Structural Differentiation of Obligately Aerobic And

BRIEF NOTES

STRUCTURAL DIFFERENTIATION OF OBLIGATELY

AEROBIC AND FACULTATIVELY ANAEROBIC YEASTS

DAN O. McCLARY and WILBERT D. BOWERS, JR. From the Department of Microbiology and the Biological Research Laboratory, Southern Illinois University, Carbondale

INTRODUCTION characteristics had varied greatly from one experi- ment to another (21). The earlier study of C. Although biochemical criteria are used in the utilis (1 i) had revealed a complex, reticular mem- taxonomic differentiation of the yeasts, the cyto- brane system in electron micrographs of anaero- logist has generally considered that the structures bically grown cells, while the later electron micro- of these organisms are essentially alike, although graphs of anaerobically grown cells of S. cerevisiae more readily discernible in some than in others. (21) revealed no such membrane system, the Actually, the fine structures of different species of cytoplasm being essentially devoid of morpho- yeast cells vary, particularly with respect to mem- logically demonstrable mitochondria and other brane systems and mitochondria, according to membrane systems. their relative capacities to rcspirc or fcrmcnt The wide diversity of physiological characteris- under aerobic conditions. tics found among the various species of yeasts of Based upon their requirements for molecular similar gross morphology and simple cultural oxygen, the yeasts are divided into obligate aer- requirements offers an ideal opportunity for obes and facultative anaerobes. The latter group relating functional and structural differences in has been subdivided into "petite positive" and individual cell types. This study was undertaken "petite negative" types, according to differences in to investigate differences, rather than similarities, their inducibility to produce respiratory deficient in yeasts which are known to represent three mutants upon treatment with acriflavine or physiological and genetic categories, namely (a) euflavine (1, 2, 4). Although certain respiratory obligate aerobes, (b) petite positive, facultative characteristics of the two types of yeasts were deter- anaerobes, and (c) petite negative, facultative mined in these studies, they were not correlated anaerobes. with structural features of the cells. Several articles have dealt with the influence of MATERIALS AND METHODS anaerobic conditions (10, 11, 21), glucose con- Because of their diversity of respiratory and genetic centrations (5, 10, 17, 22), nonfermentable cata- characteristics the following were chosen for this bolites (17), or less readily fermentable sugars study: Rhodotorula gradlis NRRL Y1091, a non- (10) On mitochondrial development, but these fermenting, obligate aerobe (12) ; Saccharomyces articles have been confined essentially to Sac- "Carbondale" culture B8502, a petite positive, charomyces cerevisiae, whose versatility to respire or facultative anaerobe; Saccharomyces fragilis NRRL ferment a variety of different substrates under Y665, a facultativc anaerobe, noninduciblc by various physical conditions is not characteristic of cuflavinc to produce respiratory-deficient mutants yeasts in general. Although Candida (Torulopsis) (4), or inducible by acriflavine to produce occasional, aberrant petites (1); and Candida utilis, a facultative utilis was used in an investigation of the origin of anaerobe not inducible by euflavine or acriflavine to mitochondria in yeasts (I1), its use was later produce respiratory-deficient mutants (1, 4). The abandoned, in favor of S. cerevisiae, with the state- above biochemical and genetic characteristics of ment that experimentation with this organism (C. each were thoroughly verified, preceding cytological utilis) had proved difficult, in that its cytological studies of these organisms.

519 FmcaP, 1 Logarithmic growth phase cell of Rhodoturula gracilis grown in ~% glucose-yeast extract- salts medium. The mitochondria (m) are typical, cristate structures. X 3~,000.

FIGURE ~ Logarithmic growth phase cell of 8accharomyces Carbondale strain B850~ grown in ~% glucose-yeast extract-salts medium. Mitochondria are not conspicuous in these cells. A broken, or intermittent membrane (arrows) lies adjacent to the plasma membrane and, in some regions, extends out into the cytoplasm. Note that no such membrane exists in the obligate aerobe, R. gracilis. X 3~,000.

