Molec. gen. Genet. 156, 93-98 (1977) © by Springer-Verlag 1977

Isolation of Metabolically Active Petite Mutants of Kluyveromyces lactis, a Petite-Negative Yeast

John Heritage and Peter A. Whittaker Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton BNI 9QG, U.K.

Summary. Conditions under which complete cultures of the petite-negative yeast Kluyveromyces lactis can be converted to metabolically active petite mutants have been found. These mutants, which lack mito- chondrial protein synthesis have been shown to be metabolically active by their ability to exclude the dye trypan blue. They appear to possess a functional protein synthesising system, which is sensitive to the inhibitor trichodermin. However, on transfer to solid nutrient medium, these mutants fail to grow normally, and give rise to microcolonies composed of up to a thousand cells. These colonies autolyse after several days.

Introduction

The petite mutation, first described by Ephrussi et al. (1949a, b, c) in baker 's yeast (Saccharomyces cere- visiae) is a cytoplasmically inherited mutat ion charac- terised by respiratory deficiency, associated with gross alteration or complete loss of mitochondrial D N A (Nagley and Linnane, 1970; Faye et al., 1973). As mitochondrial D N A carries the genetic information for mitochondrial r ibosomal R N A (Wintersberger, 1967; Casey et al., 1972) and mitochondrial t R N A (Casey et al., 1972) synthesis, mitochondrial protein synthesis is absent from petite mutants. Consequently these mutants are incapable of synthesising those protein subunits of the oxidative phosphorylat ion sys- tem which are synthesised within the mitochondria (Tzagaloff et al., 1973).

The majority of yeast species (about 70%) were found to be unable to form viable petite mutants (Bulder, 1963) and these are termed "petite-negative" yeasts. It was suggested that these yeasts are able

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to undergo the petite mutagenic process, but the loss of functional mitochondrial D N A proves lethal. On plating cultures of petite-negative yeasts exposed to petite mutagens, the cells undergo a very limited number of divisions to form microcolonies, which in most petite-negative species have never been shown to develop into normal colonies.

Many agents have been shown to cause the petite mutation, the phenanthridine dye ethidium bromide being the most potent known mutagen (Slonimski et al., 1968) preventing de novo synthesis of mito- chondrial DNA, and allowing degradation of pre- existing mitochondrial D N A (Perlman and Mahler, 1971).

When grown in 10 gg/ml ethidium bromide, the petite-negative yeast Kluyveromyces lactis has been shown to lack cytochromes b and a a3, components of which are known to be synthesised intramitochon- drially in S. cerevisiae, but true petite mutants of K. lactis are not formed under these conditions, as transfer to drug-free medium permits rapid resyn- thesis of these cytochromes (Luha, 1972).

We have studied the effects of higher concentra- tions of ethidium bromide on K. lactis and have found conditions under which large quantities of true petite mutants can be isolated. These have been subjected to a preliminary biochemical investigation.

Materials and Methods

Strain. K. lactis 1140-R was derived originally from a haploid pro- totrophic culture NRRL Y-1140 donated by Dr. A. Herman. Luha (1972) demonstrated the existence of two discreet cell types in the culture, distinguished using the tetrazolium overlay technique (Ogur et al., 1957). One cell type produces darker red colonies than does the other, implying greater respiratory activity in the darker (R) type than in the other (P). This characteristic has been shown to be stable through many generations, and it was cells from the laboratory strain l140-R that were used in this study.

Results Cultural Conditions. Cells were grown in a liquid medium contain- ing (NH4)-Hz.PO4 (10 g/l); MgSO4 (2 g/l); Difco yeast extract (5 g/l) and glucose at a final concentration of 0.2 M. The solid medium contained Difco yeast extract (10 g/l); Oxoid Bacteriolog- ical peptone (10 g/l); Oxoid agar No. 3 (15 g/l) and glucose at a final concentration of 0.2 M. Starvation medium contained the same ingredients as liquid growth medium omitting the yeast ex- tract and glucose. All media and a stock solution of ethidium bromide (10 mg/ml) were autoclaved for 20 min at 121°C prior to use. The protein synthesis inhibitors were sterilised by ethanolic solution. The final ethanol concentration of control cultures were adjusted accordingly.

