Quantitative Selection of Respiratory Deficient Mutants in Yeasts by Triphenyltetrazolium Chloride

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1 V. BACHOFEN, R. J. SCHWEYEN, K. WOLF, AND F. KAUDEWITZ 252 Quantitative Selection of Respiratory Deficient Mutants in Yeasts by Triphenyltetrazolium Chloride V. BACHOFEN *, R. J. SCHWEYEN, K. WOLF, a n d F. KAUDEWITZ Institut für Genetik, Universität M ü n c h e n, 8 M ü n c h e n 19, Maria-Ward-Strasse 1 a (Z. Naturforsch. 27 b, [1972]; received January 14, 1972) Dedicated to Prof. WERNER SCHÄFER on the occasion of his 6th birthday Triphenyltetrazolium chloride exhibits a strong growth inhibition in respiratory competent cells but shows only m i n o r effects in respiratory deficient mutants of Saccharomyces cerevisiae pombe. Use of this d y e thus allows rapid selection of rarely o c c u r r i n g respiratory deficient mutants, showing karyotic as w e l l as extrakaryotic inheritance. Mutation induction b y tetrazolium chloride was not observed. T h e results favour the hypothesis that triphenyltetrazolium chloride interferes with the terminal o x i d a s e of the respiratory chain. Since the first report on respiratory deficient mutants (petite) in Saccharomyces cerevisiae was published 1 several reliable techniques for the detection isolation of these mutants have been elaborated: characterization by colony size on solid glucose media 1, replica plating on nonfermentable substrates 2, staining of colonies with d y e s 3-6. Although most of them can be applied to the isolation of petites in other yeasts, they are efficient only with strains giving rise to high mutation rates. belongs to the group of yeasts which show only low rates of mutation to respiratory deficiency even after induction with mutagens, such as UV nitrosoguanidine 7 ' 8. The most powerful agents in the inactivation of the rho factor in Saccharomyces cerevisiae, acridines ethidium bromide, until now have failed to induce respiratory deficient mutants in 9 ' 1. Under these circumstances a technique for the enrichment of petite mutants would facilitate the study of the genetics of respiratory deficiency in pombe. duction by the dye used rather than of selective growth of petite cells. In this paper was shall show that enrichment of respiratory deficient mutants is due exclusively to selective growth of preexisting mutants, both in pombe in Saccharomyces cerevisiae. A second agent, cobalt sulphate, has been reported by HORN WILKIE 15 to inhibit selectively growth of rho + cells in Saccharomycescerevisiae. We found that both extrakaryotic karyotic petite mutants are enriched by cobalt sulphate. This technique of petite enrichment has been reexamined applied to pombe. Materials Methods Strains pombe: 972 h"; 5/1 a h+ ade7; 5 h~ ade 7. These strains were kindly provided by Prof. Dr. U. L E U P O L D, Berne. Saccharomices cerevisiae: 2,3,5-triphenyltetrazolium chloride () has been shown to inhibit groavth of respiring yeast Neurospora crassa a possible selection for poky mutants of Neurospora has been discussed. M a ile 5, try 2, ura A 1328 A a, ade 2, leu 1 (Seattle Yeast Stock Culture). RAUT pombe: EPHRUSSI observed an enrichment of petite mutants by in a mixed yeast population. According to LASKOWSKI14 this increase in respiratory deficient mutants is the result of mutation in* Present a d d r e s s : Universität Konstanz, Fachbereich Physik, Konstanz, Postfach 733. Requests f o r reprints should be sent to V. BACHOFEN, Universität Konstant, Fachbereich Physik, D-775 Konstanz, Postfach 733. Media Glucose Medium: 1 per cent yeast extract (Merck ^, 2 per cent (w/v) glucose (unless otherwise stated). Glycerol Medium: 1 per cent yeast extract, 2 per cent (v/v) glycerol. Medium: 2,3,5-triphenyltetrazolium chloride () (Merck) added as a sterile solution to the autoclaved glucose medium. Co Medium: cobalt sulphate (Merck) added to glucose medium. Dieses Werk wurde im Jahr 213 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.v. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung-Keine Bearbeitung 3. Deutschl Lizenz. This work has been digitalized published in 213 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution-NoDerivs 3. Germany License. Zum ist eine Anpassung der Lizenzbedingungen (Entfall der Creative Commons Lizenzbedingung Keine Bearbeitung ) beabsichtigt, um eine Nachnutzung auch im Rahmen zukünftiger wissenschaftlicher Nutzungsformen zu ermöglichen. On it is planned to change the License Conditions (the removal of the Creative Commons License condition no derivative works ). This is to allow reuse in the area of future scientific usage.

