JOURNAL OF BACTERIOLOGY, Oct. 1970, p. 473-481Copyright 0 1970 American Society for Microbiology

Vol. 104, No. 1Printed in U.S.A.

Respiratory Metabolism of a "Petite Negative" YeastSchizosaccharomyces pombe 972h-'

H. HESLOT, A. GOFFEAU, AND C. LOUIS

Institut National Agronomique, Chaires de Ge'netique et de Zoologie, Paris, Franice,and Euratom-UCL, Enzymology, Louvain University, Belgium

Received for publication 15 July 1970

The respiratory metabolism of Schizosaccharomyces pombe 972h-, a fission, hap-lontic, "petite negative" yeast, was studied. Glucose and glycerol are good growthsubstrates and are oxidized under appropriate conditions. L-Lactate, ethanol,malate, and succinate are oxidized but are poor substrates for growth. D-Lactateand pyruvate are neither oxidized nor used for growth. Limited growth was ob-served under anaerobic conditions. The addition of 0.3% KNO3 to a rich mediumrelieves the oxygen requirement. A continuous increase of cell respiration duringgrowth on repressive concentration of glucose was observed, suggesting thepresence of glucose repression of respiration. Reduced nicotinamide adenine dinu-cleotide (NADH), succinate, a-glycerophosphate, and ascorbate plus tetramethyl-p-phenylenediamine are oxidized by a mitochondrial fraction. NADH and suc-cinate oxidations are inhibited by antimycin A and NaCN but not by rotenone,suggesting the absence of the phosphorylation site I and the presence of sites II andIII. The effects of several mitochondrial inhibitors on growth and respiration indi-cate that the requirement of an oxidant for growth is related neither to the func-tioning of the respiratory electron transport chain nor to the formation of respira-tory energy. The previously suggested correlations between the nonviability ofvegetative "petites" mutants, the absence of repression of respiration by glucose, andthe incapacity to grow under anaerobic conditions are thus not strictly valid forS. pombe.

Most of the previous studies concerning yeastrespiration were carried out with Saccharomycescerevisiae or S. carlsbergensis. In these species,respiratory-deficient mutants with non-Men-delian segregation (rho minus or vegetative"petites") are obtained with high frequency byacriflavine and other treatments (9, 10).

Yeast species in which respiratory-deficientmutants are hard to isolate were called "petitenegative" by Bulder (4). It has been reportedthat "petite negative" species are characterizedby the absence of anaerobic growth (5) and bythe absence of glucose repression of respiration(7, 14).Schizosaccharomyces pombe is a fission hap-

lontic yeast, the genetics of which were recentlyreviewed by Leupold (13). We report in a com-panion paper that S. pombe 972h- is a "petitenegative" strain (11).The respiratory metabolism of S. pombe has

not been studied so far. In an attempt to obtaininformation on the mechanisms that control

1 Contribution no. 480 of the Euratom Biology Division.

the susceptibility towards cytoplasmic mutation,we characterized some physiological aspects ofthe respiratory metabolism of this species.

MATERIALS AND METHODSYeast strain. Cultures of S. pombe strain 972h-

were maintained aerobically at 30C on agar slantscontaining 0.5% (w/v) yeast extract (Difco), 3%(w/v) glucose, and 1.5% (w/v) agar (Difco). Theywere subcultured for 24 hr before inoculation.

Growth of aerobic cells. The medium contained 2%(w/v) yeast extract, 2% (w/v) peptone (Difco), and160 mm glucose, glycerol, or other substrate. Themedium was brought to pH 4.5 with HCl. Exceptwhen otherwise indicated, the cultures were inoculatedwith 106 cells per ml. They were vigorously shaken at30C.

Growth of anaerobic cells. Two different media wereutilized. YBG contained 2% (w/v) yeast extract, 2%(w/v) peptone, and 1% (w/v) glucose. When indi-indicated, 15 ml of a solution made of 250 mg ofergosterol, 25 ml of Tween 80, and 75 ml of ethanolwas added per liter of medium. The medium wasbrought to pH 4.5 with HCl.The other medium was modified according to Tusta-

nof and Bartley (27). It contained 1% yeast extract,

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HESLOT, GOFFEAU, AND LOUIS

0.5% acid-hydrolyzed casein (Difco), 0.9% KH2PO4,0.6% (NH4)2SO4, 0.3% KNO3 (when indicated),0.05% MgSO4-7H2O, 5 ml of Tween 80 per liter,0.15 ml of wheat germ oil per liter, and 5 ml of ethylalcohol per liter. The medium was brought to pH 4.5with HCI. Except when otherwise indicated, bothmedia were inoculated with 106 cells per ml ofmediumin stoppered flasks equipped with a mercury trapoutlet and a gas inlet through which purified nitrogenwas continuously flushed (Azote U, Air Liquide).The temperature was regulated by immersion of theculture jar in a water bath at 30 C. The culture wascontinuously stirred with a magnetic bar.

