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ON RESPIRATORY DEFICIENCY IN YEASTS

PROEFSCHRIFT

TER VERKRIJGING VAN DE GRAAD VAN oocrOR IN DE TECHNISCHE WETENSCHAP AAN DE TECHNISCHE HOGESCHOOL TE DELFT

OP GEZAG VAN DE RECfOR MAGNIFICUS JR. H. J. DE WIJS. HOOGLERAAR IN DE APDELING DER MIJNBOUWKUNDE. VOOR EEN

COMMISSIE UIT DE SENAAT TE VERDEDIGEN

OP WOENSDAG 19 JUNI 1963 DES NA MIDDAGS TE 4 UUR

DOOR

CORNELIS JAN EMIEL ADRIANUS BULDER SCHEIKUNDIG INGENIEUR

GEBOREN TE VOORBURG

CONTENTS

page INTRODUCTION

§ 1. Background of the present study. 9 §2. Review of the literature on respiratory deficient

mutants of yeast. 10

§3. Starting point of the present study. 16

CHAPTER I

CAP ABILITY OF YEASTS TO PRODUCE PETITE MUT ANTS. PETITE POSITIVE AND PETITE NEGATIVE SPECIES

§ 1. Principles of screening. 17

§2. Criteria for respiratory deficient mutants. 17 §3. Species fr om which petitè mutants were obtained. 18

§4. Species from which petite mutants could not be

obtained. 18

§ 5. Species difficult to clas sify in one of the

fore-mentioned groups. 19

§6. Validity of the characteristics petite positive and petite negative as specific properties. 21 §7. Correlation of the properties petite positive and

petite negative with taxonomie classifications. 23

§8. Saccharomyces delbrueckii. 25

§ 9. Saccharomyces fragilis, S. florentinus and S.

microellipsodes. 26

§10. Discussionofthe correlation of the petite mutation

with taxonomie classifications. Relation with

hap-loidy and diploidy. 27

§ 11. Correlation of petite mutation with fermentative dissimilation. A working hypothesis. 28

CHAPTER 11

LETHALITY OF THE PETITE MUT ATION IN PETITE NEGATIVE YEASTS

§ 1. Introduction. 30

§2. Influence of acriflavine on the synthesis of the

respiratory system. 30

§3. Lethality of the petite mutation. 33

§5. §6. §7. §8. §9. §10. §11.

Petite mutants of Saccharomyces roséi.

Petite mutants of other petite negative yeasts. Pattern of appearance.

The action of acriflavine on a petite negative yeast. Growth in an anaerobic jar.

Induction of petite mutation using elevated tem-peratures.

Summary and conclusions .

CHAPTER III

ANAEROBIOSIS, FERMENTATIVE DISSIMILATION AND GROWTH page 35 35 37 42 43 45 47 § 1. Introduction. 48

§2. Anaerobic growth of yeast. 48

§3. Consequences of extremely low oxygen tensions. 50

§4. The negative Pasteur effect. 50

·§5. Oxygen requirements of obligate anaerobes. 51

§6. Oxygen requirements of yeast. 52

§ 7 . Contributions Jf respiration and fermentation to the energy required for biosynthesis. 53 §8. Determination of the contributions of respiration

and fermentation to 'energy production in short

term growth experiments. 57

§9. Determination of the contribution of fermentation to energy production in steady state growth

ex-periments. 61

§10. Summary. 63

CHAPTER IV

RESPIRATORY DEFICIENCY AND BIOSYNTHESIS

§ 1. Pathways of biosynthesis and nutriHonal require-ments. Tricarboxylic acid cycle. ·65 §2. Glucose as sole carbon source. 66

§3. Vitamin requirements. 68

§4. Importance of succinate dehydrogenase. 69

CHAPTER V

FUNCTION OF MOLECULAR OXYGEN IN FERMEN-TATIVE DISSIMILATION

page

§ 1. Introduction. 73

§2. Functional disturbance of metabolism due to

res-piratory deficiency. 74

§3. Cytochrome b in respiratory deficient mutants. 75

§4. Reductive inactivation of metabolism caused by

the absence of the cytochrome system. 76

§5. Fermentation of glucose by intact cells under

aerobic and anaerobic conditions. 76

56. Fermentation of glucose by cell-free extracts un-der aerobic and anaerobic conditions. Absf'oce of anaerobic fermentation in petite negative yeasts. 78 § 7 . Localization of the anaerobic blocking of ferm en

-tation in Saccharomyces rosei. 81

§8. Fermentation by Saccharomyces rosei in the

pres-ence of arsenate. 83

§9. The control of the fermentation rate by the

NAD-NADH2 system. 85

§lO. Estimations ofthè concentration of the NAD-NADH 2

system. 91

§ 11. Function of sedimentable electron -transferring

en-zymes in fermentation. 92

§12. Conclusions. 96

CHAPTER VI

SOME GENERAL REMARKS

§ 1. A few comments on respiratory deficiency and on

yeast metabolism. 98

§2. Some remarks on respiratory deficiency and

car-cinogenesis. 103

CHAPTER VII

MATERIALS AND METHODS § 1. Materials.

§ 2. Analytical methods.

§ 3. Miscellaneous techniques.

§4. Abbreviations and symbols.

105 106 109 110

SUMMARY REFERENCES SAMENV ATTING page 111 115 123

INTRODUCTION

§1.

Background of the present study.

The present study was suggested by the reading of a publication of Warburg (1955;

see

also 1956). In that paper, Warburg reviewed his opinions on the origin of eaneer cells. Briefly summarized, he considers an irreversible injuring of een respiration as the eommon cause of eaneer, acting in the first phase of carcinogenesis. In the seeond phase the eens must "sueeeed in replaeing the irretrievably lost respiration energy by fermentationenergy". The fermentation energy is eousidered to be "mor~hologieally inferior" to respiration energy and as aresult 'the highly differentiated body eens are eonverted by this to undifferentiated eens that grow wildly - the cancer eens".

Irrespeetive of the eorrectness of Warburg's theory, whieh is attraetive by its simplicity , I thought it worthwhile to look for some microbiologieal analogue of the carcinogene sis as depieted by Warburg. The microbiologieal analogue should be a case in whieh a mierobial strain that possesses both respiration and fermentation becomes irreversibly deprived of the former process and is converted into a strain th at has to rely only, or nearly only, on fermentation energy. It has to be understood th at this mierobiologieal model sys-tem would apply in the first instance to Warburg's theory of carcinogene sis and not nece s sarily to aetual carcinogenesis;

but anyhow, the study of such an analogue would provide information as to the implications of the irreversible loss of respiration in general, and thereby arguments as to the validity of Warburg's theory.

