect oforganic substrate on the activity ofglucose-6-phosphate dehydrogenase and the Entner-

Doudoroffenzymes

Relative enzyme activityb

Growth substratea Glucose-6- Entner-phosphate Doudoroff

dehydrogenase enzymesc

Glucose ................ 1.00 1.00Sucrose ................ 1.08 0.06Fructose ............... 0.41 0.08Glutamate ............. 0.20 0.41Gluconate .............. 0.25 0.46

aAll organic substratesconcentration.

were added at 0.5% final

b Enzyme activity is compared to the levels found inglucose cells.

6-Phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase.

grown (heterotrophic) cells. Enzyme activitieswere generally lower in extracts from iron-growncells (Table 3); succinic dehydrogenase, a-keto-glutarate dehydrogenase, and NADH oxidasewere not detected in these extracts. A traceamount of succinic dehydrogenase was previouslyreported in iron-grown cells, but these workershad used a different enzyme assay (1). NADP-linked isocitrate dehydrogenase and aconitasewere very low, whereas NAD-linked isocitratedehydrognease and fumarase levels were high.The oxidation of glucose (iron-glucose growncells) induces succinic dehydrogenase, a-keto-glutarate dehydrogenase, and NADH oxidaseand greatly stimulates NADP-linked isocitratedehydrogenase, fumarase, and aconitase. NAD-linked isocitrate dehydrogenase levels remainedunchanged in the presence of glucose. This sameenzyme pattern was expressed in extracts pre-pared from cells grown solely on glucose; the tri-carboxylic acid cycle enzymes were operative andNADH oxidase activity was readily apparent.Radiorespirometric studies. To better under-

stand which pathway(s) was involved in glucosedissimilation, cells grown on the 9KG mediumand the glucose-salts medium were placed in ra-diorespirometer vessels, and CO2 formation wasobserved by using 14C-specifically labeled glu-cose. The observed labeling patterns indicate thenature of the pathways involved (29). Results ofthese studies with iron-glucose-grown cells aregiven in Fig. 2. A very active decarboxylationoccurred at the C-1 position, and a peak respira-tion rate was reached within 30 min. The rate ofCO2 evolution of C4-labeled glucose (deter-mined by extrapolation) was higher than from C-3-labeled glucose; C-3 was respired less rapidly,with peak evolution of respiratory CO, occurring

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338 TABITA AND LUNDGREN J. BACTERIOL.

TABLE 3. Activity of tricarboxylic acid cycle enzymes and reduced nicotinamide adenine dinucleotide oxidase incell extracts of Thiobacillusferrooxidans grown on various mediaa

Activity (10-' units/mg)Enzyme Iron-grown Iron-glucose- Glucose-grown

cells grown cells cells

Isocitrate dehydrogenase (NADP) ....... ................ 0.65 55.4 61.5Isocitrate dehydrogenase (NAD) ........ ................ 33.3 34.7 27.3Aconitase ............................................ 2.30 1050 1690Succinic dehydrogenase ............ .................... <0.19 54.3 179a-Ketoglutarate dehydrogenase.......................... <0.19 30.4 9.7Fumarase ............................................ 33.0 982 456NADH oxidase....................................... <0.65 17.0 30.4

a In each case, cells were taken at the late log phase of growth. Abbreviations: NAD, nicotinamide adenine di-nucleotide; NADP, NAD phosphate; NADH, reduced NAD.

after 90 min. The enzyme 2-keto-3-deoxy-6-phosphogluconate aldolase splits glucose betweencarbons 3 and 4; thus, carbons 1 and 4 will ap-pear as the carboxyl groups of the resulting pyru-vate molecules. If the Embden-Meyerhofpathway were operative, CO2 derived from C-3and C4 of glucose would appear before CO,derived from C-1. However, in T. ferrooxidansgrown in 9KG medium, apparently, about 37%of the glucose was dissimilated by the ED path-way, with a 63% contribution from the pentose-shunt mechanism. The high CO2 productionfrom C-6 was unexpected and suggests that thetriose phosphate produced from glucose dissimi-