Growth curves. The growth was measured either bycell counts using a Thoma counting chamber or bydry weights of cells obtained by centrifugating samplesof the culture medium. The glucose concentration ofthe culture medium was measured by colorimetric,enzymatic determination with the use of a Sigma kit(22).

Respiration. Oxygen uptake of whole cells wasmeasured at 30 C with a Clark electrode in a 3-mlclosed lucite chamber. Respiration rates were ex-pressed as Q02 (microliters of 02 consumed per minuteper milligram of dry weight). When the effects ofrespiratory substrates were measured, the respirationmedium contained 0.1 M phthalate-NaOH buffer(pH 4.5) and the indicated substrate. The respiratoryquotient (RQ) is the ratio of QCO2 (microliters ofCO2 produced per minute per milligram of dry weight)to Q02 measured at 30 C by the Warburg directmethod (28). The vials contained 2.6 ml of 0.1 Msuccinate-NaOH buffer (pH 4.2), 5% glucose, and 1to 4 mg (dry weight) of cells.A mitochondrial fraction was prepared by the

Balcavage and Mattoon (2) technique. The cells weregrown until early stationary phase on 2% yeast ex-tract, 2% peptone, and 2.5% glycerol (pH 4.5). Themitochondrial oxygen uptake was measured polaro-graphically at 25 C in 3 ml of a medium containing0.60 M mannitol, 10 mm potassium phosphate buffer(pH 6.5), 3.3 mm substrates [except for ethanol (40mM) and tetramethyl-p-phenylenediamine (1 MM)],and about 5 mg of protein of the mitochondrialfraction. When indicated, 146 Mm adenosine diphos-phate (ADP) or 1.6 MM carbonyl-cyanide-phenylhy-drazone was added. Respiratory control is defined bythe ratio of respiration in the presence ofADP (state 3)to respiration in the absence of 146 pM ADP (state 4).

Absorption spectrum. The cells were grown for 4days at 30 C in petri dishes on the medium used for theagar slants. Sodium dithionite grains were added to apaste of blotted yeast cells. The absolute absorptionspectrum was measured through 1 mm of cell pasteat liquid nitrogen temperature with a Cary 15 spec-trophotometer with a modified light source and sam-ple holder.

Electron microscopy. Glutaraldehyde was added tothe culture medium at room temperature at a finalconcentration of 3%. The cells were washed withwater and then postfixed with 4% permanganate or1% osmium tetroxide. After inclusion into agar andquick dehydration with ethanol, the samples wereembedded in Epon. Sections were stained with uranyl-

acetate followed by lead citrate staining, and were thenexamined in a Hitachi HS7 or HU 11 E microscope.When indicated, unfixed cells were pretreated with1% of an unbuffered solution of snail gut enzyme(Suc d'Helix Pomatia-Industrie Biologique Fran-,aise) for 2 hr at room temperature.

RESULTS

Growth on fermentable and nonfermentablesubstrates. Figure 1 shows the kinetics of growthof S. pombe 972h- grown aerobically at pH 4.5in the presence of different substrates at 160mM. Growth on glucose was fast, the generationtime being 2.5 hr. Active growth was also ob-tained in the presence of glycerol with a genera-tion time of 4 hr. Ethanol and DL-malate werepoorly utilized. No significant growth wasachieved in the presence of pyruvate, acetate,DL-lactate, and glutamate. Changing the pHfrom 3.5 to 5.5 or lowering the concentrationdown to 20 mm did not improve growth on thesesubstrates.