2 SELECTION OF RESPIRATORY DEFICIENT MUTANTS T T C m e d i a m u s t b e s t o r e d in t h e d a r k. M e d i a w e r e s o l i d i f i e d w i t h 1.5 p e r c e n t a g a r a n d s u p p l e m e n t e d w i t h a d e n i n e ( 1 m g / m l ) if n e c e s s a r y. Saccharomyces cerevisiae: M e d i a as f o r, cent peptone ( M e r c k ). enriched by 2 per Cultures Cells used for stationary phase. of liquid glucose i n c u b a t i o n at 3 all e x p e r i m e n t s w e r e g r o w n to e a r l y They were obtained after inoculation m e d i u m with a b o u t 16 c e l l s / m l a n d C with aeration f o r 2 4 hours (Sac- charomyces) or 36 hours (). Results I) S e l e c t i v e g r o w t h i n h i b i t i o n a) Saccharomyces b y cerevisiae Differential staining of respiratory competent (RC) respiratory deficient (RD) colonies with 2,3,5-triphenyltetrazolium chloride has widely been used as a diagnostic means., when added to glucose agar plates (5 /<g/ml) or to overlay agar (1 mg/ml), stains RC colonies pink to red but leaves RD colonies white 3 ' 4. In our experiments concentrations of 1 to 3 jug/ml agar led to similar results. With increasing concentrations in glucose agar (1, 15, 2 / «g / m l ), selective inhibition of RC colonies is observed whereas RD colonies show only minor reduction in colony size (Table 1 ). pg/m\ cells p l a t e d colonies grown * R D colonies * T a b l e 1. -tolerance of R D R C cells of strain A 1327 A. * T h e average value of 5 plates. Respiratory deficiency was proved by replica plating on glycerol agar. Under these conditions rare petite mutants were easily selected by plating 14 to 1 5 cells per plate. After 4 to 6 days of growth at 3 C, RD cells reached normal colony size. In a relevant experiment the following results were obtained after plating 1 4 liquid grown cells of strain M on glucose medium with 15//g/ml T T C : Colonies of normal size which, after replica plating, were not able to grow on glycerol medium. These were isolated as respiratory deficient cells. Their number differed from 5 to 6 per plate. 2. Barely visible, deep red coloured micro-colonies which were able to grow on glycerol medium. Their number (about 2 to 4 per plate) varied because of their small size it was difficult to score them. 3. Rare colonies of a size between those of 1 2 showed red colour were able to grow on glycerol medium. Their ability to grow on medium was not permanent after a passage on glucose medium their growth on was sometimes blocked in the same way as that of wild type cells. The inability of the colonies of type 1 to use glycerol as an energy source remained a constant property. These RD mutants were crossed to RC cells shown to be extrakaryotic (cytoplasmic) petites. Their frequency in strain M was about 4-1 " 4 as was revealed from plates. This is in good agreement with the data reported by SCHWAIER et al. 1 7 who found a frequency of for the spontaneous mutation rate of RD mutants. The same effect of was observed with cells of strain A 1327 A which shows a high mutation rate to RD. From 2 cells plated on medium about 7 colonies were visible after 4 days of incubation; more than 9% of them proved to be respiratory deficient In a control experiment on glucose medium without, 5 to 7 colonies out of 2 were unable to grow on glycerol medium. Increase of concentration to 25 «g/ml resulted in a reduction of the number size of RD colonies after 6 days of growth. The colour of petite colonies changed to pink red. With doubled concentration ( 5 / / g / m l ), no cells were able to produce colonies. When the concentration was decreased to 3 /<g/ml, a dense lawn of rose coloured colonies developed 3 days after plating 1 4 cells/plate of strain M. White RD colonies were only slightly larger their detection was difficult. b) solid media pombe, selective growth on Lower concentrations proved to be optimal for selection of RD mutants in. Comparable to the experiments with Saccharomyces, 3 classes of colonies were obtained on glucose agar