Shortly after I had started my investigations it beeame apparent from a discus sion of Warburg's theory by Weinhouse, Warburg, Burk & Schade (1956) that it may be diffieult to establish undeniably whether eaneer eells or cells of any other type have a defective respiration or not; it seemed that it is difficult to find adequate criteria. This eorroborated my view that a study of the phenomenon of respiratory defieieney in general would eontribute to a more founded discussion of the respiratory deficientnature ofeancer eells.

Among the mieroorganisms that possess both respiration and fermentation, the yeasts were chosen for the present study of respiratory deficieney. This choice offered several advantages. Respiratory deficieney in yeast had been inves-tigated already in several respects. Furthermore, the pres-ence of the yeast collection of the CentraalbureHu voor

Schimmelcultures (C. B. S.) in our laboratory made a wide diversity of taxonomically well defined yeast strains

conven-iently accessible. Finally. of the various more or less

well established types of metabolism found among the

mi-crobes th at of yeast possesses perhaps the close st .

simi-larity to animal metabolism.

§ 2.

Review of the literature on respiratory deficient mutants

of yeast

.

In the literature before 1949 only two reports on respiratory

deficient mutants of yeast are found. Stier & Castor (1941)

mention arespiratory deficient yeast mutant resulting from

the action of potassium cyanide. Whelton & Phaff (1947)

re-port on arespiratory deficient variant of yeast originating

on ethylene oxide treatment. .

Since 1949. Ephrussi and his school have reported on extensive research on respiratory deficient mutants of yeast.

As their results are the basis for the present work. a brief

account of these results will be given here.

This group has found that a yeast culture growing in the presence of acriflavine is converted under appropriate con-ditions into a population consisting almost entirely of cells which are somewhat smaller than the original cells and. furthermore. multiply slower and form markedly smaller colonies on solid media than the original cells. This con-version has been observed to be stabIe over at least 1600 generations; it occurs almost exclusively when the cells

are budding. A similar type /of cells has been encountered

with a frequency of about 1% iÎn untreated cultures. which

suggests th at the· mutation mayalso occur spontaneously.

Because of the small size of the colonies formed by the

mutant cells. the name

petite colonie

has been attached to

the mutants (Ephrussi. Hottinguer & Chimenes, 1949). In

the following. the respiratory deficient mutants will usually

be referred to as

petites.

It has been demonstrated that the mutants are respiratory

deficient. Under aerobic conditions the normal cells produce

per unit of w~ight of glucose about four times as much

living matter as the mutants do. Under anaerobic conditions

the amount of living matter produced by normal yeast is

markedly reduced, but th at produced by

p

e

tit

e

yeast remains

unchanged as compared to aerobic conditions (Tavlitzki.

1949). Manometric measurements under aerobic conditions

demonstrated that the mutants dissimilate glucose nearly

is reduced to less than 10% of the value found for the nor-mal yeast. The snor-mall respiration left is not sensitive to cyanide poisoning. Endogenous respiration' or endogenous fermentation can hardly be observed in the mutants, although these mutants are capable to synthesize reserve material from glucose. The aerobic fermentation rate of the mutants is higher than that of the normal cells. Under anaerobic conditions, the fermentation rate of the mutants is hardly changed, but th at of the normal yeast raises to about th at of the mutants (Slonimski, 1949). Slonimski (1953 a) has in a monograph given an extensive, comparative account of the physiological properties of normal and

petite

yeast. Ex-amining aerobically grown cells, he found th at in the

petites

all those enzymes are absent,. that in normal ceHs are bound to particles !?edimentable at 30, 000 x g. These included cytochrome c: 02 oxidoreductase, succinate: cytochrome c oxidoreductase, glycerol-3-phosphate: cytochrome c ox-idoreductase and NADH_{2 :} cytochrome c oxidoreductase. In

the absorption spectrum of intact cens the cytochromes a, b, c and e, the latter one now usually referred to as cy-tochrome cl'

(see

Slater, 1958) are present in normal yeast, but in the

petite

yeast the cytochromes a, b, and e are lacking. Besides cytochrome c, the

petite

cens contain a cytochrome designated as al' with an absorption band at Ga. 580m~.

The more easily soluble enzymes lactate: cytochrome c oxidoreductase, alcohol: NAD oxidoreductase and cytochrome c, are present in the

petites

to the same extent as they are in normal yeast as appears from activity measurements; a somewhat lower activity of malate: NAD oxidoreductase is found in the

petites

than in the normal cens (Slonimski, 1953 a). The activities found of citrate hydro-Iyase (aconitase). malate hydro-Iyase (fumarase) and isocitrate: NAD oxido-reductase are in the

petites

much smaller than in the normal cells, but they are still present (Slonimski & Hirsch, 1952 a; Hirsch~ :1952).

Marcovich (1953) has shown that besides acriflavine several other acridines and also some derivatives of triphenylmethane can induce the

petite

mutation. although less effectively than acriflavine does.

The gene tic al background ·of the respiratory deficient mutation has also'been exhmsively' investigated by Ephrussi and his school. It could be demohstrated that the synthesis of the respiratory enzymes concerned requires the simulta-neous presence of a cytoplasmic factor and a dominant nuclear gene. The cytoplasmic factor appears to be dependent on the nuclear factor in its function. but independent of it in

its reproduction. Mutants lacking the cytoplasmic factor are called

"vegetativepetites ",

as they may arise in vegetative reproduction. Mutants lacking the pertaining dominant rmclear gene are called "segregational

petites"

since in crossing experiments the mutant character is inherited in the normal Mendelian way. Also "double mutants" can exist, which possess neither the cytoplasmic factor nor the dominant nuclear gene (Ephrussi, 1953;

see

also Ephrussi, de Margerie-Hottinguer & Roman, 1955). As a matter of fact,

the mutants investigated by Slonimski from a biochemical point of view were vegetative

petites.

The segregational mutant!;>, however, are believed to present the same bio-chemical characteristics as the vegetative petites (Ephrussi, 1953):

The respiratory activity of yeast is determined not only by genetical factors, but also by environmental conditions. The synthesis of the respiratory system of a normal yeast is dependent upon the presence of molecular oxygen to such an extent th at a normal yeast grown under anaerobic con-ditions is practically devoid of re spiratory activity. The enzymatic constitution of'such an anaerobic yeast, however, does not in all respects correspond with that of a

petite

mutant grown unde-r aerobic conditions . Cytochrome c: O₂ oxidoreductase, succinate: cytochrome c oxidoreductase, NADH_{2 :} cytochrome c oxidoreductase and glycerol:-3-phos-phate: cytochrome c oxidoreductase, the synthesis of which cannot be performed by

petites,

are also lacking in normal cells grown under anaerobic conditions. However, lactate: cytochrome c oxidoreductase is not affected by the

petite

mutation, but is practically absent in anaerobic yeast. The activities of malate: NAD oxidoreductase, citrate hydro-Iyase

(aconitase). :u.alate hydro-Iyase (fumarase) and isocitrate: NAD oxidoreductase found in

petite

are much smaller than in normal yeast, as has been mentioned already, but in anaerobically grown normal yeast still lower activities are

found.

