The Metabolism of Organic Acids by Marine Pennate · PDF filePreparation ofCell-free Extracts. ... sodium acetate in 0.01% (v/v) Triton X-100 and treated as ... (DCPIP)2 oxidoreductase

Plant Physiol. (1972) 50, 1-6

The Metabolism of Organic Acids by a Marine Pennate Diatom1Received for publication July 30, 1971

KEITH E. COOKSEYRosenstiel School of Marine and Atmospheric Science, Division of Functional Biology, University of Miami,Miami, Florida 33149

ABSTRACT

Cocconeis diminuta, a marine benthic diatom, metabolizesacetate and lactate-'4C. In the light, the major product was lipid,whereas in the dark, C02 was the major product. Analysis ofproteins synthesized in the presence of acetate or lactate showedthat radioactivity was incorporated predominantly into the glu-tamate family of amino acids and those amino acids relateddirectly to the substrate. Light and dark assimilation of sub-strate was inhibited slightly by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea and 2 ,4-dinitrophenol. 3-(3',4'-Dichlorophenyl)-1, 1-dimethylurea caused a pattern of metabolism of acetate inthe light characteristic of that which occurs in the dark. Mono-fluoroacetic acid inhibited assimilation considerably in thedark, but less in the light. The level of enzymes of the tricar-boxylic acid cycle and NADH-oxidase were found to be aboutthe same as those in other autotrophs. The metabolism of ace-tate and lactate is discussed in relation to the autotrophic modeof nutrition of Cocconeis diminuta.

The intermediary metabolism of marine diatoms is not welldocumented except for studies concerning their silicon metabo-lism (6) and their carbon dioxide fixation reactions (3). Growthconditions for diatoms are, however, well known. Danforth(5) has discussed the nutritional diversity shown by diatoms.Diatoms have been cultured that are heterotrophic, mixo-trophic, and obligately autotrophic (5). Lewin (16) found evi-dence that this latter group could not couple the energy of ace-tate oxidation to biosynthesis. However, Danforth (5) suggestsobligate phototrophy may have different causes in differentorganisms. The basis of obligate autotrophy in bacteria andblue-green algae has been the subject of much controversy over

the last few years. The metabolism of acetate by freshwatergreen algae has also been well documented (30, 33); however,there are few reports of the intermediary metabolism of auto-trophic diatoms in relation to their inability to grow in thedark as heterotrophs. A diatom isolated by Bunt, Cocconeisdiminuta, assimilated acetate, lactate, and other compounds,but was incapable of heterotrophic growth (2). This paper pro-vides biochemical information on the heterotrophic capabilityof the diatom and attempts to explain why it cannot grow inthe dark.

'This work was supported by Atomic Energy Commission Con-tract AT-(40-1)-3795 to Dr. John S. Bunt and National ScienceFoundation Grant GB-31102 to Dr. Keith E. Cooksey. It is Contri-bution 1497 of the Rosenstiel School of Marine and AtmosphericScience, University of Miami, Miami, Fla. 33149.

MATERIALS AND METHODS

Growth of the Organism. The diatom used in this study,Cocconeis diminuta, was isolated by Bunt (2) and has beendescribed by Taylor (31). It was maintained in DC-C medium(2) on slopes, or in liquid medium in tubes illuminated withnatural light. Stock cultures were checked for heterotrophiccontaminants before each experiment by inoculating intonutrient broth (1% [w/v] in DC-C medium). Cells for experi-mental purposes were grown in DC-C medium in several ways.They were grown in a 2.5-cm layer of medium in a 2.8 literFernbach flask without aeration, in a 10-cm layer of mediumaerated at 0.4 volume of air per volume of medium per min,and in a Vibromix fermenter aerated at 0.2 volume of air pervolume medium per min (Chemapec Inc., Hoboken, N.J.). Cellsgrown in these different ways were indistinguishable in theexperiments described here. The incubation temperature was30 C and illumination was 500 ft-c from cool white fluorescenttubes. Cultures were harvested at 2000g for 10 min at 25 C forassimilation experiments, and at 0 C for enzymic preparations.

