Molec. gen. Genet. 137, 269--276 (1975) © by Springer-Verlag 1975

Cytoplasmic and Nuclear Mutations to Chloramphenicol Resistance in Aspergillus nidulans I. A. U. N. Gunatilleke, C. Scazzocchio a n d H. N. Arst, J r . Department of Genetics, University of Cambridge, Cambridge, England Received February 11, 1975

Summary. Two chloramphenicol resistance mutations out of 123 tested in Aspergillus nidulans are inherited extranuclearly as judged by transmissibility in heterokaryons, lack of segregation at meiosis, and independent segregation from all of the eight nuclear linkage groups. They do not recombine with each other. However, experiments in collaboration with G. Turner and R. T. Rowlands show that they do recombine with cytoplasmic mutations to oligomycin resistance (Rowlands and Turner, 1973) and cold-sensitivity (Waldron and Roberts, 1973). These cytoplasmic chloramphenicol resistance mutations are stable and do not affect growth or morphology on antibiotic-free media. Nuclear mutations to chloramphenicol resistance map at a minimum of three loci. At one of these loci, most, but not all, mutations lead pleiotropicallyto cycloheximide hypersensitivity, and most of these, but not all, also confer pleiotropic hypersensitivity to salicylhydroxamic acid. Introduction I n h i b i t o r s of p r o k a r y o t i c p r o t e i n synthesis h a v e been useful selective a g e n t s for o b t a i n i n g m u t a t i o n s in organelle genomes of a n u m b e r of e u k a r y o t i c o r g a n i s m s (Schlanger a n d Sager, 1974; B u n n et al., 1974; R o b e r t s a n d Orias, 1973; B u n n et al., 1970; K u t z l e b et al., 1973; T r e m b a t h et al., 1973; H o w e l l et al., 1974; Grivell et al., 1973; Beale et al., 1972; Beale, 1973; A d o u t t e a n d Beisson, 1970). H e r e we show t h a t in Aspergillus nidulans m u t a t i o n s in a t l e a s t one e x t r a n u c l e a r , pres u m a b l y m i t o c h o n d r i a l , gene a n d a t least t h r e e n u c l e a r genes can confer a subs t a n t i a l degree of resistance t o chloramphenicol. A p r e l i m i n a r y r e p o r t of t h i s w o r k was p r e s e n t e d to t h e G e n e t i c a l S o c i e t y of G r e a t B r i t a i n ( G u n a t i l l e k e et al., 1974).

Materials and Methods

1. Strains A. nidulans strains employed here carried markers in general use (Clutterbuck and Cove, 1974).

2. I n vivo and Genetical Techniques Growth testing and genetical techniques for A. nidulans have been described previously (Arst and Cove, 1969, and references therein). Resistance to chloramphenicol was determined at 2.5 to 6 mg/ml in appropriately supplemented minimal medium (Cove, 1966) containing 1% (w/v) D-glucose, 1% (v/v) ethanol or 144 mM acetate (as the sodium salt, adjusted to pH 6.5) using 10 m!V[ Ntt~ (as the (~)-tartrate) or 5 mM urea or 10 mM NO~ (Na+ salt) as nitrogen source. Unlike Saccharomyces cerevisiae (Roodyn and Wilkie, 1968), A. nidulans is not appreciably more sensitive to chloramphenicol toxicity when using a non-fermentable carbon source such as ethanol or acetate than when using a fermentable carbon source such as glucose. This is in accord with the obligately aerobic metabolism of A. nidulans. Similarly chloram-

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phenicol toxicity is not affected by whether NI-I~, urea or NO~ serves as nitrogen source. Hypersensitivity to eycloheximide was determined at 500 ~g/ml, a concentration not toxic to the wild type, in appropriately supplemented glucose-urea minimal medium. Hypersensitivity to salicylhydroxamic acid was determined at 1.5 mg/ml, a concentration very slightly toxic to the wild type, in appropriately supplemented glucose-urea minimal medium.

