MODIFICATION AND INHERITANCE OF PLEIOTROPIC CROSS RESISTANCE AND COLLATERAL SENSITIVITY IN SACCHAROMYCES CEREVISIAE G. H. RANK

AND

A. J. ROBERTSON

Biology Department, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

K. L. PHILLIPS Prairie Regional Laboratory, National Research Council, Saskatoon, Saskatchewan, Canada Manuscript received November 20, 1974 ABSTRACT

A meiotic segregant (oliPR1) was isolated with a phenotype of multiple cross resistance and collateral sensitivity. Strain oZiPRl has increased sensitivity to ethidium bromide, dequalinium chloride, acriflavin, paromomycin and neomycin, and increased resistance to oligomycin, rutamycin, venturicidin, triethyltin bromide, antimycin, carbonylcynamide-m-chlorophenylhydrazone, tetra-N-butylammonium bromide, dibenzyldimethylammonium chloride, triphenylmethylphosphonium bromide, chloramphenicol, carbomycin, tetracycline, triton-X-165 and cycloheximide. Single gene inheritance of the cross resistance and collateral sensitivity was shown by 2:2 parental ditype segregation and reversion of the complete phenotype by a spontaneous revertant. The locus conferring the oZipRl phenotype was mapped 11.7 units from an unspecified centromere. Antibiotic resistance showed incomplete dominance, with the level of hybrid resistance dependent upon the inhibitor tested, Resistant diploids that produced four resistant ascospores were the result ob mitotic recambination prior to meio'sis. A partial revertant phenotype (sensitive 'to all inhibitors except oligomycin, antimycin and carbonylcyanide-m-chlorophenylhydrazone) was s h m to be due tu a single nuclear gene causing partial suppression of oZiPRl. Anaerobic pretreatment, 37" and 0.5 M KC1 were observed to reduce the growth of oliPRl when challenged with seven diverse inhibitors (antimycin, carbonylcyanide-m-chlorophenylhydrazone,chloramphenicol, cycloheximide, oligomycin, triethyltin bromide, and triphenylmethylphosphonium bromide). Resistance to cycloheximide was not altered by the [rho-] state. A revertant of oliPRl (sensitive to the above inhibitors but resistant to ethidium bromide, paromycin and neomycin) showed anaerobic and temperature sensitization to ethidium bromide, paromomycin end neomycin. Continuous monitoring of oxygen uptake by the revertant after anaerobic pretreatmat revealed that anaerdbiosis sensitized respiratory adaptation of the revertant to neomycin. It is proposed that oliPRl is a mutation resulting in the alteration of plasma membrane permeability to many diverse inhibitors.

HERE are several reports of multiple cross resistance to various inhibitors of mitochondrial function in yeast (LINNANE, SAUNDERS, GINGOLD and L U K I N S 1968; BUNNet al. 1970; MITCHELL et al. 1973; HOWELL et al. 1974; AVNER and GIFFITHS1973a and b; GRIVELL et al. 1973). Genetics 8 0 : 483-493 July, 1975.