Preparation of Cell-free Extracts. Cells were washed with 10to 50 mm phosphate buffer, pH 7.0 or 20 mm tris-HCl, pH 8.0containing 1 mm Mg2` and then resuspended in the same bufferat a concentration of 5 X 108 cells/ml. They were broken bytreatment at 12,000 lb/in2 in a French press (20). The brokencell homogenate was centrifuged at 10,000g for 10 min, andthe supernatant liquid was used for enzyme assays.

Determination of Labeled Products. A thin end windowplanchet counter was used for nonvolatile samples. Radioactiveamino acids separated on paper chromatograms were detectedby radioautography and the areas excised. They were countedfor at least 2000 counts. Volatile samples were counted in aliquid scintillation counter using a solution containing toluene,666 ml; Triton X-100, 334 ml; PPO, 4 g and POPOP, 0.3 gper liter.

Fractionation of Labeled Products. Labeled cells were frac-tionated by the method of Roberts et al. (25). Two dimensionalpaper chromatography was performed on Whatman No. 3MMpaper using 1-butanol-acetic acid-water (12:3:5, v/v) and 80%(w/v) aqueous phenol-ammonium hydroxide (199: 1, v/v).Thin-layer chromatography was performed on ce0lulose plates(Merck, A. G.-Brinkmann Instruments, N.Y.) using 1-butanol-acetic acid-water (12:3:5, v/v) and 1-butanol-acetone-diethyl-amine-water (10:10:2:5, v/v). Autoradiographs were madewith Kodak No-screen x-ray film.

Assimilation Experiments. Cells were suspended in DC-Cmedium, pH 7.6, usually at a concentration of approximately5 X 10' cells/ml (about 100 [kg protein/ml). Of this suspension1.8 ml was placed in the main compartment of a Warburgflask. Inhibitors were added to the cell suspension whennecessary and 0.1 ml 10% (w/v) NaOH was added to thecenter well. The flasks were closed with serum caps and pre-incubated under the conditions of the experiment for 30 to 60

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Plant Physiol. Vol. 50, 1972

Table I. Metabolism of Acetate, Lactate, and GlucoseCells were incubated for 3 hr at 30 C and 500 ft-c, or in the dark.

Incubations contained 44 X 106 cells, 1 ,4c 14C and approximately1 ,umole substrate. Figures are means of two determinationscorrected for blank values.

RadioactivitySubstrate Illumination setabolized

14CO., Cells

dpnt nmoles

Acetate-2-14C Light 2,890 87,500 33.5Dark 15,600 61,000 29.4

L-Lactate-U-14C Light 685 19,200 9.6Dark 18,300 7,350 12.4

Glucose-U-'4C Light 0 640 0.3Dark 585 260 0.4

min. Substrate was added in 0.1 ml and the flasks were incu-bated for an appropriate time at 30 C in the dark or at 400to 500 ft-c. If "4CO2 was to be measured, assimilation was

stopped by the addition of 0.2 ml 50% (w/v) trichloroaceticacid. After 25 min, 0.05 ml of the center well contents were

removed and counted. The cells were tipped into 10 ml of10 mM-carrier substrate in 0.01% (v/v) Triton X-100 containedin a Millipore filtration assembly, fitted with a 2-cm HAWPfilter (Millipore Corporation, Bedford, Mass.). The filter was

washed twice with 5 ml of the same solution, 5 ml of water,dried, and counted. When '5C02 was not measured, the additionof trichloroacetic acid was omitted.

In some experiments, labeled acetate or lactate was addedto growing cultures of the diatom in the logarithmic phase ofits growth. The cultures were incubated for 24 hr before thecells were harvested and fractionated.To measure the uptake of acetate-2"4C in the absence of CO2,

air washed twice with 10% (w/v) NaOH was bubbled throughsaturated Ba(OH)2 solution and then through a cell suspensionof C. diminuta. A control with unwashed air was run at thesame aeration rate of 1 volume of gas per volume medium per

min. After 1 hour of incubation to remove any bicarbonatethat had not been removed from the cell suspending mediumbefore the experiment, radioactive acetate was added. Samplesremoved at convenient times were pipetted into ice-cold 10 mMsodium acetate in 0.01% (v/v) Triton X-100 and treated as

described above. Aseptic precautions were taken in all assimi-lation experiments. The absence of heterotrophic bacteriafrom the initial cultures was demonstrated by pipetting 1 mlinto 5 ml of 1% (w/v) nutrient broth in DC-C medium andincubating for one week. When growth of bacteria occurred inthe broth, the results of the experiment were discarded.Enzyme Assays. The following enzymes were measured by