3 V. BACHOFEN, R. J. SCHWEYEN, K. WOLF, AND F. KAUDEWITZ 254 with 3 m g T T C per liter: 2 to 3 colonies of type 1 CO1 UJ (large, white colonies, R D ), 1 to 15 colonies of o 9 type 2 (very small, dark red colonies, RC) 2 to o CJ 3 colonies of type 3 (medium size, red coloured, o cc R C ), each per 1 5 cells plated. In a control experiment, respiratory competent cells (ade 7 ), auxotrophic f o r adenine, were mixed 7 in a ratio of about 1 to 1 with respiratory deficient cells of the same mating type, which were prototrophic for adenine. On the average 4 cells/plate 5 were plated on T T C agar. After 6 days about 4 3 large, nearly white R D colonies were observed per plate, none of which was a d e -. During prolonged incubation about 5 to 8 small red coloured RC colo- 1 nies per plate developed which were a d e - with f e w exceptions. After plating cells f r o m the same mixture on glucose agar after replica plating on glycerol medium, the same percentage of R D mutants was found. Glucose concentrations of 2 to 3% were optimal. In solid media with 1% glucose the R D mutants no longer were able to form colonies of normal size. Incubation times lasting more than 1 days turned out to be of no advantage because the small red RC colonies grew larger; after another 1 days they appeared as large, pink colonies with a red area CONCENTRATION [mg/ml] Fig. 1. Enrichment of R D cells in liquid media dependent on glucose concentration. A mixture of about 95 per cent R C (h~, ade 7) 5 per cent R D (h~ ade + ) cells of were grown to early stationary phase (6 to 8 generations in exp. 1 3, 8 to 1 generations in exp. 2 ). 1).5 per cent glucose, without aeration; 2) 1 per cent glucose, without aeration; 3).5 per cent glucose, rapid aeration. Stationary phase cells were plated on glucose agar the colonies were replicated on glycerol agar to test respiratory deficiency. in the center only. c) pombe, enrichment of RD mutants in liquid media glycerol agar the percentage of R D colonies was determined. With optimal concentrations (5 to 6 mg per litre) the percentage of R D cells in- The selective growth inhibition,of RC cells by creases f r o m about 5 to 75 per cent (Exp. 1 ). Both T T C might be expected to lead to an enrichment of high R D cells in a mixed population growing in liquid eliminate the effect of T T C in liquid cultures (Exp. glucose concentrations aeration nearly culture in the presence of. In the experiment 2 3 ). Since all R D colonies were adenine proto- described in Fig. 1 a mixture of R D RC cells trophs, this increase of R D cells was due to enrich- (about 5 per cent R D, ade + 95 per cent RC, ment of preexisting mutants not to mutation ade 7, cells, both with mating type h - ) was grown induction in RC cells requiring adenine. under the following conditions: A possible enrichment of RD cells caused by in- 1. in a medium containing.5 per cent glucose varying concentrations, static culture activation of RC cells could be excluded. In an experiment with an RC strain, the percentage of RC cells forming colonies on glucose agar after growth without aeration, 2. in a medium containing 1 per cent glucose in T T C concentrations f r o m to 7 mg per litre culture was nearly constant in all cases. This implies that 3. in a medium containing.5 per cent glucose, to growth inhibition not to inactivation of RC cells. varying T T C concentrations constant rapid The degree of enrichment depends on the number aeration. of cell generations. In the case of a lower percentage varying concentrations, static enrichment of R D cells in mixed populations is due without aeration, Early stationary phase cultures (6 to 8 generations in 1 3, 9 to 1 generations in 2 ) were plated on glucose agar. After replica plating on of R D cells as described above, a prolonged growth in T T C medium is required for an efficient enrichment.