Less data exist on the enzymic equipment of anaerobically

grown

petites;

the data available do not establish any

dif-ference between the enzymic equipment of anaerobically

grown normal and

petite

yeast. For instance, cytochrome

c is not affected by the

petite

mutation, but it is not present

in anaerobic

petite

and the activities found for citrate

hydro-lyase (aconitase), malate hydro-Iyase (fumarase) and

isocitrate: NAD oxidoreductase in anaerobic

petite

are as

low as they are in anaerobically grown normal yeast.

The absorption spectra also reflect the influence of oxygen

ab-sorption spectra of norm al yeast and

petite

are identical. The bands of the cytochromes a. b. c and e have disap-. peared from the spectrum of normal yeast as have the bands of cytochrome c from that of

petite

yeast. In the spectrum of anaerobically grown normal yeast a band at ca. 580

mi.t.

designated as cytochrome al and one at 557-559 m~ desig-nated as cytochrome bi. have appeared. The same bands are visible in the spectrum of anaerobically grown

p

.

etite

yeast. Of these. the band of cytochrome al has been present also in the spectrum of aerobically grown

petite

whereas the band of cytochrome bi appeared only after anaerobic cultivation (Slonimski. 1953 a; Slonimski & Hirsch. 1952 a. 1952 b; Hirsch. 19052).

Aeration of anaerobically grown yeast can restore the aerobic phenotype to a large extent even in the absence of appreciable growth. The absorption spectrum of normal as well as th at of

petite

yeast change into th at characteristic

of aerobically grown normal or

petite.

The normal yeast

shows an increase of respiratory activity from practically zero to about the value usually found after aerobic cultivation and all enzymic activities increase from the anaerobic to the aerobic level (Slonimski. 1953 a, 1955; Hirsch. 1952).

Slonimski (1953 b) has demonstrated that this "adaptation to oxygen" can be blocked by acriflavine. The concentration required is ab out equal to the concentr.ation that gives rise to respiratory deficient mutants in a growing population. This inhibition is specific for the synthesis of the respiratory system. The functioning of enzymes already present. either respiratory or others. is not inhibited. nor is the synthesis of enzymes not belonging to the respiratory system. Com-parison of different acridine derivatives has demonstrated th at a quantitative correlation exists between induction of mutation to

petite

and inhibition of adaptation to oxygen. Since the inhibition of adaptation precedes the appearance of mutant cells. the mutation cannot be the cause of this inhibition. Slonimski (1953 b) concludes th at there exists a stage. or a reaction. that is common to the phenotypic development and to the hereditary trans.mission of the res-piratory enzymes.

The enzymatic activities that are known to be adversely affected by the mutation to

petite

can be divided into two groups. One group includes the difficultly soluble enzymes cytochrome c: O2 oxidoreductase. succinate: cytochrome c

oxidoreductase. NADH2 : cytochrome c oxidoreductase and

glycerol-3-phosphate: cytochrome c oxidoreductase; the other group includes the rather easily soluble enzymes malate: NAD oxidoreductase. citrate hydro-lyase (aconitase). malate

hydro-lyase (fumarase) and isocitrate: NAD oxidoreductase. The enzyme s of the first group are practically absent in

petites

but as regards the second group. the activities found

are only more or less reduced. As these latter activities

(see

Hirsch. 1952) are also more or less reduced in the

course of growth under anaerobic conditions. not only of normal yeast. but also of

petite

yeast. Slonimski & Hirsch (1952 a) suppose that these decreased activities in aerobic

petites

as compared to norm al yeast are a consequence of the

disappearance of the cytochrome system. and that the

petite

mutation primarily affects the four sedimentable enzymes. namely cytochrome c: 02 oxidoreductase. succinate: cyto-chrome c oxidoreductase. NADH2 : cytochrome c reductase and glycerol-3-phosphate: cytochrome c oxido-reductase.

In view of the complex nature of these enzymes. each including a hemoprotein electron carrier. it has been sug-gested that this primary effect consists in interfering with the synthesis of only a few hemoproteins. namely cytochro~e

a (including a3). cytochrome b and perhaps cytochrome e; probably not the synthe sis of the hemin moiety. but that of the protein part is affected (Slonimski. 1953 a. 1955). It is of interest to note here that hemoprotein. enzymes like cyto-chrome b₂• associated with lactate: cytochrome c oxido-reductase. and cytochrome c. the synthesis of which is not affected by the mutation. are easily soluble.

As a matter of fact. arespiratory deficient mutant has been described that did not synthesize glycine at a sufficient rate and as a result was also unable to synthesize catalase and cytochrome c besides cytochrome a and cytochrome b (Ycas & Starr. 1953). This mutant was obtained after ir-radiation and it is obvious th at

petite

mutants obtained after exposure to a non-specific mutagenic treatment may exhibit sorne other deficiencies additional to the

petite

pattern. Such deficiencies might include the loss of easily soluble hemo-protein enzymes. but they are not related to the

petite

mutation.

No answer can be given to the question whether the hemo-protein enzymes involved in the·

petite

mutation are totally absent in the

petites.

Slonimski (1953 a) estimates that the

petites

possess not more than 1: 1000 of the cytochrome

oxidase (= cytochrome c: 02 oxidoreductase) of the normal yeast. which means that this enzyme is practically absent.

After the publications of Ephrussi and coworkers. many other investigators have studied respiratory deficiency in yeast. As a review has been given recently by Nagai. Yanagishima & Nagai (1961). only some results th at have a

direct bearing upon the present work will be mentioned in the following.

Linnane & Still (1956) briefly report on a re-examination

of the enzymatic constitution of

petite

yeast. U sing the

petite

strain investigated most extensively by Slonimski, they "have been unable to confirm that succinic and a-glycerophosphate dehydrogenases and DPNH-cytochrome c reductase are absent from these organisms". Further they "find th at aconitase, malic and isocitric acid dehydrogenases and cytochrome c

are all present in the granules sedimentable at 25,000 x g.

In fact, in respect to the above mentioned enzymes, granules

from normal and 'petite ' yeast cells have a similar enzymic content and concentration". They conclude "that at present the only well known defined enzymic deficiencies in 'petite '

yeast are cytochrome band cytochrome c oxid8.se".