01

'4.

lation via the ED scheme, and comprising glu-cose carbon atoms 4, 5, and 6, may be metabo-lized in an unorthodox manner. Controls wererun with E. coli, and these gave normal radiores-pirometric patterns, indicating that the "4C-la-beled glucose compounds were authentic and thatthe radiorespirometric procedure was standard.Table 4 shows the analysis of the cumulative"4CO2 recovery from glucose and is the basis forthe calculation of the relative contribution of EDand pentose-phosphate pathways.A similar radioactive inventory was performed

on glucose-grown cells (Table 5). In this me-dium, the ED pathway contributes 77% to glu-

9E0120 10 160306 INUTES

- GLUCOSE-1- C.....GLUCOSE-2jC--O OWCOSE-3-1C.-.-x GWCOSE-3.4-1C----o GLUCOSE-6-*C

60 90 120 150 180

MINUTESFIG. 2. Radiorespirometric pattern for the utilization of specific 14C-labeled glucose by iron-glucose-grown

cells of Thiobacillus ferrooxidans. Cells [19.2 mg (dry weight)] were suspended in the basal salts solution makingup the iron-containing medium (pH 2.5).

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TABLE 4. Utilization of "4C-labeled glucose by iron-glucose-grown Thiobaillus ferrooxidansa

Radioactive distribution"Total

Substrate . .Respi- Super- l 4Cratory natant Cells recoveryCO2 fluid (%) (%)(%) (%)

Glucose-1-14C ..... 89.0 1.5 5.6 96.1Glucose-2-14C ..... 15.2 8.7 57.5 81.4Glucose-3-"4C ..... 6.1 3.8 81.0 90.9Glucose-3,4-14C ... 31.7 1.2 60.9 93.8Glucose-6-14C 37.3 4.0 46.4 87.7

a A 19.2-mg amount (dry weight) of cells was addedto 20 ml of the salts of the (9K) medium.

b Counts collected at the end of the experiment.

cose catabolism; the pentose-phosphate shuntwas responsible for 23% of the glucose dissimi-lated.

If glucose is metabolized through the ED path-way, the triose phosphate formed must be di-verted into hexose phosphate. The hexose phos-phate is then degraded by the pentose cycle (11).If this explanation were correct, "4CO, recoveredwould be high from carbon 1 and significant fromcarbon 6. The per cent "4CO2 recovered (Table 4and 5) substantiates that carbon 1 was indeedevolved at a high rate, and CO2 evolved fromcarbon 6 was substantial but evolved at a lowerlevel. This glucose metabolism scheme wouldalso necessitate that carbon 2 be retained as ace-tate, possibly leading to the synthesis of PHB, astorage product known to accumulate in thisorganism (29).

DISCUSSIONThe iron-oxidizing bacteria are unusual among

the chemolithotrophs in that they are capable ofoxidizing two dissimilar inorganic substrates forenergy-reduced iron and sulfur compounds.Cells grown on ferrous iron are able to adapt tosimple organic compounds such as glucose (19,25). The change in growth substrates causes aprofound effect on both iron oxidation as well asCO, fixation; the latter was tested by deter-mining repression of the enzyme ribulose-1,5-diphosphate carboxylase (25). The effect of or-ganic substrates on the oxidative machinery in-volved in sulfur oxidation is not as pronounced,even at high glucose concentrations (21); how-ever, these effects have not been extensivelystudied.Growth on glucose affords the cell an alternate

mechanism for energy generation through a formof heterotrophic metabolism. From the presentresults, based upon enzyme studies and "4C-la-beling data, the Embden-Meyerhof pathway does

TABLE 5. Utilization of "4C-labeled glucose ofglucose-grown Thiobacillusferrooxidansa