4 SELECTION OF RESPIRATORY DEFICIENT MUTANTS II) C h a r a c t e r i z a t i o n s e l e c t e d by of mutants Both extrakaryotic karyotic mutants were selected in Saccharomyces cerevisiae. Enrichment of karyotic mutants was proved with cells of the mutant pet-6 pet-7, which complement to rho~-"petite" cells. In pombe, genetic analysis of about 5 spontaneous UV-induced RD mutants selected by revealed that all mutants showed Mendelian inheritance of respiratory deficiency. From the fact that we failed to find extrakaryotic mutants after isolation by replica plating (3 mutants tested), it may be assumed that this type of RD mutants if present, is rare in. Biochemical characterization of selected mutants proved that a wide range of mutations lead to a growth advantage in the presence of. These include mutations affecting complex I, II, III IV of the respiratory chain as was shown by studies on respiratory enzymes cytochromes 8 ' 1 8. III) S e l e c t i o n o f R D m u t a n t s b y CoS4 HORN WILKIE 15 reported that in a cobalt medium cytoplasmic respiratory deficient mutants of Saccharomyces cerevisiae show a selective advantage. Although we too found selective growth of respiratory deficient cells in cobalt medium, the results were not as clearcut as in experiments with. Depending on yeast strain culture conditions, the selective advantage of RD cells was variable. In pombe RD cells also exhibit a selective advantage in cobalt media. In the experiment shown in Table 2, a mixed population of karyotic RD cells wild type cells was plated on agar containing varying CoS 4 concentrations. With increasing concentrations wild type colonies are inhibited to a higher degree than RD cells. As with, a growth advantage is also observed in liquid cobalt media. Rapid aeration diminishes the effect. Since only karyotic RD mutants have so far been found in, the observation made by HORN WILKIE 15 that the selective advantage is to be restricted to extrakaryotic RD mutants (rho - ) is not generally true. 255 CSO4 MM cells plated N o. o f colonies g r o w n per plate No. o f R D cells p e r plate T a b l e 2. C o S 4 tolerance of R D R C cells in pombe. A mixture of R C R D (karyotic) cells was plated on glucose agar containing varying C o S 4 concentrations. T h e plates were incubated at 3 C for 5 days. Respiratory deficiency was proved by replica plating. Discussion The results presented above demonstrate that selective growth of RD colonies is achieved by plating a mixed population of RD RC cells on agar containing triphenyltetrazolium chloride. The RD colonies arising on agar are not due to mutations induced by the dye but grow from preexisting mutant cells. This is shown by two experiments : A) The percentage of RD cells revealed by plating a cell population on agar was not increased compared to that revealed by plating on glucose agar by replica plating on glycerol. B) A mixture of auxotrophic RC prototrophic RD cells was grown in the presence of. All RD mutants selected by the dye were found to be prototrophic, i. e. no mutation to respiratory deficiency had occurred in RC cells. C) Neither was there any mutation induced with concentrations lower than those used for selection. W. LASKOWSKI 14 reported a mutagenic effect for on a strain of Saccharomyces. A concentration higher than induced 1 per cent "petite" mutants. This incompatibility of results has not yet been explained. Our experiments demonstrate the selective effect of to be due to growth inhibition of respiring cells. A decrease in respiratory activity due to high glucose concentrations reduces the selective effect of. There was no inactivation of cells, even with highest concentrations of the dye.