I am of the opinion that these results are not at such

variance with those of Slonimski as it might seem at first sight. The methods used in cell disintegration largely deter-mine whether enzymes are found sedimentabie or not, and

the techniques used by Linnane & Still obviously have been

less destructive than those applied by Slonimski. Their con-clusion as to the only well known enzymic deficiencies of the

petite

mutation is almost identical with the view of Slonimski that the primary effect of the mutation bears on

some hemoprotein enzymes, namelyon cytochrome a (+ a3),

on cytochrome band probably also on cytochrome e. The reported disagreement as regards enzymic content and con-; centration is thus a matter of secondary nature; it can only be evaluated by comparing the methods used and the actual results obtained.

A high frequency of mutation to respiratory deficiency can re sult, besides from the treatment with a chemical mutagen like e. g. acriflavine, also from growth at temper-atures higher than the temperature optimal for growth. The absorption spectrum of the mutants obtained is that

character-istic of

petite

(Ycas, 1956) and the mutants possess a

fer-mentative dissimilation only (Gutz, 1957).

In the cases hitherto described, growth is essential for

the mutation to occur (Ephrussi, Hottinguer & Chimenes,

1949; Ycas, 1956). In the absence of growth, respiratory

deficient mutants may result from irradiation (Raut, 1953.

1954; Paretzky & Clark, 1953), and from exposure to lethal

temperatures (Sherman, 1957). In these cases the mutagenic

effect is accompanied by a killing effect and mutations other

than to

petite

mayalso re sult, at least from irradiation

respiratory deficient mutants obtained af ter irradiation may either result from gene mutations (Raut: "mutant"; Ephrussi: "segre*ational

petite")

or from a cytoplasmic mutation (Raut:

"petite

I; Ephrussi: Ilvegetative

petite").

§3.

Starting point of the present study

.

Nearly all work on respiratory deficiency in yeasts has been done with

Saccharomyces cerevisiae.

Other species, of which respiratory deficient mutants have been reported are:

Sac

-charomyces ellipsoideus(syn.

S.

cerevisiae

var.

ellipsoideus).

S.

car.lsbergensis,

S.

mandshuricus

(syn. either S.

cerevisiae,

S.

earlsbergensis

or S.

uvarum ).

S.

italicus

and S.

validus

(syn. either S.

willianus

or S.

uvarum)

(Ephrussi, Hottinguer & Chimenes. 1949; the synonyms given here are the names accepted by Lodder & Kreger-van Rij, 1952); S.

chevalieri

(Spiegelman. DeLorenzo & Campbell. 1951) S.

earlsbergensis

(Gutz, 1957). and

Candid.a albicans

(Alvarez & Mckinnon. 1957). I thought it would be worthwhile, to investigate whether the

petite

mutation could occur in all yeast species th at possess

both arespiratory and a fermentative metabolism. It was

imaginabIe, that the ability to form

petite

mutants might be .

limited to certain species. Such a limitation, if found, would probably throw some more light on the problem of respiratory deficiency.

Therefore, the present study started with a survey ·of the capability of a wide diversity of yeast species to produce

petite

mutants.

CAPABILITYOFYEASTS TO PRODUCE PETITE MUTANTS.

PETITE POSITIVE AND PETITE NEGATIVE SPECIES

§1.

PrinciPles of screef'ling.

As set forth in the Introduction, a number of yeast species

were examined for their ability to produce

petite

mutants.

For practical reasons, work was limited at first to those

species which are described in "The Yeasts" (Lodder &

Kreger-van Rij, 1952) and are reported to ferment one or more sugars; and fro'm these species only the type strains of the C. B. S. -collection were tested. Acriflavine was used as a mutagenic agent. The work of Ephrussi and his co-workers has shown th at this compound, in concentrations which are not markedly lethal, gives a high frequency of mutation, as a matter of fact up to 100%.

§2.

Criteria for respiratory deficient mutants.

A description of the method used in the present work t~.

obtain respiratory deficient mutants is given in the chapter entitled "Materials . and Methods ", p.109. As a rule, from

every strain which yielded

petite colonie

mutants upon this

treatment, two mutant strains were isolated as pure cultures. The cytochrome deficiency of these mutant strains was checked by visual observation of the absorption spectrum; ifthe absorption spectrum observed left some doubt regarding the respiratory deficient character, a final decision was based on the results of manometric determination of oxygen uptake arid carbon dioxide production. In trying to distinguish arespiratory deficient strain from a normal one, it has to be taken into account that the array of respiratory enzymes in yeast cells is determined not only by hereditary factors but also by the previous history of the cells. (Slonimski,

1953 a; Ephrussi et al., 1956). For this reason, all relevant

conditions have to be such that a normal strain upon spec-troscopie observation will clearly show the absorption bands

ofcytochromea(ca. 603m~). cytochromeb (562 and 529 m~)

and cytochrome c (550 and 520m~). Ornly under such conditions

the absence of one or more cytochromes can be considered as an indication of respiratory deficiency. Accordingly, a

manometric check on respiratory deficiency can be regarded as decisive only if performed with cells of the same type as those used in the spectroscopie test.

Suitable cell material, both for spectroscopy of the cyto-chromes and for manometric measurements, was obtained by cultivating the strains concerned for two days on malt agar. Details on spectroscopy and manometry are given in the chapter entitled "Materials and Methods", p.106, 108.

§3.

Species from which petite mutants were obtained.

Respiratory deficient mutants could be obtained from the species listed below. Af ter treatment with acriflavine, the popuIa tións contained 50% to 100%

petite

mu tants . In the following, these species will be designated as

''petite

posi-tive" .

Schizosaccharomyces pombe.

Saccharomyces cerevisiae;

S.

pastorianus;

S.

exiguus;

S.

logos;

S.

bayanus;

S.

willianus;

S.

uvarum;

S.

delbrueckii

;

S.

carlsbergensis;

S.

chevalieri;

S.

heterogenicus;

S.

ovi-formis;

S.

italicus;

S.

steineri;

S.

fructuum.

Torulopsis holmii; T. glab.rata*).

Candida robusta.

Kloeckera africana

.

Brettanomyces claussenii; B. lambicus; B. anomalus.

§4.

Species from which petite mutants could not be obtained.

In these attempts no respiratory deficient mutants could be obtained from the species mentioned bel ow , designated in the following as

"petite

negative":

Schizosaccharomyces octosporus

.

Saccharomyces rouxii;

S.

marxianus;

S.

bailii;

S.

lactis;

S.

rosei;S. bisporus; S.

'

pastori**);

S.

fermentati;

S.

micro-e) Cytochrome c could not be distinguished with certainty in the absorption spectrum of the mutants. .

.. ) Now generaHy regarded as belonging to the genus Pichia (Lodder. Slooft & Kreger-van Rij. 1958).

ellipsodes;S. mellis;S. acidifaciens; S. elegans; S. veronae. Hansenula anomala; H. saturnus; H. suaveolens; H. schneg-gii; H. subpelliculosa; H. mrakii; H. silvicola.

Pichia fermentans.