the methods indicated: citrate synthase (29); isocitrate:NADPoxidoreductase (21); malate:NAD oxidoreductase (7); succi-nate: dichlorophenolindophenol (DCPIP)2 oxidoreductase (13);a-ketoglutarate: NAD oxidoreductase (24); orthophosphate:oxalacetate carboxylyase (P-enolpyruvate carboxylase) (1, 14);and P-enolpyruvate synthetase (4). Pyruvate:CO2 ligase was

measured by a similar method to that used for P-enolpyruvatecarboxylase, but P-enolpyruvate was replaced by pyruvate, andacetyl CoA by 1.5 mm ATP. Reduced pyridine nucleotide oxi-dase was measured by the method of Smith et al. (28), but witha substrate concentration of 50 ytM instead of 66 btM. Spectro-

'Abbreviations: DCPIP: 2. 6-dichlorophenol-indophenol; DNP:2, 4-dinitrophenol.

photometric assays were performed at 30 C in a split beamspectrophotometer fitted with scale expansion so that changesin absorbance of 0.0005 per min could be measured. Anaerobicassays were made in Thunberg cuvets evacuated and flushedwith prepurified nitrogen. All values of enzyme activitiesquoted are the means of at least two determinations. The ac-tivity measured was proportional to the protein concentrationin the cuvets.

Protein Determination. Soluble protein was determined bythe method of Lowry et al. (18) and referred to a standard ofbovine plasma albumin. Protein in whole cells was determinedin the same manner after digestion at 90 C for 20 min in 0.5N NaOH.

Chemicals. Biochemicals were obtained from Sigma Chemi-cal Company; acetate-2-11C, acetate-i-"4C, glucose-U-'C, andlactate-U-14C were obtained from Amersham-Searle, andNaH25CO2 from New England Nuclear Corporation. All otherchemicals were reagent grade.

RESULTS

Conditions for the Assimilation of Acetate and Lactate. Cellsin the logarthmic state of growth were used for these experi-ments. Table I shows that acetate and lactate, but not glucose,were assimilated and oxidized. The total substrate metabolizedwas similar in the light and dark. The uptake mechanism foracetate became saturated at 10 mm acetate (Fig. 1). Assimila-tion was linear with time (Fig. 2) and was not dependent on anexogenous supply of assimilable nitrogen. DC-C medium with-out NaNO3 (0.5 g per 1) was used to wash and resuspend dia-tom cells for assimilation experiments. Incorporation of acetateinto cell structure or its oxidation to CO2 was not significantlyreduced in the absence of nitrate. The uptake of acetate was,however, inhibited in the absence of CO2 (Fig. 2).

Fractionation of Labeled Cells. Cells labeled by the assimi-lation of acetate-2-'4C or L-lactate-U-'4C were fractionated bythe method of Roberts et al. (25). The designation of the iden-tity of the various fractions was taken from the work ofRoberts et al. (25) who studied Escherichia coli (Table II).Darkness increased the fraction of the cell that was rapidlyturning over. This would be expected as stimulation of respira-tion by darkness was demonstrated in this alga (Table I). Oneother difference between light and dark incubated cells wasthat less lipid synthesis took place in the dark.The protein residue referred to in Table II was hydrolyzed

for 24 hr at 110 C in 6 N HCl and the products were examinedby paper and thin-layer chromatography. Table III shows theresults from cells which had been labeled for 4 hr with ace-tate-2-'4C, lactate-U-14C in the light or dark, and NaH14CO3 inthe light. The labeling of the cells from NaH'5CO2 should begeneral and thus the radioactivity in the proteins should re-flect their amino acid composition. In most cases the labelingof the amino acids from radioactive lactate and acetate didnot match that from NaH'5CO. Furthermore, amino acidslabeled in light and dark were not the same for a single radio-active substrate. For example, the basic amino acids werelabeled far less from acetate and lactate in the dark than theywere in the light. Cells incubated for 24 hr with radioactiveacetate or lactate showed the same amino acid labeling patternas cells incubated for 4 hr. After rechromatography of the iso-leucine-leucine spot in two dimensions on a cellulose thin layerplate, the majority of the radioactivity was found in leucine.