484

G . H. R A N K ,

A. J. ROBERTSON

AND

K. L.

PHILLIPS

Mutants with cross resistance to closely related antibiotics have been shown to result from mutations in mt DNA (LINNANE,LAMB,CHRISTODOULOUand LUKINS1968; GRIVELL,REIJNDERSand DEVRTES1971; GRIVELLet al. 1973). Resistance of these mutants is the result of an antibiotic-resistant protein synthesis in the mitochondrion (LINNANE, LAMB,CHRISTODOULOU and LUKINS 1968; GRIVELL et al. 1973) and has been suggested to be the result of an alteration in mitochondrial rRNA (GRIVELL 1974). However, the inheritance of multipile cross resistance to chemically unrelated inhibitors in unclear. AVNERand GRIFFITHS(1973 a and b) reported on “class I” mutants resistant to diverse inhibitors of mitochondrial energy conservation and protein synthesis; tetrad analyses indicated a nuclear mode of inheritance but their observations also suggested the involvement of cytoplasmically inherited factors. A mutant resistant to four unrelated mitochondrial protein inhibitors and to oligomycin was shewn to have the cytoplasmically inherited properties of somatic segregation and [rho -1 marker loss (BUNN et al. 1970; MITCHELL et al. 1973); however, both sensitive and resistant diploids gave a nuclear segregation for resistance. The level of resistance to mikamycin was shown in one isolate (HOWELL et al. 1974) to be dependent upon an interaction between a nuclear mutation conferring wide cross resistance and an additional resistance mutation in mt DNA. Anaerobic sensitization and in vitro sensitivity to inhibitors (BUNN et al. 1970; MITCHELL et nl. 1973) d ere suggested as evidence for a resistance mechanism at the level of the mitochondrial inner membrane. We have isolated a spontaneous mutant with single nuclear-gene inherited: (i) cross resistance to 12 diverse mitochondrial inhibitors and (ii) collateral sensitivity to 5 additional mitochondrial inhibitors (RANKand BECHHANSEN 1973). Additional observations on the environmental and genetic modification of this pleiotropic phenotype are presented here; all our data can be explained by the modification in expression and segregation of a single pleiotropic nuclear gene that affects the permeability 9f the plasma membrane. M A T E R I A L A N D METHODS

Yeast strains: Strain GR359 (isolated as a chance meiotic segregant) was previously referred to as 33Z7c (RANKand BECH-HANSEN 1973) and is of genotype a his6 met t r p l o l i p R l [eryRl rho+] The designation o W R l (oligoniycin pleiotropic resistance) is used to indicate a single nuclear gene conferring multiple cross resistance and collateral sensitivity (RANKand BECHHANSEN 1973). Markers enclosed by brackets ( [ 1) refer to cytoplasmically inherited erythromycin resistance ( [ e r y K l ] )and to the cytoplasmically inherited determinant for respiratory sufficiency ([rho*+]).Strains GR350 and GR361 are spontaneously derived isolates of GR359 that have a complete and partial reversion of the pleiotropic phenotype. Strain GR317 is wild type for antibiotic resistance and has the genotype cuude2 ural OLIPSI [ERYS rho+]. Strain 18A is of genotype auru3 O L I W [rho+] and 11-1-40 is an isolate of 18A lacking mitochondrial DNA (Goldring et aE. 1970). Media: YPD: 1% yeast extract, 2% bactopeptone, 2% agar and 2% dextrose; YPG: as YPD with 4% glycerol substituted for dextrose; YPE: as YPD with 4% ethanol in place of dextrose. HUANG and LINNANE1968) containing WAM and AAM: a minimal salts medium (WALLACE, 0.5% yeast extract and supplemented with 5% glucose (WAM) or 1% ethanol (AAM) as an energy source.