S chwanniomyces occidentalis. Saccharomycodes ludwigii. Hans eniaspora valbyensis.

Nadsonia fulvescens; N. elongata.

Torulopsis colliculosa; T. molischiana; T. dattila; T. sPhaeri-ca; T. sake; T. globosa; T. versatilis; T. etchellsii; T. anomala; T. magnoliae; T. stellata; T. stellatr var. cam-bresieri; T. baciUaris.

Candida utilis; C. pulcherrima; C. pelliculosa; C. inter-media; C. stellatoidea; C. pseudotropicalis; C. cate11:ulata; C. parapsilosis; C. tenuis; C. claussenii; C. solani; C. albicans; C. tropicalis; C. reukaufii; C. macedoniensis; C. guilliermondii.

Kloeckera aPiculata; K. antillarum; K. corticis; K. javanica; K. jensenii;

K.

lafarii; K. magna.

Brettanomyces bruxellensis.

§ 5. Species difficult to classify in one of the fore-menti "lned groups.

Some species could. for different reasons. not be classi-fied in one of these two groups. In the attempts . to obtain petite colonie mutants of Saccharomyces fragilis. there was once found on a plate one colony with characteristics sug-gestive of those of a petite mutant. Af ter isolation. it ap-peared to have very strong absorption bands of cytochrome c (550 and 520 mJ,l) and weak bands of cytochrome b (562 and 529 mJ,l); cytochrome a could not be observed. Manometric experiments showed that this mutant was respiratory deficient

(Q 0

=

o.

Q ~~

=

363; for the normal strain had been

measured: Q

°

=

168, Q~i~

=

192). It is therefore considered

2 2

to be a mutant that appeared incidentally, with characteristics different from those of the classical

petite

mutants, which show only the absorption bands of cytochrome c.

(see

also §9, p. 26).

Petite colorlie

mutants of

Saccharomyces florentinus

were" found on plates after ten or more days only. After isolation of these mutants, their growth remained very slow. Other properties, however, were characteristic of

petite

mutants. In the absorption spectrum, only the absorption band of cytochrome c at 550 mf,J could be distinguished. Manometric measurements showed that the mutant was respiratory deficient

(Q = 2· Q air 105; for the normal strain has been

ob-02 ' cO

2

tained: Q02

=

67; Qg~2

=

192).

Schizosaccharomyces versatilis

proved to be a remarkable species. All seven strains present in the collection of the C. B. S. did not show any visible absorption bands in their spectrum, or only a weak band at 558 mf,J. Manometric measurements have shown that th is species is respiratory deficient (Kwik, 1957). Respiratory deficient mutants have never been observed to form ascospores (Ephrussi, Hot-tinguer & Tavlitzki, 1949) and are considered to be unable to produce them because of the very lack of arespiratory dissimilation (Ephrussi, 1953). This species differs from the classical

petite colonie

niutants not only by its absorption spectrum, but also by its ability to form spores (Wicker-ham & Duprat, 1945). In addition, growth of this species seems to be adversely influenced by oxygen. In shake tubes there is hardly any growth near the surface (Wickerham & Duprat, 1945). I found th at growth in Kluyver flasks was inhibited by aeration and set in only after aeration was stop-pedo It is curious that Wickerham & Duprat (1945) were al-ready aware of the peculiar status of

S. versatilis

in the genus

Schizosaccharomyces

but were inclined to see relation-ships with species of the genus

Endomyces,

which are obli-gately aerobic.

Another species, of which the type strain and probably all " strains are respiratory deficient, is

Torulopsis

lactis-con-densi.

No absorption bands could be distinguished with cer-tainty in the spectrum, but a trace of a band seemed to be located at about 558 mf,J. Manometric measurements have shown that this species is respiratory deficient (Kwik, 1957). In addition, it must be mentioned that respiratory deficient strains do not seem to be uncommon in nature. Out of the

sixty odd strains of

Saccharomyces cerevisiae

present in the C. B. S. collection, three proved to be respiratory deficient. Also, some yeasts isolated from warm blooded animals and sent to the C. B. S. showed this deficiency. These respiratory deficient isolates have been described as

Torulopsis

pintolo-pesii

(van Uden, 1952), and

Candida slooffii

(van Uden &

do Carmo Sousa, 1956).

§6.

Validity of the characteristics petite positive and petite

negative as specific properties.

In the foregoing it has tacitly been assumed th at the result, as it was obtained with the type strain of a species, is valid for th at species. Now it is well known th at strains considered to belong to one species are not identical with respect to all properties. It might be, that the ability to yield

petite

colonie

mutants is a property not shared by all known strains of a species. In that case, it is not admissible to attach the property

petite

positive or

petite

negative to a species. Only occasionally other strains than the type strain have been e~­ amined in the present study. These cases will be mentioned here.

Saccharomyces rosei.

'

Strains 7, 9 and 18, which looked somewhat different from the other strains of this species, were

petite

negative, like the type strain.

S. marxianus;'

Strain 5, which differed a little from the type strain, was

petite

negative as this strain.

S. elegans:

Strains 2, 3 and 10, thought to be different from the other strains of this species, were

petite

negative, like the type strain.

S. fructuum:

A haploid strain, isolated by the C. B. S. on heat treatment of a sporulating culture of the diploid type strain, was

petite

positive like the type strain.

Candida robusta:

All six strains available proved to be

petite

posltlve.

These findings suggest that it is justifiabIe to designate a species as being

petite

positive or

petite

negative, when only the type strain or any other strain of this species has proved to be

petite

positive or

petite

negative.

Only one species has been encountered,

Saccharomyces

delbrueckii,

which includes both

petite

positive and

petite

negative strains. It will be shown below (§8, p. 25) that an analysisof the situation in this species will rather give additional evidence in favour of the view that the property of being

petite

positive or

petite

negative is characteristic for a whole species and not just for a strain.

Additional support for the above view is afforded by the following considerations.

Many species of asporogenous yeasts are considered as imperfect forms of sporogenous species .(Lodder & Kreger-van Rij, 1952). Several pairs of species ·are known, of which the one species is sporogenous, the other species asporogenous; in all other respects the species of such a pair are thought to differ no more from each other than do two strains of the same species. If this is true, and if all known strains of a species are either

petite

positive or petite negative, then there must be no exception to the rule that a sporogenous species and the species regarded as its imper-fect form are identical with respect· to being either

petite

positiveor

petite

negative.

In Table I are listed all known pairs, according to Lodder

& Kreger-van Rij (1952), of sporogenous species and their

TABLE I

Comparison of the ability of sporogenous yeast species to form petite colonie mutants with that of the corresponding asporogenous (imperfect) forms.