Comparison of the Metabolism of 1- and 2-24C Acetate.Both 1- and 2-'4C-labeled acetate were assimilated into cellcontents and oxidized to '4CO2; however, acetate-1-'4C gaverise to more '4CO2 in the dark than acetate-2-'5C (Fig. 3). Theopposite was true of the assimilation into cell structure in the

2 COOKSEY




201

U)15

z

1< 5

5--D

METABOLISM OF ORGANIC ACIDS BY A DIATOM

LIGHT

DARK

0

5 I0CONCENT7MTATION OF ACETATE (mNl)

FIG. 1. Assimilation of acetate-2-'4C by Cocconeis diminuta at differing acetate concentrations. Cells were incubated in DC-C medium con-taining acetate at the concentrations shown on the abscissa and 1 uc acetate-2-14C. The ordinate is the product of radioactivity in the cells after3 hr and acetate concentration.

15

b 12-

0x

(9

0U)-JLLH 6-

z2

a-(9 3-

-o,

_-

100TIME IN MINUTES

CELLS -LIGHT: Ac-2-l4CCELLS- LIGHT: Ac- -'4C14C02 -DARK: Ac-I-'4C

CELLS-DARK: Ac-2-'4C

'4C°2 -DARK: Ac-2-'4C

CELLS-DARK: Ac- I-'4C

200

FIG. 3. Metabolism of acetate-2-'4C compared with acetate-1-"4C.Cocconeis diminuta was incubated with specifically labeled acetates.Incorporation into cell structure and production of '4CO2 was meas-ured. Incubations contained 5 mM acetate and 2 Ac acetate-"4C. Aset of flasks was incubated in the light and another in the dark.Metabolism was stopped at the times shown by the addition of 5%trichloroacetic acid.

00 200TIME IN MINUTES

300

FIG. 2. Assimilation of acetate in the absence of CO2. Twosamples of cells were suspended in bicarbonate-free DC-C medium(15 ml). The flasks containing the suspensions were attached to agassing manifold and sterile COfree nitrogen was passed throughthe system to remove all air. Sterile air (control, 0) or CO,-freeair (test, 0) was then passed through the illuminated suspensionfor a period of 1 hr to remove any remaining bicarbonate from thesystem. Acetate (150 /cmoles and 7.3 Ac) was added to both thecontrol and test flask and samples were removed at the times indi-cated. The samples were treated as described in the text.

dark. In the light, no 14CO, production from either form ofacetate was detected, nor was there any significant differencebetween the quantities of acetate assimilated.

Experiments with Inhibitors. Table IV shows that 10 mmmonofluoroacetate inhibits the incorporation of acetate bothinto cell structure and "4CO2. These figures also indicate thatthe inhibition of incorporation into whole cells is proportional

Table II. Fractiontation of Labeled CellsCells of C. diminuta were labeled with acetate-2-14C or lactate-

U-14C and fractionated by the method of Roberts et al. (25). Theidentity of fractions is also based on that of Roberts et al.

Acetate-2-'4C Lactate-U-14CCell Fraction

Light Dark Light Dark

dpm

Soluble fraction 19,200 61,600 7,370 9,340Lipids 151,000 40,400 29,200 5,780Nucleic acids 3,560 3,920 820 820Protein and insolu- 67,300 57,400 40,100 19,870

ble matter

to the logarithm of the monofluoroacetate concentration.DCMU is thought to prevent noncyclic photophosphorylationin green plants and algae (17). Thus, assimilation dependent

3

i5 20

15-

U)

-LJ

2

0L

5-




Table III. Amino Acids from Lactate-U-14C, Acetate-2-t4C, andNaH'4CO3 Labeled Cells

Cell protein residues prepared by the method of Roberts et al.(25), were hydrolyzed in 6 N HCl for 24 hr at 110 C. The aminoacids were separated by two-dimensional chromatography, de-tected by radioautography, and counted. At least 2000 disintegra-tions were counted for each amino acid. Cysteine and tryptophanwere not determined.