Except where stated, cells were grown at 30°C in a Gallenkamp Orbital incubator rotating at 250 rpm. Plates were incubated at 30°C for three days, after which time the viability was scored. Anaerobiosis was achieved using an anaerobic jar with a Gaspak system (BBL. Cockeysville, Maryland 21030, U.S.A.).

Cell Counting. Total cell counts were made by counting suitable dilutions using a haemocytometer. Total viable counts were made by plating 0.1 ml aliquots directly or after dilution. When counting colonies, all those with less than a thousand cells (microcolonies) Were disregarded, as these have never been shown to recover to give normal colonies. Numbers of metabolically active cells were assessed using a modification of the technique of Moore et al. (1966). Equal volumes of cell suspensions and 0.025% trypan blue were incubated together for five minutes, and then compared mi- croscopically with heat killed cells.

1013

The Effects of Varying Ethidium Bromide Concentrations

Figure 1 shows the effect of ethidium bromide at four concentrations on the viability of K. lactis measured over 24 h. It can be seen that as the concentration of ethidium bromide increases so the percentage ~iability increases. It is also apparent that the mean cell doubling time increases with increasing drug concentration.

When S. cerevisiae is incubated in high concentra- tions of ethidium bromide for a relatively short time, it has been shown that after a rapid initial mutagen- esis, there is a recovery to wild-type, followed by an irreversible mutation (Hall et al., 1976; Criddle et al., 1976). This effect may also be observed in K. lactis, but only when ethidium bromide concentra- tions of 200 ~tg/ml are used. This compares with an ethidium bromide concentration of 100 gg/ml re- quired to bring about the same effect in S. cerevisiae (Fig. 2).

Cytochrome Spectra. These were measured on washed cells pre- pared in the following way:

2 x 109 cells were washed and suspended in a sodium hypochlo- rite solution (0.005% w/v available chlorine), and then resuspended in 4 mls of 50% glycerol. The reduced sample was prepared by washing the same number of cells twice in distilled water and then resuspending them in 4 mls of 50% glycerol. A few crystals of sodium dithionite were added to the reduced sample. The spectra were measured using a Perkin Elmer model 356 dual wavelength recording spectrophotometer operating in the "sp l i t " mode with both monochromators scanning the same wavelength.

Ethidium Bromide Treatment under Non-Growing Conditions

It seemed likely that the increased viability observed with increased drug concentration may have been due to the increase observed in the mean cell doubling time. Assuming that, unlike with S. cerevisiae, cell proliferation was necessary for efficient ethidium bro-

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94 J. Heritage and P.A. Whittaker: Petite Mutants of Kluyveromyces lactis

Fig. 1 a and b. Growth kinetics of K. lactis in ethidium bromide: cells were grown in liquid nutrient medium containing ethidium bromide at the following concentrations: e - e - e 25 gg/ml, A-zx-zx 50 gg/ml, A - , t - A 75 lag/ml, • " - = 100 Ixg/ml. a total cell count; b % cells remaining viable

J. Heritage and P.A. Whittaker: Petite Mutants of Kluyveromyces lactis 95

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cells were grown in liquid medium containing ethidium bromide at the following concentrations: • • - • 251~g/ml, zx zx-zx 50gg/ ml, • -A • 75txg/ml, = - • - = lOOgg/ml, v -v 7 150~tg/ml, o '.~ ~ 200 ~tg/ml. % petites was calculated from the difference between the haemocytometer count and the plate count

mid• induct ion o f petite mutants , decreasing the cell doubl ing time would decrease the muta t ion rate. This was investigated by incubat ing the cells under a vari- ety o f non-growing condit ions, and scoring viability after 24 h (Fig. 3).

We have shown that the more stringent the growth prevent ion is, the less likely are cells to mutate. The difference observed between freestanding and shaking cultures is due to oxygen depletion of the freestanding cultures causing cessation of g rowth in this yeast, an obligate aerobe. There would also be a localised depletion o f other nutrients and a build up o f toxic waste products as the cells settle to the b o t t o m of the culture vessel.