Sporogenous species petite Corresponding imperfect species petite Saccharom yces cerevisiae + Candida robusta +

S. exiguus + Torulopsis holmii +

S. marxianus - Candida macedoniensis

-S. fragilis ? §5, p.19 C. pseudotropicalis

-S. lactis - Torulopsis sphaerica

-S. rosei - T. stellata var. cambresieri

-S. fermentati - T. colliculosa

-Hansenula anomala - Candida pelliculosa

-Hanseniaspora valbyensis - Kloeckera apiculata

-corresponding: .:imperfect forms. The data in this table con-firm the Msumption stated at the beginning of this para-graph, namely th at the result as obtained with an arbitrary strain of a species, is valid for the species as a whoIe.

§7.

Correlation of the properties petite Positive and petite

negative with taxonomie elassifieations.

In §6 (p. 21) it has been made probable th at a whole species, and not only the strain tested, may be designated as being

petite

positive or

petite

negative.

In order to find a possible, causal relation between the property

petite

positive or

petite

negative, and other specific properties, as a first attempt correlations were looked for with properties already known.

Since taxonomie classifications are expressions of known properties of the species classified, it seemed worthwhile to compare the classification of yeasts into

petite

positive and

petite

negative species (§3 and §4, p. 18) with existing

taxonomie classifications. It could be that not o'1ly a whole species is either

petite

positive or

petite

negative (§6, p. 21), but that the same is valid for a whole genus or even a whole family.

The nomenclatUre used in the present study is taken from the system of Lodder & Kreger-van Rij (1952), and from the names listed in §3 and §4 (p. 18) it appears that this system does not provide valuable viewpoints in the respect concerned. There are genera of which all species are

petite

negative, but homogeneously

petite

positive generp. are not found here, nor can it be found that all

petite

positive species belong to one genus.

Now many species described by Lodder & Kreger-van Rij

as belonging to the genus

Saeeharomyees

have synonyms in

which the generic name is

Z ygosaeeharomyees

or

Torulaspo-ra.

As

Torulaspora

species have been described yeasts

be-longing to S,

delbrueekii,

S.

rosei,

and S.

fermentati.

Krumbholz (1933) rejected the genus

Torulaspora,

and brought

these yeasts into

Zygosaeeharomyees.

As

Zygosaeeharomyees

species have further been described yeasts belonging to S.

rouxii,

S.

marxianus,

S.

bailii,

S.

laetis,

S.

bisporus,

S.

pastori,

S.

mellis,

S.

florentinus

and S.

aeidifaeiens.

In

the species th at had been brought to the genus

Torulaspora,

as a rule conjugation tubes are formed previous to asqus formation, but conjugation itself is seldom observed. The

other

Zygosaeeharomyees

species show usually a manifest

conjugation of two cells immediately before ascus formation.

These phenomena indicate that in

Zygosaeeharomyees

(in-cluding

Torulaspora)

the vegetative cells are predominantly haploid. A comprehensive discussion of th is matter is given by Lodder & Kreger-van Rij (1952). In the context of the

present study, a subdivision of the genus

Saeeharomyees

"Zygo-TABLE II

The possibility to obtain petite mutants from species belonging to [he genus Saccharomyces s.l. in relation to a subdivision of this genus into Saccharomyces

s. s. I Zygosaccharomyces and Torulaspora ..

Species Subdivided as Petite

Saccharomyces cerevisiae Saccharom yees +

S. pastorianus Saccharomyces +

S. rouxii Zygosaccharomyces)

.

-S. exiguus Saccharomyces +

S. marxianus Zygosaccharomyces

-S. bailii Zygosaccharomyces

-S. logos Saccharom yees +

S. bayanus Saccharom yees +

S. willianus Saccharomyces +

S. uvarum Saccharomyces +

S. deJbrueckii Torulaspora + (see !8. p. 25)

S. earlsbergensis Saccharomyces +

S. fragilis Saccharomyces 1 (see \9. p.26)

S. lactis Zygosaccharomyces -S. rosei Torulaspora -S. chevalieri Saccharomyces + S. bisporus Zygosaccharomyces -S. pastori Zygosaccharomyces -S. fermentatl Torulaspora -S. heterogenicus Saccharomyces + S. microellipsodes Saccharomyces - (see !9. p. 26) S. oviformis Saccharom yces +

S. mellis Z ygosaccharom yces

-S. italicus Saccharomyces +

S. florentinus Z ygosaccharomyces 1 (see 19. p. 26) S. acidifaciens Zygosaccharomyces

-S. steineri Saccharomyces +

S. fructllum Saccharomyces +

S. elegans") Z ygosaccharom yces")

-S. veronae") Z ygosaccharom yces")

-.) A synonym "Zygosaccharomyces rouxii" does not exist in the literature. The generic synonym Zygusaccharomyces is used here since many strains, regarded now to belong to S. rouxii, have synonyms in which the gener ie name is Zygosaccharomyces (Lodder & Kreger-van Rij. 1952) .

.. ) The descriptions of these new species S. elegans and S. veronae (Lodder & Kreger-van Rij, 1952) show that in these species ascus formation is immediately preceded by conjugation. as in species th at have been de -scribed as Zygosaccharomyces.

-25-saccharomyces"

appears to be useful. This does not mean that the author disagrees with the rejection öf this subdi-vis ion by Lodder & Kreger-van Rij (1952).

Table II shows a comparison between the classification of the species of the genus

Saccharomyces

s.l. into

"Zy-gosaccharomyces ",

"Torulaspora",

and

"Saccharomyces

s. s. 11 species, and the classification into

petite

positive

and

petite negative yeasts.

It appears th at as a rule

petite

positive species belong to

"Saccharomyces

s. s." and

petite

negative species to

"Torulaspora"

or

"Zygosaccharomyces"

as far as the genus

Sa:ccharomyces

s.l. is concerned. The exceptions are S.

delbrueckii,

S.

fragilis,

S.

microellip-sodes

and S.

florentinw;.

These yeasts will be dealt with in the following.

§8.

Saccharomyces delbrueckii.

Th~ species S.

delbrueckii includes not only strains which

can be regarded as

"Torulaspora",

but also strains, among others the type strain, that can surely not be considered as belonging to the genera "Torulaspora" or

"Zygosaccharomy-ces"

(Kreger-van Rij, personal communication). Therefore all thirteen strains present in the C. B. S. collection, together with two strains of the variety S.

delbrueckii

var.

mongoli-cus,

were subjected to the acriflavine treatment, in order to see which strains yielded

petite colonie

mutants. It was found, that the strains I, 3, 4, 5, 6, 7. 8, 10, 11, 12, 13 of S.

delbrueckii

and strain 2 of its variety S.

delbrueckii

var.

mongolicus

were

petite

positive; these were the very strains that could not be regarded as

"Zygosaccharomyces"

or "Torulaspora". The st rains 2 and 9 of S.

delbrueckii

and strain 1 of the variety S . .

delbrueckii

var.

mongolicus

were

petite

negative; the latter strain could be regarded as a

"Zy~osaccharomJces", the strains 2 and 9 of the species as '

Torulaspora'.