MULTIPLE RESISTANCE MUTATION

485

Anaerobiosis: Anaerobiosis was achieved by the use of an anaerobic jar (BBL Gaspak Anaerobic System) except for the anaerobic growth of GR350 prior to continuous monitoring of 0, uptake; in the latter case anaerobiosis was achieved by flushing with nitrogen prior to capping of the growth flask with a fermentation lock. Growth inhibition: Cultures were initially taken from agar slants and grown in liquid YPD for 16-18 hours at 30". Anaerobic treatment was achieved by placing cells in W A M at an optical density (640 nm) of 0.1 unit per ml; cultures were then incubated at 30" for 24-48 hours in an anaerobic jar. After anaerobic incubation the culture was cooled to O", cells were collected by centrifugation, washed once with H,O, and resuspended to an optical density of 0.4 units/& i n 8 x 150 mm tubes containing AAM plus inhibitor. Cultures were incubated at 30" on a rotary action shaker (250 rpm) for 25 hours. After 24 hours growth was measured by determining the optical density and expressed as the percentage of growth in AAM lacking the inhibitor. The effect of inhibitors on aerobic growth was determined in a similar manner except that cells were adapted for 24 hours in AAM prior to inoculation. The effect of temperature and salt on aerobic growth was determined by placing adapted cells in AAM a t 37" and in AAM plus 0.5 M KC1 at 30". Antibiotic resistance: Resistance of parental strains and ascospore isolates was routinely scored as the ability to grow on YPG supplemented with one of: antimycin (0.05&g/ml), carbonylcyanide-m-chlorophenylhydrazone(CCCP) (15 ag/ml), chloramphenicol (4mg/ml), cycloheximide (0.5 @g/ml),erythromycin (3 mg/ml), ethidium bromide (1 pg/ml), neomycin (2 mg/ml), oligomycin (5 pg/ml), triethyltin bromide (TET) (0.004 al/ml), tetracycline (2 mg/ml) , triphenylmethylphosphonium bromide (TPMP) (0.5 mg/ml) . A sensitive isolate gave negative growth after one week at the above levels. The source of inhibitors is as reported previously (RANKand BECH-HANSEN 1973) with the addition that cycloheximide was purchased from Nutritional Biochemicals Corporation and triton-X-165 was donated by ROHMand HAAS. Continuous monitoring of oxygen uptake: A constant volume of air was passed through a growth flask shaken at 250 rpm at 30". At 250 rpm the oxygen supply to cells exceeds the total oxygen requirement. The oxygen content of the effluent gas was measured with a Beckman model F-3 paramagnetic oxygen analyzer (with a suppressed range of l6-21% oxygen) or a Servomex model OA184 (with a suppressed range of 20-21% oxygen; PHILLIPS 1963). RESULTS

Single gene inheritance: Antibiotic disks were previously used to score the resistant phenotype (RANK and BECH-HANSEN 1973) ; the inefficiency of this method has led to our use of replica plating onto YPG media supplemented with various inhibitors (see MATERIALS AND METHODS). Twenty eight complete tetrads from the GR359/GR317 hybrid were selected that gave 2+: 2- segregation for all 5 auxotrophic markers. Replica plating onto indicator media indicated a clear nuclear segregation for antibiotic resistance since there was a 2 resistant: 2 sensitive PD segregation for oligomycin, TET, antimycin, CCCP, TPMP, chloramphenicol, tetracycline, cycloheximide and triton-X-165. The [eryRZ]marker did not interfere with o2iPRlexpression since 4 resistant: 0 sensitive and 0 resistant: 4 sensitive tetrads both segregated 2 resistant: 2 sensitive for the other inhibitors. Unfortunately the inheritance of collateral sensitivity was not as clear. Of the 28 tetrads tested, 14 of 28 segregated 2 : 2 for ethidium bromide resistance: sensitivity, and 20 of 28 segregated 2:2 for neomycin resistance: sensitivity; the remaining tetrads segregated 4:0 or 3: 1 for resistance: sensitivity. However, the ethidium bromide- o r neomycin-sensitive ascospores from 2: 2 tetrads were always observed to be those with multiple resistance to the nine other inhibitors.

486

G. H. R A N K , A. J. ROBERTSON A N D K. L. P H I L L I P S

Thus the PD segregation shown by 2: 2 tetrads confirms the single-game inheritance of collateral sensitivity (RANKand BECH-HANSEN 1973); the lack of penetrance for collateral sensitivity in 4:0 and 3:1 tetrads has not been investigated but is presumably the result of modification in phenotypic expression by nuclear and/or mitrochrondrial loci. Further evidence fo rthe collateral sensitivity of oliPRl ise seen by the growth of GR359 and GR350 is varying concentrations of ethidium bromide, neomycin and paromomycin (Figure 1 and 2). The revertant GR350 (simultaneous loss of cross resistance and collateral sensitivity) is seen to be more resistant to ethidium bromide, neomycin and paromomycin than GR359 ( o l i p R l ) . Environmental modification of the oliPRlphenotype: The environmental modification of growth of strains GR359 and GR350 is given in Table 1. In the ab-