Amino Acid

AlanineGlutamateAspartateProlineMethionine/valineIsoleucine, leucine,phenylalanine

Histidine, lysine, argi-nine

ThreonineGlycineTyrosineSerineUnidentified com-pounds

Radioactivity in Amino Acids

Acetate Lactate NaHl4CO3

Light Dark Light Dark Light

1303,0904051703,6

3,560

00000

1,

170,500565285100,530

1100o400

dp,n

170875245150185475

1),3300

000

0

11545085450

110

20

00000

6,2308,9007,2005,68010,50014,200

12,250

3,0603,4904,650

70010,850

Table IV. Effect of Moniofluoroacetate oni the Metabolismof Acetate-2-54C

Cells were incubated in the presence of different concentrationsof monofluoroacetate and acetate-2-'4C (4,c, 11.4 ,moles) for3 hr. Assimilation and oxidation of acetate was measured. Oxida-tion in the light was not detected.

Assimilation into Cells Oxidation to 14CO2Concentration ofMonofluoroacetate

Light Dark Dark

}?z.51 dpmn0 12,500 6,300 2,8000.5 14,800 5,290 3,0205 10,750 2,430 2,67010 8,900 2,030 1,040

on ATP produced by this pathway would be reduced in thepresence of DCMU. It was found that 5 [kM DCMU reducedassimilation in the light but did not prevent it completely(Table V). Light respiration was increased to the normal darklevel but dark respiration in the presence of DCMU was un-altered. Assimilation in the dark was only slightly affected byDCMU.

In cells permeable to DNP, assimilatory processes whichare dependent on oxidative phosphorylation as a source ofenergy are likely to be inhibited by 50 [kM DNP (26). Howeverphotophosphorylation is usually not affected by this concentra-tion (17). Respiration in the light was not increased by 50 bcMdinitrophenol but respiration in the dark was increased slightly.Assimilation in both light and dark was diminished. When cellswere treated with both DCMU and dinitrophenol at the sametime, assimilation was considerably reduced over the control

level and also over the level of assimilation found in the indi-vidual presence of the inhibitors.Enzyme Assays. The inability of C. diminuta to grow in the

dark may be a result of a deficient carbon or energy metabo-lism. To investigate possible enzymic lesions, the enzymesshown in Table VI were assayed. Most of them were measureddirectly in crude extracts, but NADH-oxidase was assayedspectrophotometrically after dialysis against 20 mm phosphatebuffer, pH 7.0, containing 1 mm Mg'+. a-Ketoglutarate de-hydrogenase, an enzyme postulated to be redundant in anautotroph (28), was present. P-enolpyruvate carboxylase wasdetected, but not pyruvate carboxylase or P-enolpyruvate syn-thetase.

Dialyzed cell extracts catalyzed the removal of NADH fromthe experimental cuvets as shown by the fall in extinction at340 nm. This fall was dependent on the presence of oxygenso that we may refer to the activity as an NADH-oxidase.

DISCUSSION

C. diminuta metabolized acetate and lactate both in thelight and dark although the organism is incapable of hetero-trophic growth. Photoassimilation of acetate was not depend-ent on an assimilable nitrogen source in a short term experi-

Table V. Effect ofDCMU anld DNP on the Metabolismof Acetate-2-14C

Cells were incubated with DCMU and/or DNP, and acetate-2-14C (19.1 ,umoles, 0.95,uc). Assimilation into cells, oxidation to14CO2, and quantity of acetate metabolized was measured.

14 ~~~AcetateCells tC02 MetabolizedAdditions _____________________

Light Dark Light Dark Light Dark

dprn nrnolesNone 3,440 2,805 0 3,270 28 605 ,uM DCMU 2,440 2,670 3,690 3,150 56 5350 ,uMDNP 2,840 2,130 0 3,900 26 555,uMDCMU + 1,230 990 2,900 3,460 38 41

50,M DNP

Table VI. Specific Activities of Enizymes in AutotrophicallyGrown Cells

Cell-free preparations of C. diminuta were made with the aid ofa French press (20) and enzyme activity was measured in the10,000 g supernatant solution. Figures are mean values from atleast two determinations.