Radioactivity distribution"Total

Substrate Respi- Super- 14Cratory natant Cells recoveryCO2 fluid (%) (%)(%) (%)

Glucose-l-"IC ..... 42.4 54.9 1.1 98.4Glucose-2-"4C ..... 20.2 51.7 12.1 84.0Glucose-3,4-14C ... 18.7 75.8 3.3 98.8Glucose-6-14C ..... 17.5 50.8 10.1 78.4

a A 53.2-mg amount (dry weight) of cells was addedto 20 ml of the salts of the 9K medium.

b Counts collected at the end of the experiment.

not appear to be the major route of glucose dis-similation in T. ferrooxidans. Instead, glucose isdissimilated through the ED pathway involvingG6PD, 6-phosphogluconate dehydrase, and 2-keto-3-deoxy-6-phosphogluconate aldol,.,e orthrough the pentose-phosphate shunt. TheEmbden-Meyerhof pathway probably plays aminor role in providing carbon skeletons for bio-synthesis. Thus, this chemolithotroph whengrown solely on glucose is similar to T. interme-dius (15), Hydrogenomonas eutropha (12), andHydrogenomonas H 16 (8) in using the EDscheme for glucose dissimilation.

Pyruvate and 3-phosphoglyceraldehyde are theproducts of glucose dissimilation through the EDpathway. For energy to be derived from thebreakdown, these products must be channeledthrough the tricarboxylic acid cycle. In thioba-cilli grown on glucose, an increase in activity oftricarboxylic cycle enzymes was noted. It hasbeen suggested that the tricarboxylic acid cyclemerely serves a biosynthetic function in che-moautotrophs (24), since there is an apparentabsence of a-ketoglutarate dehydrogenase andlow levels of succinic dehydrogenase. A lack ofNADH oxidase was also reported and given asthe reason for the inability of the complete tri-carboxylic cycle to function (24). Several au-thors, however, have found NADH oxidasepresent in extracts from several chemolithotrophs(5, 15, 23, 27). From results of these studies, T.ferrooxidans appears to have an incomplete tri-carboxylic cycle when grown autotrophically aswell as an apparent absence of NADH oxidase.However, when grown heterotrophically (on glu-cose) the tricarboxylic cycle is functional, andthe cells exhibit an increase in NADP-linkedisocitrate dehydrogenase, aconitase, fumarase,succinic dehydrogenase, and a-ketoglutaratedehydrogenase. NADH oxidase was also readilydetected in glucose-grown cells.The presence of two isocitrate dehydrogenases

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(NAD- and NADP-linked) may have importantregulatory functions for this organism. T. inter-medius, which grows autotrophically on thiosul-fate (rather than iron) also grows heterotroph-ically on complex media (supplemented with glu-cose); this organism also has two isocitrate dehy-drogenases (15). Evidence is accumulating sup-porting the idea that the tricarboxylic cycle inchemolithotrophic bacteria is incomplete andfunctions as a biosynthetic mechanism when au-totrophic conditions exist. The presence or ab-sence of NADH oxidase in autotrophicallygrown cells probably reflects a function of theregulation of its activity (16).

Labeling experiments with "4C-specifically la-beled glucose show an early release of C-l- andC4-labeled C02, indicating that the ED pathway

I H§O2 H OH3 HOqH _

5 H6_

is operating. In these experiments, CO2 from C-6-labeled glucose was released earlier than respi-ratory CO2 from C-3-labeled glucose. This wasunexpected based upon established dissimilationschemes for bacteria (29). Figure 3 represents ascheme which could account for the way thatglucose is dissimilated in T. ferrooxidans. Oneexplanation for high C-6 evolution is the factthat all autotrophic organisms, both photosyn-thetic and chemosynthetic, form fructose-I , 6-diphosphate by joining one molecule of 3-phosphoglyceraldehyde and one molecule ofdihydroxyacetone phosphate, essentially a re-versal of the aldolase step of the Embden-Mey-erhof pathway (4). In addition, autotrophs usephosphoglyceric acid kinase and phosphoglyc-eraldehyde dehydrogenase in the reverse direc-