90 -

80 -

70 -

5r

60-

90 +-

50-

tu 5 40n 30 -

-

oGR350 IN A A M

20 -

~GR359IN A A M

10

L

01

0

I

0.50

I

1.00

I

1.50

I

2 .oo

ETHIDIUM BROMIDE (Icg/ml>

FIGURE 1.-Anaerobic

sensitization of GR350 to ethidium bromide. Strain GR350 was grown anaerobically ( ) in WAM and strains GR350 (0)and GR359 (A)were grown aerobically in AAM prior to aerobic growth in AAM with different concentrations of ethidium bromide. Growth is expressed as the percentage of aerobic growth in AAM in the absence of inhibitor. Horizontal bars (I) represent the standard error of the mean of five observations.

48 7

MULTIPLE RESISTANCE MUTATION

FIGURE 2.-Collateral sensitivity of GR359 to neomycin and paromomycin. Strain GR350 was grown for 24 hours in AAM in the presence of (i) neomycin ( A ) ; similarly GR359 was grown for 24 hours in AAM in the presence of (i) neomycin (0)and (ii) paromomycin ( a ) . Growth is expressed as the percentage of growth in AAM in the absence of inhibitor.

TABLE 1

Effect of anaerobiosis, salt and temperature on growth in the presence of various inhibitors Percent m o d *

Strain GR350

Strain GR359 Inhibitor

Antimycin CCCP Chloramphenicol Cycloheximide Oligomycin TET Triton-X-I6 TPMP Rutamycin Ethidium bromide Paromomycin Neomycin

Conc./ml

0.025 pg 1.50 pg 4.5mg 0.50pg 50.0 pg 0.DoOyF pl 100.0mg 1.0 mg 50.0pg

0.50p g 0.25mg 0.25 mg

Anaer- 0.5 M Aerobic obbic+ KC1

42

14 18

85

80 100 7 6

90 31 57 -

13 13 66

53 47

63 74 9 98

68 39

100 72 92

4 3

12 9 68

42

100 100 72

37’

22

34 31 36

72

34 -

-

-

5 - - 1 - - -

AnaerAerobic obicf

37’

- 58 - 12 20 - 57 47 - 2 8 - a3 - 36 48 - 86 78 56 88 34 47 92 58 56

-

* Cells were grown for 24 hours in AAM in the presence of inhibitor; growth is exprased as the percent d growth in AAM under aerobic conditionsin the absence of inhibitor. t Cells were grown anaerobically for 24 hrs in WAM prior to placing in AAM plus inhibitor.

488

G. H. R A N K , A. J. ROBERTSORi A N D K. L. PHILLIPS

sence of any treatment (anaerobiosis, 37" or 0.5 M KCI) GR359 is more resistant to antimycin, CCCP, chloramphenicol, cycloheximide, oligomycin, TET, tritonx-165, TPMP or rutamycin, and more sensitive to ethidium bromide, paromomycin and neomycin than strain GR3SO. In addition anaerobiosis, 37" and 0.5 MKCl sensitized GR359 to all inhibitors except triton-X-I 65. Similarly, anaerobiosis and 37" sensitized GR350 to ethidium bromide, neomycin and paromomycin. Challenge of GR350 to varying concentrations of ethidium bromide after anaerobic sensitization resulted in a phenotypic expression similar to GR359 (Figure 1). The effect of neomycin on 0, uptake of GR350 after an anaerobic pretreatment was measured (Figure 3). In the ibsence of neomycin there was a steady increase in 0, uptake to a maximum value of 1.529 mM/hr/g dry wt. at 23 hours. In contrast, neomycin dramatically interfered with respiratory adaptation if present at the onset of aerobiosis; there was no 0, uptake for 5 hours. followed by a sharp increase at 6 hours that resulted in a final 0, uptake of 0.280 mM/hr/g dry wt. at 23 hours. Addition of neomycin after respiratory adaptation (8 hours of aerobiosis) had no immediate effect on 0, uptake but resulted in a gradual decline in 0, uptake to 0.616 mM/hr/g dry wt. at 23 hours, Cycloheximide resistance: Resistance to cycloheximide (an inhibitor of cytoplasmic protein synthesis) appears not be be due to an alteration of the mitochondrion since: (i) the maximum non-inhibitory concentration for GR359 was