Enzyme Specific Activity

Citrate synthaseIsocitrate: NADP oxidoreductaseSuccinate: DCPIP oxidoreductaseL-Malate: NAD oxidoreductasea-Ketoglutarate: NAD oxidoreductasea-Ketoglutarate: NAD oxidoreductase in

the presence of 0.2 mm NaCNOrthophosphate: oxalacetate carboxylase

(P-enolpyruvate carboxylase)NADH-oxidase:aerobicanaerobic

nmoles/1mg protein,s,in401619

239l 0

2

98

10

4 COOKSEY

JoJ JJ



METABOLISM OF ORGANIC ACIDS BY A DIATOM

ment. It is possible that the necessary nitrogen compoundscould have been derived from the cells themselves, as proteinturnover is known to be enhanced in starved algae (12). Car-bon dioxide was necessary, however, for efficient assimilationof acetate, which is in agreement with the results of Hoare etal. (10) and in contrast to those of Goulding and Merrett (9).

The fraction of the cell related to acetate which was turningover was much greater in the dark than in the light. The majorproduct in the light was lipid, whereas in the dark it was CO.,.If "CO2 from the oxidation of acetate-2-"C in the light wassubsequently fixed by photosynthesis, then the labeling patternof cell amino acids from acetate-"C could be expected to besimilar to that from NaH"CO,. This was not so (Table III),which indicated that the oxidation of methyl labeled acetate to4CO, in the light was insignificant. Goulding and Merrett (9)came to the same conclusion with Chlorella pyrenoidosa. Lipidsynthesis requires NADPH, which is produced by light drivennoncyclic electron transport. In the dark, NADPH is not pro-duced in this manner so that this result was predictable. Ingeneral, the amino acids labeled from acetate and lactate weresimilar, but the extent of labeling differed widely, especiallybetween light and dark. For instance, the label in the basicamino acids fell considerably in the dark. ATP is more heavilyinvolved in the biosynthesis of these amino acids than many ofthe others, so that the fall in labeling suggests that ATP wasnot available for their synthesis in the dark.

a-Ketoglutarate dehydrogenase was present in this organismin contrast to the situation in many other autotrophs (28).However, its low level probably accounts for the large propor-tion of the radioactivity found in the glutamate family ofamino acids. The absence of P-enolpyruvate synthetase andpyruvate carboxylase precludes labeling of aspartate from lac-tate-derived pyruvate. The specific activities of some of theother enzymes shown in Table VI were somewhat higher thanone would expect for an obligate autotroph (1 1, 22, 28): how-ever, the levels were not as high as those in facultative auto-trophs (19, 27).The process by which an organic compound enters a micro-

organism is often energy dependent. In Cocconeis, the uptakeof acetate was not entirely dependent on photophosphoryla-tion because there was considerable uptake in the dark. Incu-bation with DCMU in the light produced a dark type metabo-lism of acetate. As significant uptake and metabolism ofacetate occurred in the presence of both DCMU and DNP,it was unlikely that its uptake was an active process, unlessATP was provided by cyclic phosphorylation. The small inhi-bition of assimilation produced by DCMU and DNP; there-fore. is likely to be caused by a scarcity of ATP for thefurther metabolism of acetate, rather than for a transportmechanism. This situation is in contrast to that in the blue-greenalga, Anacystis nidlulans, in which acetate assimilation is pre-vented completely by DCMU or darkness (10).The amino acid labeling pattern from acetate-2-"C, the en-

zyme assays, the greater production of "CO2 from acetate-I -"Cthan from acetate-2-"C, and the inhibition of assimilationproduced by monofluoroacetate showed that the tricarboxylicacid cycle was responsible for the initial steps in the metabo-lism of acetate. Although 5 mM monofluoroacetate inhibitedassimilation slightly in the light, a concentration of 0.5 mMstimulated it. A possible explanation is that the metabolicallyformed fluorocitrate activated acetyl CoA carboxylase, theenzyme catalyzing the first step in fatty acid synthesis (32).

Recently, Smith et al. (28) suggested that obligate auto-trophy depended on an absence of NADH oxidase and thatthe lack of this system made the possession of a completetricarboxylic acid cvcle redundant. The subsequent loss of

5

a-ketoglutarate dehydrogenase was not detrimental to the cell.C. diminuta most likely has a complete tricarboxylic acid cycleand has an NADH-oxidase system. Several other autotrophshave NADH oxidases and in one, Anabaena variabilis (15),it has been shown to be coupled to a low level of ATP produc-tion. It was suggested (15) that this low level of oxidativephosphorylation, while being too low to provide the require-ment for growth, was enough to provide the "energy of main-tenance" referred to by Dawes and Ribbons (8). The oxidationof NADH needs to be examined more carefully before suchconclusions can be drawn concerning its role in C. diminuta.However, it seems possible that an inadequate system forcoupling substrate oxidation to ATP production is the reasonthat this organism cannot grow in the dark.