Figure 2 shows that only limited growth wasobtained under anaerobic conditions on YBGmedium. After a lag of about 6 hr, only threedivisions were carried out, the generation timebeing 3 hr. In these conditions, yields higherthan 0.8 g of dry weight per liter of medium werenever obtained. Neither the presence of ergosteroland Tween nor an increase of the glucose con-centration up to 1.6 M (30%) improved theanaerobic growth. The rich medium (R) andsynthetic medium (Go) used by Sels et al. (24)for anaerobic growth of S. cerevisiae did notgive better results. However, when a Tustanoffand Bartley medium (27) supplemented with0.3 % KNO3 was inoculated with 20,000 cells/ml,more than seven anaerobic generations wereobserved (Table 1). Moreover, inoculation of afresh medium with previously anaerobicallygrown cells enables anaerobic growth to resume.Under these conditions, a total of at least 14anaerobic generations can be obtained.

Ultrastructure. Although the ultrastructure ofS. pombe has not been studied very much, severalfixation procedures have been described (20, 23).After several trials, we decided to use the doublefixations glutaraldehyde-potassium permanganateor glutaraldehyde-osmium tetroxide.

Figure 3 illustrates the ultrastructure of S.pombe 972h- cells under different conditions ofgrowth. Figure 3A shows aerobic cells in sta-tionary phase, fixed with glutaraldehyde andpotassium permanganate. Several structures canbe observed: cell walls, plasmic membranes,nucleus, vacuoles, endoplasmic reticulum, andmitochondria. At least two types of vacuolescan be distinguished; some have high electron

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METABOLISM OF S. POMBE

5

E

E

._1D

>1

05

0.1

nn0116 20 2. 28 32 36 /0

Hours after inoculation

FIG. 1. Aerobic growth of Schizosaccharomycespombe 972h- on 160 mM substrates. Inoculation wasat a concentration of 106 cells/ml, except for glucosewhich was inoculated at 105 cells/ml.

Hours after inoculation

FIG. 2. Anaerobic growth of Schizosaccharomycespombe 972h- on YBG medium.

density, others have a granular and less densecontent.

In Fig. 3B, the same cells were fixed withglutaraldehyde and osmium tetroxide. The cellwall was removed with snail gut enzymes. Underthese conditions, the membranous structures areless apparent than in Fig. 3A, but the ribosomesare better preserved. Typical mitochondria withtwo membranes and cristae are observed. Their

diameter varies from 0.35 to 0.6 ,tm and theirlength may reach 2 ,um. More than 12 mito-chondria can be found in the same cell section.Figures 3C and D show anaerobic cells grownon YBG medium and fixed with glutaraldehydeand potassium permanganate. Although onlythree generations were carried out, anaerobicallygrown cells show modifications compared toaerobic cells. Endoplasmic reticulum-like struc-tures seem to be more frequent than in aerobiccells. Numerous vacuoles are present (more than12 in some cell sections). The vacuoles are oftencontiguous to the nuclear membrane. They con-tain either dark grains or a clear area. Althoughmitochondrial profiles could not be detected inmost of the anaerobic sections, more than 10mitochondria were observed in some of them(Fig. 3C). The staining of these mitochondrialstructures is generally less contrasted than inaerobic cells, but some cristae can be seen. Whatappears to be dividing mitochondria (20) wasalso observed (Fig. 3D).

Respiration. Table 2 shows fermentation andrespiration rates of S. pombe aerobically grownon 40 mm glucose. Fermentation rates are highduring exponential growth and decrease sharplyduring early stationary growth. Under theseconditions, the respiration rates do not varysignificantly and the decrease of the RQ from3.8 to 4.3, to 1.6, is due only to the QCO2 de-crease. Table 3 shows the QO2 of cells growneither anaerobically in the Tustanoff and Bartleymedium containing 110 mm glucose or aerobicallyin media containing 320 mm glucose or 320 mmglycerol. The respirations were measured in thepresence of different substrates at 320 mm. Glu-cose, ethanol, L-lactate, and malate are signifi-cantly oxidized under both aerobic conditions,cells grown on glycerol being three to four timesmore active than cells grown on glucose. D-Lactate, glycerol, and pyruvate, which are goodrespiratory substrates for S. cerevisiae grown onglucose (3), are not oxidized by S. pombe grownunder similar conditions. Glycerol is oxidizedonly when S. pombe is grown on glycerol, whereassuccinate is oxidized only by cells grown onglucose.

TABLE 1. Anaerobic growth in Tustanoffand Bartleymedium supplemented with 0.3% KNO3

Inoculum Stationary phase Anaerobic generations

cells/lnl cells/ml2 X 104 5 X 106 7 to 82 X 105 16 X 106 6 to 72 X 106 45 X 106 4 to 5

- I1 0

0

0

0 AoA

A aMtt

0 oU

-

GL TC.