FmUR~ 3 Logarithmic growth phase cell of Saccharomycesfragilis grown under the previously described conditions. Numerous, typical mitochondria (m) appear in this non-aerobic fermenter and only vestiges of the intermittent membrane (arrows), which is a prominent feature of the highly fermentative Sac- charomyees B850~, are seen. X 32,000. The yeasts were grown in glucose-yeast extract- lactate was substituted for the fermentable glucose in salts medium (13). To insure identical culture the growth medium, and cells from logarithmic conditions for all the yeasts, cells from agar slants of growth were prepared for electron microscopy in the the medium were used to inoculate 10 ml quantities manner described above. of the broth medium in 250 ml Erlenmeyer flasks and The respiratory activities of the yeasts were shaken on a reciprocal shaker for 24 hr at 30°C. determined by conventional Warburg manometric These cultures were used to inoculate 200 ml of the methods. Cells from cultures in exponential phase medium in 1 liter Erlenmeyer flasks. The flasks were were washed and suspended in 0.5% glucose in incubated at 30°C on a rotary shaker, and samples 1/~ 5 M KH2POd. Respiratory quotients (RQs) were were take~n at 2-hour intervals to measure growth, as calculated from the #10~ and ul CO2 measured at indicated by turbidity with a Klett-Summerson 15-rain intervals. colorimeter equipped with a 420 #m filter. When plots of the turbidity readings showed that the cultures RESULTS were well within the exponential growth phase, samples were removed, washed twice in distilled Electron Microscopy of the Cells water, fixed in 2% KMnO4 for 4 hr at 4°C, washed A section of a cell of R. gracilis taken during three times with water, stained with uranyl acetate, logarithmic growth in 2% glucose medium is dehydrated in a graded alcohol series, and embedded shown in Fig. 1. The distinctive features of this in methacrylate. Sections were cut with a Porter- cell are the large, well-developed, cristate mito- Blum microtome equipped with a diamond knife, chondria. Shown in Fig. 2 is a section through a and attached to Formvar-coated grids. Some of the logarithmic phase cell of Saccharomyces Carbondale sections were stained with lead citrate, and all were examined and photographed with an RCA EML culture B8502 grown under the same conditions. electron microscope. For some experiments with the Although various membranes and vesicles are Carbondale culture, Saccharornyces B8502, 1% sodium visible, mitochondria are rudimentary and in-

FIGURE 4 Logarithmic growth phase cell of Candida utilis grown as previously described. The features of this culture are essentially like those of S. fragilis. X 3~,000.

FIGURE 5 Logarithmic growth phase cell of Saccharomyces Carbondale strain B850~ with sodium lactate substituted for glucose in the medium. Under these conditions well-developed, cristate mitochondria (m) appear in appreciable numbers. The intermittent membrane (arrow) is clearly defined in these cells. X 3~,000.