Acknowledgments-I thank my colleagues for their discussion of this work andBarbara Cooksey for technical and editorial assistance.

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2. BC-NT, J. S. 1969. Observations on photoheterotrophy in a inarine diatom. J.Phycol. 5: 37-42.

3. CHAPIA&N, G. AND A. C. RAE. 1969. Excretion of photosynthate by a bentlhicdiatom. MIar. Biol. New York 3: 341-351.

4. COOPER, R. A. AN-D H. L. KORNaBERG. 1969. Phosphoenolpyruvate synthase.I??: J. 'M. Lowenstein, ed., Methods in Enzymology, Vol. 13. AcademicPress, New York. pp. 309-314.

5. DA'NFORTH, W. F. 1962. Substrate assimilation and heterotrophy. In: R. A.Lewin, ed., Physiology and Biochemistry of the Algae. Academic Press,New York. pp. 99-123.

6. DARLEY, W. 'M. AN-D B. T. VOLCANI. 1969. Role of silicon in diatom metabo-lism: a silicon requirement for deoxyribonucleic acid synthesis in the diatomCylindrotheca fusiformis Reimann and Lewin. J. Exp. Cell Res. 58: 334-342.

7. DAVIES, D. D. 1969. Malate dehydrogenase from pea epicotyls. In: J. M.Lowenstein, ed., Methods in Enzymology, Vol. 13. Academic Press, New

York. pp. 148-150.8. DAWES, E. A. AN-D D. W. RIBBONS. 1962. The endogenous metabolism of micro-

organisms. Annu. Rev. Microbiol. 16: 241-264.9. GOULDING, K. H. AND M. J. MERRETT. 1166. The photometabolism of acetate

by Chlorella pyrenoidosa. J. Exp. Bot. 17: 678-689.10. HOARE, D. S., S. L. HOARE, AND R. B. MOORE. 1967. The photoassimilation of

organic compounds by autotrophic blue-green algae. J. Gen. MIicrobiol. 49:351-370.

11. HOOPER, A. B. 1969. Biochemical basis of obligate autotrophy in Nitrosomonaseuropaea. J. Bacteriol. 97: 776-779.

12. JOHN, P. C. L., C. F. THI;RSTON, AND P. J. SYRETT. 1970. Disappearance of iso-citrate lyase enzyme from cells of Chlorella pyrenoidosa. Biochem. J. 119:913-919.

13. JURTSHUK, P., A. K. _MAY, L. 'M. POPE, AND P. R. ASTON. 1969. Comparativestudies on succinate and terminal oxidase activity in microbial and mamma-lian electron-transport systems. Can. J. Microbiol. 15: 797-807.

14. LAN-E, M. D., H. MARUGAMA, AND R. L. EASTERDAY. 1969. Phosphoenolpyru-vate carboxylase from pea cotyledons. In: J. M. Lowenstein, ed., Meth-ods in Enzymolog-, Vol. 13. Academic Press, New York. pp. 277-283.

15. LEACH, C. K. AND N. G. CARR. 1970. Electron transport and oxidative phos-phorylation in the blue-green alga Anabaena variabilis. J. Gen. Mlicrobiol.64: 55-70.

16. LEwIN-, R. A. 1954. The utilization of acetate by wild type and mutantChlamydomronas dysosmos. J. Gen. MIicrobiol. 11: 459-471.

17. LoSODA, M. AN-D D. I. ARN-ON-. 1963. Selective inhibitors of photosynthesis.In: R. 'M. Hochster and J. H. Quastel, eds., Metabolic Inhibitors, Vol.2. Academic Press, New York. pp. 559-593.

18. LOWRY, 0. H., N. J. ROSEBROLTGH, A. L. FARR, AND R. J. RANDALL. 1951. Pro-tein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

19. MATIN, A. AND S. C. RITTEENBERG. 1970. Regulation of glucose metabolism inThiobacillus intermedius. J. Bacteriol. 97: 776-779.

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6 COOKSEY



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