° /+

AEROBIOSIS

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HESLOT, GOFFEAU, AND LOUIS

N~~~~~~~~

pomp~~~~~~01k _05

:,' 't*

<} ,tt o ,, t s t ''i-4I-,

Fry cm) t .

X7,~X72A4A

Wit/l glutaraldehyde-potassium permanganate. (B) Aerobic stationary cells. The celluilar wail was removed Wit/lsnail gut enzzyme. Fixation? was Wit/l glutaraldehyde-osmium tetroxide. (C anid D) Aniaerobic stational.y cells.Fixation Wit/t glutaraldelhyde-potassium permanganate.

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RESPIRATORY METABOLISM OF S. POMBE

TABLE 2. Respiration anid fermentaiSchizosaccharomyces pombe at diff

of aerobic growth on 40 mM gli

Stage Q02

Exponential, 16 hr, 0.62 mg 51(dry weight)/ml

Late exponential, 20 hr, 1.39 41mg (dry weight)/ml

Early stationary, 24 hr, 1.64 45mg (dry weight)/ml

a Inoculum: 105 cells/ml.

TABLE 3. Oxidation of different sSchizosaccharomyces poml

Respiratory substrates(320 mm)

None ............

Glucose..........Ethanol..........Glycerol.........L-Lactate........D-Lactate........Malate...........Succinate ........

Pyruvate.........

Respiration i

Aerobic cells grownon

320 mmGlucosec

1.816201.1

123.316172.6

320 mmGlycerold

336075526029662933

a Inoculum: 106 cells/ml for all ccbExpressed as microliters of 02 P

milligram of dry weight.c Exponential cells, 18-hr culture

cells/ml.d Exponential cells, 24-hr cultur

cells/ml.e IRtntinnnrv -P1lic Tivztqnnff sqnd

tion rates of concentrations of these inhibitors, whereas the?erent stages respiration of cells aerobically grown on glycerolfucosea is totally inhibited. No inhibition of respiration

QC02 RQ by rotenone could be demonstrated under any

condition.

191 3.8 In aerobic culture on glucose, the respirationof "petite positive" S. cerevisiae sharply increases

176 4.3 when growth proceeds from the logarithmic tothe stationary phase. These results probably

71 1.6 reflect the relief of the glucose repression ofrespiration and of mitochondrial genesis (17, 21,

l 26, 27). De Deken (7) observed that the respira-tion of "petite negative" yeast species is notrepressed by glucose. McClary and Bowers (14)

vubstrates by confirmed this correlation for Saccharomycesbea fragilis, a "petite negative" species which exhibits

in Q02b no repression of respiration or of mitochondrial

development by glucose. Figure 4 shows therespiration on 320 mm glucose of a culture of

Anaerobic cells S. pombe 972h- inoculated with 10,000 cells/ml.grown on 110 ms The Q02 was 5.5 after 17 hr of culture (about

glucose,' seven generations) and increased gradually to36 during the next eight generations. The respira-

0.9 tion continued to rise to a Q02 of 58 during the8.0 stationary phase of growth. Although these data1.2 suggest the existence of glucose repression of1.1 respiration or mitochondrial development, there4.2 is no strict quantitative correlation between the6.1 disappearance of glucose and the increase of2.2 respiration. The glucose concentration of the0.9 medium significantly decreases only during the0.6

last three generations, and this exponentialnditions. decrease is not quantitatively reflected in theer minute per slow, steady, linear increase of respiration during

the last eight generations.DP 160 X 106 Figure 5 shows a low-temperature spectrum

of whole cells of S. pombe strain 972h- in sta-e, 40 X 106 tionary phase of growth on glucose. The res-

Rnrthlv mi-_ piratory cytochromes a + a3, b, c, and cl are- at.tLeeldly LUlul U lVialU 1a&LICy I U-

dium + 0.3% KNO3, 24-hr culture, 19 X 106cells/ml.

Cells grown on glycerol show a high rate ofrespiration in the absence of added substratesduring the respiration measurement. This en-dogenous respiration is further increased by theaddition of glucose, glycerol, ethanol, malate,and L-lactate but not by D-lactate, succinate, andpyruvate.