B R I ~ F NOTES 521 conspicuous. A structural feature, just beneath the Saccharomyces, harvested from 2% glucose under cytoplasmic membrane of this highly facultative aerobic conditions, ferments with an RQ greater anaerobe, which is not observed in the obiigately than 4.0, while C. utilis and S. fragilis, although aerobic, R. gracilis, is a discontinuous reticular both are producers of gas under the reduced membrane. In Figs. 3 and 4, respectively, are oxygen tension of a fermentation tube, simply shown sections of logarithmic phase cells of the respire, as indicated by their average RQs of 1.1. two other facultatively anaerobic yeasts, S. R. gracilis does not grow anaerobically and fails fragilis and C. utilis. These figures are very similar to produce gas from glucose broth in fermenta- to each other and, with respect to the rather well- tion tubes. Respirometer tests with this organism, developed mitochondria, resemble that of R. like those described above, revealed an RQ of 1.2. gracilis. Small fragments of other membranes are arranged parallel to the plasma membrane, as DISCUSSION seen in the highly fermentative, Carbondale Saccharomyces, but to a much less extent than in that It is clear from the reports of others (5, 10, 17, organism. Fig. 5 shows a cross-section through a 22) who used S. cerevisiae, and from our results logarithmic phase cell of Carbondale Saccharo- with the Carbondale Saccharomyces culture, that glucose concentrations as low as 2 % repress the formation of mitochondria, as well as respiration, TABLE I in the highly fermentative bakers' yeasts, i.e., Aerobic RQ and Mitochondrial Integrity of Four those which respire with a high RQ even under Species of Yeasts during Logarithmic Growth in 2% highly aerobic conditions. However these effects Glucose Medium and Their Inducibility by Acri- flavine to Produce Petite Mutations are not apparent in the obligate aerobes which assimilate glucose by the oxidative mechanism Well- and regularly exhibit the most highly morpho- formed Acriflavine Aerobic mito- induced logically developed mitochondria. Between these Culture RQ chondria petites two extreme groups are many species, as exempli- fied by S. fragilis and C. utilis, which ferment, or S. Carbondale B8502 4.3 -- + produce gas, vigorously in the reduced oxygen S. fragilis 1.1 + -- tension of a Durham or Smith fermentation tube C. utilis 1.1 + -- and in aerated culture show little if any glucose R. gracilis 1.2 + N.D.* repression, either on respiration or on the morpho- * Not done. R. gracilis is an obligate aerobe. It is logical development of mitochondria. This study, extremely unlikely that an agent could induce though limited to but few species, suggests that aerobic independence and respiratory deficiency the species which ferment glucose under highly in an organism at the same time. aerobic conditions with a high RQ and possess poorly defined mitochondria under these condi- myces, grown under the conditions described tions fall into the group which DeDeken (4) and above, except that 1% sodium lactate was sub- Bulder (1) found inducible by euflavine and acri- stituted for glucose in the medium. Under this flavine to form viable respiratory-deficient strictly aerobic condition, as Schatz (17) has mutants, while those which respire and possess shown in S. cerevisiae, this culture also produces well-developed mitochondria under these condi- morphologically well-developed mitochondria. tions are rarely induced by these agents to form The discontinuous membrane in the peripheral the viable mutants. region of these cells is retained, indicating that its The work of Slonimski (18) and Bulder (2) existence does not depend uoon the fermentable offers an explanation for the differences in petite substrate. inducibility in yeasts. Slonimski found that euflavine-treated ceils of S. cerevisiae did not them- The Respiratory Activities of the Cells selves become respiratory deficient but were The relationships among the respective RQs inhibited in their production of certain of the of the cultures, their formation of morphologically respiratory enzymes and of the hereditary factors well-developed, cristate mitochondria, and their essential for transmission of respiratory com- inducibility to petite formation by acriflavine are petence to their buds. Since S. cerevisiae and related shown in Table I. Logarithmic phase cells of bakers' yeasts ferment strongly, either aerobically

522 BRIEF NOTES or anaerobically, respiratory deficient buds are not appear in our preparations of the strict aerobe, viable through the fermentative mechanism and R. gracilis, and is less prominent in the weaker continue to reproduce and form petite colonies. fermenting S. fragilis and C. utilis than it is in the Both DeDeken (4) and Bulder (2) found that this strongly fermenting Carbondale Saccharomyces. agent blocked the synthesis of respiratory enzymes Not having concentrated any cytochemical study in the petite negative, as well as in the petite on the membrane, we are not prepared to attribute positive yeasts, and Bulder found, in fact, that to it any particular biochemical function. How- petite negative yeasts were induced to produce ever, the structure seems of interest in view of the microcolonies of respiratory-deficient cells capable large number of papers associating the peripheral of reproducing a few times but rarely developing zone of the yeast cell with the locations of enzyme viable petite colonies. He also found that some systems such as those of the Embden-Meyerhof- petite negative yeasts, although capable of fer- Parnas system of alcoholic fermentation (16) and menting glucose under reduced oxygen tension, various hydrolytic systems (3, 6-9, 14, 15, 19, 20). would not grow anaerobically, and that the in- frequent, viable petites obtained from the petite This work was supported by the research grant negative yeasts in general required some molecular (GB4608) from the National Science Foundation; oxygen for their growth. training grant (T1 GM 593-05) from the National Our cytological studies on representative groups Institutes of Health; and the Graduate School, Southern Illinois University. of these yeasts thus indicate that the capacity for The authors are indebted to Mrs. Gertrude aerobic fermentation, glucose repression, and Lindegren for obtaining and furnishing the cultures petite inducibility are related to, or are paralleled and for her general interest in the work, and to Mr. by, the mutability or stability of the mitochondria John Richardson for his photographic work. of the respective types. A preliminary report of this paper was presented Returning briefly to the intermittent electron- at the 66th Annual Meeting of the American Society opaque membrane located in the peripheral region for Microbiology, 4 May 1966. of the facultative anaerobes, this structure does Received for publication 5 July 1966.

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52~ BRIEF NOTES