NO NEOMYCIN NEOMYCIN AFTER 8HR. AEROBIC GROWTH

1.25 -

A

NEOMYCIN DURING ANAEROBIC 8 AEROBIC

1.529

GROWTH

I

d 3l

21.00-

z

~

,--..

\.'~

E

d

-

'~ b.

-* '.

..

-._ --.

\. I.

0.616

FIGURE 3.-Effect of anaerobiosis on the resistance of GR350 to neomycin. Cells were grown 48 hours anaerobically in WAM prior to aeration in YPD. The oxygen content of effluent gas was continuously monitored by a paramagnetic oxygen analyzer (PHILLIPS 1963). All values entered for all three treatments are the mean of two experiments. Numerical values given at 23 hours are mMO,/hr/g dry wt. 0 : no addition of neomycin; U: neomycin (1 mg/ml) added after 8 hours of aerobic growth; A: neomycin present during anaerobic and aerobic growth.

489

MULTIPLE RESISTANCE MUTATION

TABLE 2 Parental and hybrid resistance* M a x i m u m non-inhibitory concentrations Inhibitor

Medium

Antimycin Chloramphenicol Cycloheximide Cycloheximide Oligomycin

YPE YPE WD YPE

0.005 p g / d 0.1 m g / d 0.025 p g / d 0.025 p g / d

YPE

0.10

GR317

GR359

pg/ml 5.0 m g / d 0.25 pg/ml$ 0.25 p g / d >IO pg/d >O.l

GR317/GR359+

0.025 pg/ml 3.0 m g / d 0.25 p g / d 0.10 p g / d 1.0 P d m l

* Parental and hybrid cells were g r o w n on solid medium for 72 hours. Individual colonies were diluted in water; a sample of 2.5 x 1 6 6 O.D. (640nm) was then placed onto media containing different concentrations of inhibitor. Inhibitor levels are the maximum non-inhibitory concentratims after 3 days d growth. C Strain GR359 was used in both the rrho+l and r r b - ( n )1 state: since fiftv " zygote - - colonies from each cross gave essentially the samgresdti, onIy bne @'of' data i s &en. $me[rho+] and [rho-] isolates of GR359 were resistant to the same concentrations of cycloheximide on YPD. the same if a fermentable (YPD) or non-fermentable (YPE) energy source was used, and (ii), [rho+] and [rho-] isolates of GR359 had the same level of resistance on YPD containing cycloheximide (Table 2). Dominance: Dominance was found by subjecting the GR359/GR317 hybrid to varying concentrations of inhibitors. The level of hybrid resistance was dependent upon the inhibitor used (Table 2) ; on YPE the hybrid was 5,30,4 and 10 times more resistant to antimycin, chloramphenicol, cycloheximide and oligomycin than the sensitive parent (GR317). In all cases the hybrid was not as resistant as the resistant parent (GR359). Centromere linkage: A detailed analysis of 63 tetrads from the GR359/GR317 hybrid showed that the cytoplasmically inherited marker [ e r y R l ] segregated 4R:OS as expected. Centromere linkage for chloramphenicol and oligomycin resistance was clearly demonstrated since all 3 known centromere-linked markers (trpl, his6 and a) produced F-distributions (PD=NPD, lT