Anaerobically grown cells have a low butsignificant oxygen uptake in the presence ofglucose, D- and L-lactate, and malate. This oxygenuptake differs from normal respiration since it isnot inhibited by 1.66 $AM antimycin A and is only33% inhibited by 1 mm NaCN (Table 4). Therespiration of cells aerobically grown on glucoseis strongly, but not totally, inhibited by identical

TABLE 4. Inhibition of oxygen uptake by Schizo-saccharomyces pombe grown under

different conditions

Respirationa of cells grownb on

Inhibitors Glucose Glycerol Glucose(aerobiosis) (aerobiosis) (anaerobiosis)

Q02 % Q02 % Q0s %

Normal. .1 5 100 60 100 7.0 1001.2,UM Rotenone. 15 100 61 101 7.1 1001.66 Mm Antimy-

cinA. 3.4 23 0.1 0 7.5 1071 mM NaCN. 2.0 13 0.1 0 5.0 67

a Measured in the presence of 320 mm glucose.b Culture conditions as for Table 3.

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HESLOT, GOFFEAU, AND LOUIS

Cells

Q002

60

50

.40

.30 0

.20

.10

O

51

Hours of culture

FIG. 4. Growth, respiration, and glucose concentra-tion of the medium during aerobic growth of Schizo-saccharomyces pombe 972h-.

C

-4

- ,,)E.-

c CLS u EnC4 >% Lnis) U

\1d,. L

O.D.=TO.I.

6-

cr

0

a

>U

490 520 550 580 610 640WAVELENGTH -nm

FIG. 5. Low-temperature spectra of Schizosaccharo-myces pombe 972h-. Aerobic stationary cells.

observed. As described by Claisse (6), the cyto-chrome c of S. pombe is particular. It is the onlyknown yeast species in which an a2 band ofcytochrome c is observed at 541 nm. None ofthese bands is observed after three anaerobicgenerations. Disappearance of the cytochromespectrum and inactivation of respiration byanaerobiosis is reversible since respiratory adapta-tion is observed when the anaerobic cells are

put in the presence of oxygen and glucose undernondividing conditions. A mitochondrial fractionwas isolated from glycerol-grown cells by amechanical procedure described for S. cerevisiaeby Balcavage and Mattoon (2). As shown inTable 5, the mitochondrial fraction oxidizesreduced nicotinamide adenine dinucleotide(NADH), succinate, a-glycerophosphate, andtetramethyl-p-phenylenediamine (TMPD) plusascorbate. However, no significant activity wasobserved with a-ketoglutarate, ethanol, L- andD-lactate, malate plus pyruvate, acetate, citrate,and 13-hydroxybutyrate. The oxidation of NADHand of succinate was stimulated by 146 ,UM ADPand by 1.6 ,uM of the uncoupler carbonylcyanide-phenylhydrazone. Respiratory control of about2.0 was obtained with succinate and NADH.However, the method used yields damagedmitochondria. State 3 respiration cannot berestored after the addition of small amounts ofADP, indicating the existence of an active adeno-sine triphosphatase. Moreover, the specificactivity of respiration is 5 to 10 times lower thanthis of Saccharomyces mitochondria (2, 15, 18,19). Respiration of all oxidizable substrates isrotenone-insensitive and antimycin A sensitive,with the exception of TMPD plus ascorbate,which is antimycin A-insensitive.

Differential effects of inhibitors. Table 6 showsthat several substances selectively inhibit growthon glycerol, whereas growth on glucose is muchless affected. Most of these inhibitors are knownto inhibit one or another of the mitochondrialfunctions, indicating that these functions arenot strictly required for S. pombe growth onglucose.The respiration and growth of cells in the

TABLE 5. Oxygen uptake by a mitochondrial fractionof Schizosaccharomyces pombe 9721-

Inhibi- Inhibi- Respira-Respira- tion by tion by tory

Substrates tion 10-6 M 10-4 M control(state 3)a anti- rote- (state

mycin A none 3/state 4)

Succinate........... 39 Yes No 1 .8NADHb........... 170 Yes No 2.1a-Glycerophosphate. 39 Yes No 1 .0Ascorbate + TMPDC 83 No No 1.0

Activity observed with ethanol, pyruvate, L-lactate, citrate, and acetate was <3; with malate,pyruvate plus malate, a-ketogluterate, 3-hydroxy-butyrate, and L-lactate was <1. Results expressedas nano atoms of 02 respired per minute per milli-gram of protein.

b Nicotinamide adenine dinucleotide, reducedform.

c Tetramethyl-p-phenylenediamine.