5 256 SELECTION OF RESPIRATORY DEFICIENT MUTANTS 256 Cobalt sulphate, which was reported to select extrakaryotic RD mutants in Saccharomyces cerevisiae, also leads to a growth advantage of karyotic RD cells in pombe. However, the absence of physiological adaption of RC cells to, the low rate of -resistant mutants, as well as the wide range of concentrations allowing selective enrichment of RD cells so far render to be the most efficient means for rapid selection of rarely occurring "petite" mutants. After rapid aeration no enrichment of RD cells by in liquid culture was ascertained. This is in agreement with the results obtained by SLATER et al. 19, who noticed that the succinate : reductase system was blocked by rapid shaking of the suspension. This inhibition is thought to be due to the fact that the reduction of to formazan in the electron pathway competes with the reduction of oxygen 19 ' 2. In addition to this interaction with the electron transport system, an uncoupling effect of tetrazolium salts on the oxidative phosphorylation has been described 21. The electron potential (E' = 46 mv) reported for 22 as well as the fact that CN e completely blocks the succinate: reductase favour the hypothesis that couples with the respiratory chain via cytochrome oxidase 19 ' 2. Further support comes from the analysis of RD mutants selected by. In, mutants showing defects in different parts of the respiratory chain, especially in complex IV, have been isolated 8 ' 18. The genetic block in the electron transport chain leads to a decrease in the reduction of to formazan, as can be seen from reduced colour production of the RD colonies, thereby allows these cells to growfaster than RC cells. No share of the uncoupling effect of was to be noticed in the selection of RD cells by the dye. This investigation was supported by the Deutsche Forschungsgemeinschaft. We are indebted to the European Molecular Biology Organization for the award of a short-term fellowship to K. W. The skilled technical assistance of Mrs. HEINERICH Mrs. SCHICHTEL is gratefully acknowledged. 1 B. EPHRUSSI, Ann. Inst. Pasteur 76, 351 [1949]. 2 M. OGUR R. ST. JOHN, J. Bacteriol. 72, 5 [1956]. 3 C. RAUT, J. Exp. Cell Res. 4, 295 [1953]. 4 M. OGUR, R. ST. JOHN, S. NAGAI, Science [Washington] 3,928 [1957]. 5 S. NAGAI, J. Bacteriol. 86, 299 [1963]. 6 J. H. PARKER J. R. MATTOON, J. Bacteriol. 1, 647 [1969], 7 C. J. E. A. BULDER, Antonie v. Leeuwenhook 3, 1 [1964]. 8 K. WOLF, M. SEBALD-ALTHAUS, R. J. SCHWEYEN, F. KAUDEWITZ, Molec. Gen. Genetics 11, 11 [1971]. 9 R. SCHWAB, M. SEBALD, F. KAUDEWITZ, Molec. Gen. Genetics 11,361 [1971]. 1 H. HESLOT, C. LOUIS, A. GOFFEAU, J. Bacteriol. 14, 482 [197]. 11 T. D. BROCK, Naturwissenschaften 45, 216 [1958]. I. ZSOLT, Naturwissenschaften 47, 355 [196]. 13 J. WILSON, Genetics 56, 597 [1967]. 14 W. LASKOWSKI, Heredity 8, 79 [1954]. 15 P. HORN D. WILKIE, Heredity 21, 625 [1966]. 1 S. N. KAKAR R. P. WAGNER, Genetics 49, 213 [1964]. 17 R. SCHWAIER, N. NASHED, F. K. ZIMMERMANN, Molec. Gen. Genetics, 29 [1968]. 18 K. WOLF, unpublished results. 19 T. F. SLATER, B. SAYWER, U. STRÄULI, Biochim. biophysica Acta [Amsterdam] 77, 383 [1963]. 2 M. M. NACHLAS, S. J. MARGULIES, A. M. SELIGMAN. J. biol. Chemistry 235, 2739 [196]. 21 J. B. CLARK A. L. GREENBAUM, Biochem. J. 94, 651 [1965]. 22 S. S. KARMARKER, A. G. E. PEARSE, A. M. SELIMAN. J. org. Chemistry 25, 575 [196].