These results are strongly in favour of considering the strains, for the present brought together into the species S.

delbrueckii,

as belonging to different species. 'lf this is accepted then it can be concluded that no

"Toru-,laspora"

is petite positive and also th at all strains belonging

to one species have been found to be either

petite

positive or

petite

negative.

In connexion with the discussion on S.

delbrueckii

it has to be mentioned that in the attempts to get

petite colonie

mutants from S.

delbrueckii

var.

mongolicus

strain I, a strain was isolated from a small colony, which strain proved to be arespiratory deficient mutant different from the

clas-sical

petite

colonie

mutants. This strain showed the

absorp-tion bands of cytochrome band cytochrome c; the manometric

checkgaveQo =0, Q~~ = 133. Another peculiar feature

2 2

of this strain was the tendency of cultures on agar slants to give rise, after one month or more, to cells which are

no longer respiratory deficient; the respiring cells outgrow

the respiratory deficient population. This latter could only be retained by reisolation from the mixed population. Be-cause of these observations, S.

delbrueckii

var.

mongolicus

strain 1 will still be regarded as

petite

negative.

§9.

Saccharomyces fragilis,

S.

florentinus and

S.

microel-lipsodes.

TM species

Saccharomyces

fragilis

has after the

publi-catio.n of the monograph of Lodder & Kreger-van Rij (1952)

been separated from the genus

Saccharomyces

by

Kudrjaw-zew (1954) and placed into his new genus

Fabospora.

Wicker-ham (1955) has proposed a genus

Dekkeromyces

which should include S.

fragilis,

S.

marxianus

and S.

lactis.

Thus the fact that from S.

fragilis

no regular

petite

colonie

mutants could be obtained, correlates well with the recent removal

of this species from the genus

Saccharomyces.

The species S.

florentinus.

which has been described as a

Zygosaccharomyces,

has yielded respiratory deficient mutants. Unlike the respiratory deficient mutants of S.

fragilis ,

the mutants of S.

flor,entinus

could be obtained at

will, like those .of the

petite

positive species. Nevertheless, it was felt already at an early stage, th at these mutants differed from the usual respiratory deficient mutants by their late appearance on the isolation plates and their very slow growth (§5, p. 20). Further experiments, reported in Chapter II (§8,

p.

42) will throw more light on the status of S.

florentinus.

S.

microellipsodes

has never been described as a

Zygo-saccharomyces

or a

Torulaspora.

The data on sporulation in this species are very scanty (Lodder & Kreger-van Rij, 1952); they do not exclude th at the life cycle, if it could be completely observed, would be like that of yeasts

de-scribed as

Zygosaccharomyces

or

Torulaspora

species. In

fact. S.

microellipsodes

closely resembles the species

To-ntlaspora nilssonii

recently described by Capriotti (1958;

Kreger-van Rij, personal communication).

Thus, it may be concluded tnat m the genus

Saccharo-myces

s.l., respiratory deficient mutants are only easily

produced by species which neither have the characteristics of the former genera

Torulaspora

or

Zygosaccharomyces,

nor can be brought in the new genera

Fabospora

or

Dek-keromyces.

§10.

Discussion of the correlation of the petite mutation with

taxonomic classifications. Relation with haploidy and

diploidy.

Outside the genus

Saccharomyces, petite

positive species have been found in the genera

Schizosaccharomyces,

Toru-lopsis, Candida, Kloeckera

and

Brettanomyces.

Of these,

Torulopsis holmii

and

Candida robusta

are the imperfect forms of

petite

positive

Saccharomyces

species

(see

§ 6, p. 22). Of

Torulopsis glabrata

no perfect form is known yet. In any case, the

petite

positive species belonging to the genera

Schizosaccharomyces, Kloeckera

and

Brettanomyces

can not be considered as imperfect forms of species belonging to the genus

Saccharomyces*).

For this reason one cannot conclude th at the possibility to yield

petite

mutants is restricted to species ofthe genus

Saccharomyces

and their imperfect forms. One can say that the possibility to yield

petite

mutants is determined by properties which may be found frequently but not exclusively in yeasts of the genus

Saccharomyces,

and which are, and probably rightly so, considered by taxono-mists as having no primary taxonomic importance.

As has been mentioned in §7 (p. 23), the genera

Zygo-saccharomyc

e

s

and

Torulaspora

differ frorn

Saccharomyces

s. s. by the fact that their vegetativ.e cens are predominantly in the haploid phase. One could be inclined to suppose that haploid cens cannot give rise to

petite colonie

mutants. This is in flagrant contradiction to the results of experiments of Ephrussi. Ephrussi, Hottinguer & Chimenes (1949) obtained

petite colonie

mutants from haploid strains of

Saccharomyces

cerevisiae

;

the haploid state of these mutants is also con-firmed by the possibility to cross these mutants with other haploid cens (Ephrussi, Hottinguer & Tavlitzki, 1949). Also in the present study examples were encountered, where a predominantly haploid strain of a

petite

positive species gave respiratory deficient mutants as easily as a predominantly diploid strain

(Saccharomyces fructuum,

§6, p. 21); further, two predominantly diploid strains of

S. marxianus,

strains 1 and 5, gave no respiratory deficient mutants (§4, p. 18; §6, 0) Van der Walt & van Kerken (1960) havé observcd srorulat~)I1 in thc genus

p. 21). Therefore, one may only conclude that the tendency of a strain tö show conjugation shortly after ascospore ger-mination is strongly positively correlated, at lèast in the genus

Saccharomyces,

with the possibility to yield

petite

mutants easily.

§11.

Correlation oj petite mutation with jermentative

dis-similafion. A working hypothesis.

The correlation found above between the possibility to produce

petite

mutants and some characteristics used in taxonomy may be striking. It offers, however, no satisfac-tory explanation as to what renders the formation of

petite

mutants possible or as to what prevents it. Therefore, this question was now approached from a more direct physiolo-gical point of view.

As pointed out already in the Introduction, the dissimilation in

petite

mutants is practically exclusively a fermentative one. Therefore,

petite

mutants were expected to be obtain-able only from yeasts that possess a fermentative dissimi-lation besides their respiration; eventually produced respir-atory deficient mutants of a non-fermenting yeast could hardly be imagined to be viable. The latter situation might also apply to yeasts that possess a fermentation that would, in some way or another, be insufficient to compensate for the loss of respiratory dissimilation.