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RESPIRATORY METABOLISM OF S. POMBE

TABLE 6. Inhibition ofgrowth and oxygen uptake of aerobic Schizosaccharomyces pombea

Relative inhibition of

Inhibitorsb Concn Probable siteGrowthc on Growthcon Re glucsprtglucose glycerol gowngelucs

mg/liter % % %Rotenone................. 0.3 Respiratory chain 7 0 0

(site I)Antimycin A.............. 0.135 Respiratory chain 24 98 85

(site II)Synthaline ................ 20 Respiratory chain 0 75 71

(site III?)2,4-Dinitrophenol......... 25 Uncoupler 0 71 19NaN3 ..................... 1 Uncoupler? 18 100 0COS04.100.1lo Uncoupler? 6 82 11

a Culture conditions as in Table 3.bInhibitors were added in the medium before inoculation.c Measured by cells counting.d Measured by QO2 in the presence of 320 mm glucose.e Decamethylene diguanidine.

presence of rotenone are not affected. Theseresults are in accord with the lack of instantaneousinhibition of respiration obtained with cells andisolated mitochondria.Antimycin A inhibits the respiratory chain

between cytochromes b and c (25). Synthalin isreported to inhibit site III of oxidative phos-phorylation in mammalian mitochondria (25).When added to the growth medium, both anti-mycin A and synthalin strongly inhibit the res-piration of S. pombe cells grown on glucose, withonly a small decrease of the growth rate. Bothrespiratory inhibitors inhibit growth on glycerol.2, 4-Dinitrophenol is a classical uncoupler ofoxidative phosphorylation; it inhibits the forma-tion of adenosine triphosphate without inhibitingoxygen uptake (25). Dinitrophenol stronglyinhibits growth on glycerol, whereas growth onglucose is not affected. The respiration of cellsgrown on glucose is only slightly inhibited byhigh concentrations of dinitrophenol. In vitro,sodium azide may have several sites of action onrespiration, depending to its concentration (25).When added to the growth medium of S. pombe,it acts like the uncoupler 2,4-dinitrophenol.The effects of CoS04 on cell metabolism are

not clear. It may inactivate metabolites whichchelate cobalt, such as amino acids, purines,and porphyrins (1), or alter deoxyribonucleicacid properties (8). Horn and Wilkie (12) showedthat respiratory-deficient cells of S. cerevisiaeare able to grow in the presence of concentrationsof cobalt sulfate which inhibit the growth ofrespiratory-competent cells. In S. pombe 972h-,COS04 acts preferentially on processes involvedin the utilization of glycerol, since, at 100 mg/

liter, the growth on glycerol is 82% inhibited,whereas the growth on glucose is inhibited byonly 6%. As for dinitrophenol and sodiumazide, oxygen uptake by glucose-grown cells isnot much inhibited by this concentration ofCoSO4. These data suggest that much of theoxygen uptake and of the energy produced byrespiration is not necessary for growth on glucoseof S. pombe. However, respiratory energy isstrictly required for growth on glycerol.

DISCUSSIONLike S. cerevisiae, S. pombe 972h- contains

numerous mitochondria with two membranesand cristae. Cytochromes a + a3, b, c, and clare present, and Q02 higher than 50 ,liters of02 per hr per mg of dry weight can be observedwith intact cells. The inhibition of respirationby antimycin A, inhibitor of the respiratorychain between cytochromes b and cl (25), indi-cates the presence of site II of phosphorylation.The sensitivity of cell respiration to NaCN,inhibitor of cytochrome oxidase (25), and alsothe ability of mitochondria to respire in thepresence of ascorbate plus TMPD indicate thepresence of site III. In mammalian mitochondria,rotenone inhibits the oxidation of nicotinamideadenine dinucleotide-linked substrates withoutinhibiting the oxidation of succinate (25). Theinsensitivity of growth and of oxygen uptake bycells and mitochondria of S. pombe suggestthat this species, like S. cerevisiae and S. carls-bergensis, does not possess site I of phosphoryla-tion (19). Other yeast species such as Torulautilis and Endomyces magnusii seem to possessthis site I (19).