Isolation of Petite Mutants

Treatment o f K. lactis with 10 gg/ml ethidium bro- mide causes the cells to become respiratory deficient, but true petite mutants are not fo rmed under these conditions. Remova l f rom ethidium bromide allows a rapid regenerat ion o f respiratory activity (Luha, 1972). F r o m our data we have found that when much higher concentra t ions o f ethidium bromide are used, its efficacy as a petite mutagen for this species is markedly reduced. It is consequent ly necessary to use an intermediate ethidium bromide concent ra t ion to achieve high levels o f muta t ion. The following proto- col was developed in order to obtain a culture com- posed a lmost entirely o f petite cells.

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Ethidium bromide concn.(IJM) Fig. 3. Effects of ethidium bromide on the viability of K. lactis under varying physiological conditions: o-©-o cells incubated aerobically in liquid nutrient medium at 4 ° C, ~7-v v incubated anaerobically at 30 ° C in liquid nutrient medium, • - - - = incubated anaerobically at 30°C in starvation medium, A-A • incubated aerobically at 30 ° C in starvation medium, zx-~-zx incubated aero- bically at 30 ° C in liquid nutrient medium without shaking, • e - • incubated aerobically at 30 ° C in liquid nutrient medium with shak- ing

Liquid growth medium containing 25 gg/ml ethi- d ium bromide was inoculated with 105 cells per ml and incubated with aerat ion at 30°C. After 24 h, all but a very small residue o f cells have been converted into petite cells. The remaining wild type cells were eliminated by re-inoculating the culture in fresh me- d ium containing the same concentra t ion o f ethidium bromide and incubating for a further 24 h. After this treatment, a lmost all o f the cells are judged to be petite mutants by their inability to give rise to any normal sized colonies when plated out on to nutrient agar containing glucose as a sole ca rbon source. In a typical experiment, only 1 in 106 cells remain wild- type under these circumstances. All the cells at this stage have a normal appearance in the light micro- scope, and were all judged to be metabolical ly active by their ability to exclude the dye t rypan blue.

Biochemical Characterization of Petite Mutants of K. lactis

It has long been known that cy tochromes b c ci and a a 3 disappear f rom S. cerevisiae during anaerobiosis, to be rapidly resynthesised during aerobic adapta t ion

96 J. Heritage and P.A. Whittaker : Petite Mutants of Kluyveromyces lactis

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Fig. 4a-f. Drug effects on the cytochrome spectrum of K. lactis: a wild-type cells, b petite mutants before removal from ethidium bromide (25 gg/ml), c petite mutants suspended in liquid nutrient medium for 11/2 h, d petite mutants suspended in liquid nutrient medium containing chloramphenicol for 11/2 h, e petite mutants suspended in liquid nutrient medium containing erythromycin for 11/2 h, fpetite mutants suspended in liquid nutrient medium con- taining triehodermin for 11/2 h

(Ephrussi and Slonimski, 1950). The presence of the antibiotics chloramphenicol (Huang et al., 1966) and erythromycin (Clark-Walker and Linnane, 1966) has been shown to cause loss of cytochromes b and a a3, although cytochrome c is maintained under these conditions. This work was extended to the petite nega- tive yeast Candidaparapsilosis (Kellerman et al., 1969) and K. lactis (Luha, 1972). These workers showed that the same antibiotics prevented synthesis of proteins produced at least partly within the mitochon- dria.

The cytochrome spectrum of petite mutants of K. lactis (Fig. 4b) has been compared with the spec- trum of wild-type cells (Fig. 4a). This shows that pe- tite cells contain cytochrome c (550 nm peak), but the bands for cytochrome b (560 nm peak) and cy- tochromes a a3 (605 nm) are missing. An extra peak at 503 nm has also appeared in the petite cells. It

seems likely that this is due to the presence of protote- trahydroporphyrin IX (P-503), a haem precursor (Poulson et al., 1974).

When petite mutants of K. lactis are transferred to drug free medium, no resumption of synthesis of cytochromes b or a a 3 occurs and the levels of cyto- chrome candP-503 drop slightly (Fig. 4c). This suggests that these cells are true petite mutants, unlike cells treated with lower concentrations of ethidium bro- mide (Luha, 1972). As would be expected from petite cells lacking mitochondrial protein synthesis, the pres- ence of chloramphenicol (4 mg/ml) or erythromycin (2 mg/ml) during the recovery from ethidium bromide do not affect the pattern of cytochromes (Figs. 4d and e). Inclusion of trichodermin (5 gg/ml) in the recovery medium (Fig. 4f) causes a total loss of cy- tochrome c and P-503. (K. lactis is resistant to the effects of cycloheximide, and hence the use of tricho- dermin to inhibit cytoplasmic protein synthesis).