In this connexion, results of a survey made by Lafon (1956) were elucidating. She had measured the respiration rate and the rates of aerobic and anaerobic fermentation in a number of yeast species. A comparison of her data with the classificati0n of yeasts in

petite

positive and

petite

neg-ative species as given in §3 (p. 18) and §4 (p. 18) showed that all eleven

petite

positive species examined by her have a high aerobic fermentation rate, Q~~ (F)

>

100. With

2

only one exception all

petite

negative yeasts tested by Lafon have a low aerobic fermentation rate, Q ~ib (F)

<

100. The

2

correlation which apparently exists between the values of the fermentation rates as measured by Lafon and the

petite

clas-sification described in §3 and §4 does not neèessarily imply that a fermentation rate above a certain limit, e. g. higher than Q ~~ (F) = 100, would be required to sustain life in the

2

absence of arespiratory dissimilation. Such a conclusion as to some quantitative causal relation'wollld be premature, but the correlation strongly suggests t,hat fermentation plays a

less important role in the

petite

negative species than it does in the

petite

positive species. Hence, the loss of the respir-atory system might have 'more severe consequences for the

petite

negative yeasts than for ilhe

petite.

positive ones, in so far as the

petite

negative yeasts cannot grow when deprived oftheir respiration system, whereas the

petite

positive yeasts apparently can.

Thus, the following working hypothesis may be proposed: Acriflavine may in yeasts genenally induce a mutation man-ifesting itself as respiratory defièiency. Many yeast species cannot grow without a .respiratory system and therefore these so-called

petite

negative species cannot produce viable res piratory deficient mutants.

LETHALITY OF THE PETITE MUTATION IN PETITE

NE GA TIVE YEASTS

§ 1.

Introduction.

In Chapter I it was established that not all species of yeasts are able to give

petite colonie

mutants on treatment with riflavine. Further, a working hypothesis was formulated. ac-cording to which the

petite

mutation may occur in

petite

neg-atiye species, but does not produce any viabie mutants. In trying to prove the correctness of this hypothesis, I first examined whether and to what extent the mutagenic agent af-fects the

petite

negative yeasts. This problem was approached as follows.

Acriflavine exerts not only a mutagenic effect, but it may also specifically inhibit the synthesis ofthe respiratory system

(see

Introduction, p. 13). As this inhiDitionprecedes the mu-tagenesis and as, to all probability, some stage or reaction is common to mutagene sis and inhibition, the eventual presence of such an inhibition in

petite

negative yeasts would render the assumption of an initiated, but abortive mutation more plau-sible.

Slonimski (1953 b) has demonstrated this inhibition using anaerobically grown cells suspended in a medium that did not support growth; in a growing population, it would have been impracticable to distinguish between the inhibition of enzyme synthesis in .uready existing cells and the mutagenic effect, which involves an inhibition of the production of normal (i. e. not respiratory deficient) cells.

Such a distinction, however, needs not to be made when looking for the very occurrence of any inhibition of the syn-thesis of the respiratory system. Moreover; I thought it most interesting to investigate whether any such inhibition occurs with a

petite

negative yeast under conditions that lead with

petite

positive yeasts to the production of mutants. but not with

petite

negative yeasts.

§ 2.

Influence of acriflavine on the synthesis of the

respira-tory system.

res-piratory system in populations of a

petite

negative yeast growing in malt extract was compared with its influence on a

petite

positive yeast under the same conditions . The con-centrations of acriflavine used were the same that lead un-der these conditions to the production of

petite

mutants in

petite

positive yeasts and not in

petite

negative yeasts. As

a representative of

petite

positive yeasts

Saccharomyces

cerevisiae

was used. For the

petite

negative yeasts

Sac-charomyces rosei

was thought to be a suitable model strain; it grows weil and

a

pseudomycelium which might cause trou-bIe in several experiments is never formed.

The influence of acriflavine on the synthesis of respiratory enzymes was examined by observing manometrically the up-take of oxygen. The production of carbon dioxide was measured too, in order to get an idea of an eventual effect of acri-flavine on the synthesis of the fermentative system. Care was taken to use populations that were actively growing from the beginning of the measurements. As a rule, ceils from an agar slant were inoculated in 30 mI of medium. After ca. 16 hr of incubation, 3 mI of this culture was used as an inoculum for 30 mI of fresh medium; 0.9 mI of this suspension was brought into the Warburg vessels; 0.1 mI of acriflavine solution, or water in the controls, was tip-ped from the side bulb' immediately before the first read-ings. The results are given in Figures 1 - 4. The ex-periments show that acriflavine inhibits the increase in the rate of oxygen up take in growing cultures of the

petite

neg-ative species

Saccharomyces rosei

as weil as in growing cul-tures of the

petite

positive species

Saccharomyces

cerevis-iae

j under conditions th at are equal to those under which

petite

positive yeasts produce

petite colonie

mutants and

petite

negative yeasts do not. The respiratory activity al-ready present is notmarkedly inhibited. These results obtained with,growing cultures are completely in accordance with the conclusionsdrawnbySlonimski(1953 b) from his experiments with non-growing populations adapting to oxygen, namely that acriflavine inhibits the synthesis of the respiratory system but not its functioning.

The increase of the fermentative activity in S.

cerevisiae

under aerobic as weil as anaerobic conditions is somewhat depressed by 102 _{ppm acriflavine, but not by 10 ppm. In} S.

rosei,

the increase of the fermentative activity under aerobic conditions appears to be stimulated by acriflavine. Under anaerobic conditions, 10 ppm acriflavine has no in-fluence on the increase of the fermentation rate in this yeast and 102 _{ppm a slightly adverse influence. One is} in-clined to conclude that the stimulation observed under

aero-bic conditions is not caused directly by acriflavine; pro-bably it is a consequence of the impaired synthesis of the respiratory system . 11 5 4 a 2 o ... ACOz(FJ resp. In A0 2 2

)\

'"

...... 1:f' ti"" in hh _r ... " _~~-,o 3 4 5 6 5 4 3 - --. -g. ... 0. ... -0-___ 0"# .... 0 tim. in hr 2 o 2 3 4 5

Fig.I. Saccharomyces cere\ ;iae; 10 ppm aCriflavine.

Fig. 2. S. cerevisiae; 102 ppm acriflavine.

Increase in rates of fermentation and respiration in populations of S. cereV1Slae growing in malt extract with 10 ppm and 102 ppm acriflavine. Fermentation and respiration

rates. l.COz(F) a.\d Ll02. respectively. are expressed in IJl per 30 min and plotted as their natural logarithms against time •

• - - - • respiration without acriflavine 0-- - - - 0 respiration in presence of acriflavine IJ,. - - - I J , . aerobic fermentation without acriflavine

Ll-- - --Ll aerobic fermentation in presence of acriflavine

• - - - . anaerobic fermentation without acriflavine

0 - - - 0 anaerobic fermentation in presence of acriflavine

Where the presence of acriflavine did not influence the resuÎts the pertaining data have been omitted from the figures.