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Growth on several nonfermentable substratessuch as ethanol, D-lactate, L-lactate, pyruvate,acetate, succinate, and malate is much morelimited for S. pombe 972h- than for S. cerevisiae.Ethanol, L-lactate, malate, and succinate areactively oxidized and thus penetrate the cell.These results suggest that the interconversion ofcarboxylic acids into carbohydrates is not func-tioning in S. pombe 972h-. On the other hand,glycerol is an efficient growth substrate for S.pombe. Growth on glycerol requires its oxidationthrough the respiratory chain since it is com-pletely inhibited by respiratory inhibitors.The oxidation of malate, ethanol, and L-

lactate by intact cells are not necessarily in con-tradiction with the lack of oxidation of thesesubstrates by mitochondrial fractions, sincenonmitochondrial dehydrogenases of these sub-strates have been reported in other cells. It hasbeen reported that anaerobic decarboxylationof malate is particularly active in S. pombe (16).Some data indicate that, in S. pombe, oxygen

is required for a function other than being theterminal acceptor of the respiratory electrontransport chain. When S. pombe is grown aero-bically on glucose, a significant part of the oxygenuptake remains insensitive to the instantaneousinhibition of antimycin A and cyanide. Thiscyanide- and antimycin A-insensitive oxygenuptake is not observed during growth on glyc-erol, but is particularly prominent in anaero-bically grown cells which show a cyanide-insensi-tive Q02 of 5 to 6 in the presence of glucose andoxygen. Cells aerobically grown on glucose inthe presence of antimycin A also show a residualoxygen uptake. On the other hand, addition ofantimycin A in the growth medium completelyinhibits growth on glycerol. Antimycin A-sensi-tive respiration is thus not required for growth onglucose, whereas antimycin A-insensitive oxygenuptake is not required for growth on glycerol.The oxygen requirement for growth on glucosecan be relieved by another oxidant, KNO3, whichis not a substrate for cytochrome oxidase. Thenonrespiratory oxidative function of oxygen ornitrate seems not to be restricted to the synthesisof sterols or unsaturated fatty acids as in S.cerevisiae, since, in absence of KNO3, the additionof sterols and unsaturated fatty is without effecton anaerobic growth.

It has been previously pointed out that glucoserepression of mitochondrial respiration anddevelopment is absent in "petite negative"species (7, 14). This glucose effect is usuallyreflected by the RQ (ratio of glucose fermented,measured by CO2 evolved, to glucose oxidized,measured by 02 uptake). This ratio has been

reported to be high in glucose-repressed "petitepositive" species (7, 14). S. pombe shows highRQ and, according to this criterion, behaveslike typical "petite positive" yeast species. Res-piration is always higher in exponential cellsgrown on glycerol than in exponential cellsgrown on high concentration of glucose. It isthus likely that some form of glucose repressionof respiration exists in S. pombe. On the otherhand, the steady increase of respiration duringgrowth on glucose is not symmetrical to theglucose disappearance. It begins before the glu-cose concentration decreases significantly andcontinues for several hours after glucose ex-haustion. In this respect, one could say that theglucose repression is atypical in S. pombe.

In conclusion, we have found conditions underwhich S. pombe 972h-, a "petite negative"species, carries out at least 14 generations underanaerobiosis and shows some form of glucoserepression of respiration. The generalization that"petite negative" species do not grow underanaerobiosis (5) and do not show repression ofrespiration by glucose (7, 14) is thus not strictlyvalid for all yeast strains and all experimentalconditions.

ACKNOWLEDGMENTS

The skillful technical assistance of M. Lambert and E. Courbeis gratefully acknowledged.

This investigation was supported by EURATOM and theCommissariat a l'Energie Atomique (grants no. 10437-II/B-6and SC 01-049 BIA F), the Delegation Generale a la RechercheScientifique et Technique (Convention no. 68-01-375), and theInstitut National de la Recherche Agronomique.We also thank M. Claisse for the absorption spectra and to

J. Mattoon for stimulating discussions.

LITERATURE CITED

1. Albert, A. 1960. Selective toxicity. Methuen. London.2. Balcavage, W. X., and J. R. Mattoon. 1968. Properties of

Saccharomyces cerevisiae mitochondria prepared by amechanical method. Biochim. Biophys. Acta 153:521-530.

3. Bizeau, C., and P. Galzy. 1965. Etude de la respiration dequelques substrats non fermentescibles par Saccharomycescerevisiae. Ann. Technol. Agr. 14:275-289.

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