Discussion

In contrast to the situation in S. cerevisiae, where ethidium bromide induces petite mutants in non-di- viding cells, when growth of K. lactis is prevented, the potency of ethidium bromide as a petite mutagen declines, and also the concentration dependant in- crease in viability becomes abolished.

In order to explain the increase in percentage viability of K. lactis with increasing concentrations of ethidium bromide, it is necessary to consider also the increase in the mean cell doubling time. The impli- cation of this is that during the same time interval, cells with a high growth rate will have a greater oppor- tunity to produce petite mutants than will cells with a slow growth rate (assuming the mutation rate to be similar in each case). Our results confirm that cell division is essential for mutation in K. lactis caused by ethidium bromide. This is not the case for S. cerevisiae where non-growing cells undergo ra- pid mutation in the presence of ethidium bromide (Slonimski et al., 1968). However, acriflavine has been shown to require cell proliferation in S. cerevisiae and only daughter cells become mutated (Ephrussi and Hottinguer, 1950).

This viability effect is, however, quite different from the recovery from high concentrations of ethi- dium bromide (Fig. 2). This has been explained (Hall et al., 1976; Criddle et al., 1976) as stimulation in S. cerevisiae of a nuclease at low concentrations of ethidium bromide, causing fragmentation of mito- chondrial DNA (manifest as the rapid initial muta- genesis), and inhibition at high concentrations of ethi-

J. Heritage and P.A. Whittaker : Petite Mutants of Kluyveromyces lactis 97

dium bromide allowing repair to occur (and hence recovery). This is made possible because of the slow penetration of ethidium bromide into cells (Mahler and Bastos, 1974). This effect can also be seen in K. lactis when incubated in extremely high concentra- tions of ethidium bromide.

The petite mutants we have isolated have been shown to possess metabolic activity by their exclusion of trypan blue. As well as this activity, the results from cytochrome measurements of petite mutants during recovery from ethidium bromide infers that some de novo synthesis of cytochrome c may be oc- curring alongside degradation of pre-existing cyto- chrome c. The degradation is suggested by the small drop in levels of cytochrome c and P-503 during re- covery. Support for the hypothesis that cytochrome c and P-503 synthesis occurs during recovery comes from the study of the effects of protein synthesis in- hibitors during recovery. These act selectively on the cytoplasmic or mitochondrial protein synthesising system. Trichodermin was shown to cause a total loss of cytochromes implying that turnover of cyto- chrome c and P-503 may be occurring in the absence of this drug.

The lack of appearance of any cytochromes b or a a3 and the absence of an effect of mitochondrial protein synthesis inhibitors lead to the conclusion that petite mutants of K. lactis have lost any functional mitochondrial protein synthesis, but retain a func- tional cytoplasmic protein synthesising system and metabolic activity.

This result is at variance with the results of Zen- naro et al. (1977) and Algeri et al. (1977) who, using a different strain of K. lactis, have shown that loss of functional mitochondrial protein synthesis causes loss of cytoplasmic protein synthesis. However, our results are supported by the work ofLuha et al. (1971) and Dobelli (1976) and the difference has been resolved (Algeri et al., 1977) as being due to differ- ences between the strains of yeasts used.

It has been demonstrated previously that loss of viability of petite mutants of K. lactis is not merely the result of a loss of oxidative phosphorylation activ- ity (Luha, 1972). The ability to prepare in large quan- tities, pure cultures of metabolically active petite mu- tants of Kluyveromyces lactis provides the possibility of investigation of the lesion which causes inviability in these mutants.

We are grateful to Prof. D.A. Long, District General Hospital, Eastbourne, for giving John Heritage the opportunity to carry out this work. John Heritage is attached to the Microbiology Labo- ratory, Department of Pathology, District General Hospital, East- bourne. Grant support from the Royal Society is gratefully ack- nowledged.

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98 J. Heritage and P.A. Whittaker: Petite Mutants of Kluyveromyces lactis

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Communicated by F. Kaudewitz

Received June 2, 1977