FIG. 3. Proposed pathway for glucose dissimilation in Thiobacillus ferrooxidans. Numbers refer to position ofcarbon atoms in glucose. Abbreviations: G-6-P, glucose-6-phosphate; 6-PG, 6-phosphogluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; OAA, oxaloacetate; 3-PGAL, 3-phosphoglyceraldehyde; DAP, dihydroxyacetonephosphate; F-i ,6-diP, fructose-1 ,6-diphosphate; F-6-P, fructose-6-phosphate; PHB, poly-,j-hydroxybutyrate; PP,pentose phosphate; Ac CoA, acetyl-CoA; aKG, a-ketoglutarate; P,1 inorganic phosphate; A TP, adenosine tri-phosphate; NAD, nicotinamide adenine dinucleotide (oxidized); NADH, reduced nicotinamide adenine dinucleo-tide; NADP, nicotinamide adenine dinucleotide phosphate (oxidized); NADPH, reduced nicotinamide adeninedinucleotide phosphate.

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VOL. 108, 1971 HETEROTROPHIC METABOLISM OF T. FERROOXIDANS

tion (4). Thus, in the case at hand, when phos-phoglyceraldehyde is formed through the EDpathway, the organism is able to isomerize thisintermediate to dihydroxyacetone phosphate(Fig. 3); triose-phosphate isomerase has been de-tected ia T. ferrooxidans (1). Aldolase then cata-lyzes the joining of these two molecules into amolecule of fructose-1,6-diphosphate; carbon 6from 3-phosphoglyceraldehyde is now the numberone carbon atom of fructose diphosphate. Fruc-tose diphosphate can be easily converted to fruc-tose-6-phosphate and then to glucose-6-phos-phate by means of fructose diphosphatase andphosphoglucose isomerase. Phosphoglucose isom-erase has been detected in extracts of T. ferro-oxidans (1). Glucose-6-phosphate then recyclesthrough the pentose-phosphate pathway withthe subsequent liberation of CO2 from C-6 ofglucose.

Glucose is dissimilated primarily (80%)through the ED scheme when T. ferrooxidans isgrown on glucose. The presence of the auto-trophic substrate iron (iron-glucose cells) ap-pears to repress the ED pathway enzymes, sincethis pathway accounts for only 40% of the glu-cose dissimilated in these cells. T. ferrooxidans isthus similar to Hydrogenomonas H-16 (8) andT. intermedius (15) in that the ED pathway isrepressed by the chemolithotrophic energy source.One of the interesting observations found was

the formation of large masses of PHB granulesin cells of T. ferrooxidans grown on glucose (28).The synthesis of this storage polymer requiresNADH. NADH is readily available throughG6PD, and isocitrate dehydrogenase (NADlinked) activity. NADH availability for possiblePHB synthesis is also shown in Fig. 3. NADP-linked isocitrate dehydrogenase and G6PDsupply needed NADPH for fatty acid synthesis.Preliminary results from this laboratory indicatethat the isocitrate dehydrogenases are two dis-tinct enzymes, since the NAD- and NADP-linked activities vary to different degrees de-pending upon how the cells are grown. The ratioof NADP-NAD-linked activity for G6PD is thesame regardless of how the cells are grown, thusindicating there is only one protein catalyzingthis activity. This subject is treated in a separatepaper (26).

ACKNOWLEDGMENTSWe acknowledge the many helpful discussions with J. R.

Vestal regarding the radiorespirometric experiments. The radi-orespirometric studies were done by J. J. Perry, Department ofMicrobiology, North Carolina State University, Raleigh, N.C.;his contribution is appreciated. This work was supported bygrant 14010 DAY, from the Federal Water Quality Adminis-tration, U.S. Department of the Interior.

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