3. Selection o/Mutants Chloramphenieol resistant mutants were selected after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis (Alderson and Hartley, 1969) in a strain of genotype yA-2 pyroA-4 cnxC-3 (yellow conidial colour, pyridoxine-rcqniring, unable to utilise nitrate or hypoxanthine as nitrogen source) as able to grow on minimal medium containing 4 mg/ml chloramphenieol, 5 mM urea as nitrogen source, and 50 ng/ml pyridoxine hydrochloride. Mutants with allele numbers 1-100 were selected using 1% D-glucose as carbon source, 101-150 using 1% ethanol as carbon source and 151-200 using 144 mM acetate as carbon source.

4. Heterolcaryon Test The heterokaryon test for cytoplasmic inheritance was developed by Jinks (1963). In order to screen a large number of mutations for cytoplasmic inheritance, we developed the following version: The resistant mutant strain to be tested and the sensitive tester strain differ in conidial colour, carry complementing mutations resulting in inability to utilise nitrate as nitrogen source, and require at least one different nutritional supplement each. Here, for example, chloramphenieol resistant mutants selected from the yA-2 pyroA-4 cnxC-3 strain were tested in heterokaryons with the sensitive tester strain of genotype biA-1 luA-1 cnxH-5 (biotinrequiring, L-leucine-requiring, unable to utilisc nitrate or hypoxanthine as nitrogen source), which has green (wild type) conidiospores. Nitrate non-utilising strains readily form balanced heterokaryons when inoculated at the same site on glucose-minimal medium containing nitrate as sole nitrogen source and supplements for the nutritional requirements (Rever, 1965). Heterokaryons obtained in this way can generally be tested directly, but an occasional unbalanced heterokaryon may require an additional transfer to glucose-nitrate minimal medium to ensure cytoplasmic mixing. Conidiospores from balanced heterokaryons are collected in sterile distilled water containing 0.1% (v/v) twreen 80. A loopful of conidiospore suspension is then put on each of the following four media to test for extranuclear inheritance and incubated at 37 ° C for 2-4 days: A. Glucose-urea minimal medium + nutritional supplements for both strains. B. Glucose-urea minimal medium ~- nutritional supplements for both strains + inhibitor. C. Glucose-urea minimal medium -{- nutritional supplements for resistant strain -t- inhibitor. D. Glucose-urea minimal medium Jr nutritional supplements for sensitive, tester strain + inhibitor. If the resistance mutation is nuclear, growth can be seen on media A, B and C but not D. If the resistance is due to a cytoplasmic mutation, then one of two results is possible: (1) If the mutation is lost during heterokaryosis, no growth can be seen on media B, C and D. (2) If the mutation reassorts during heterokaryosis, growth can be seen on all four media. I t is necessary to verify, using conidial colour and growth habit, that growth on inhibitor-containing media does not result from diploids formed during heterokaryosis and able to grow because of partial or complete dominance of the resistance marker. Results and Discussion

1. Cytoplasmic Mutations T w o o u t of 123 c h l o r a m p h e n i c o l r e s i s t a n c e m u t a t i o n s t e s t e d w e r e f o u n d t o be c y t o p l a s m i c a l l y i n h e r i t e d b y t h e h e t e r o k a r y o n t e s t . B o t h of these, (camA-112) a n d (camA-141) w e r e s e l e c t e d u s i n g e t h a n o l as c a r b o n source, b u t w e do nob k n o w w h e t h e r t h i s h a s a n y s i g n i f i c a n c e since t h e y also c o n f e r r e s i s t a n c e o n g l u c o s e a n d

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acetate. (camA-112) and (camA-141) were subjected to more extensive heterokaryon tests using several different sensitive tester strains. I n each case, in addition to strains having each of the parental genotypes, the progeny of the heterokaryons included the two recombinant classes in which the (camA) marker had reassorted with respect to the nuclear genome. Table 1 compares progeny obtained from three independent heterokaryons formed from the same pair of strains. Velveteen replica plating following Rowlands and Turner (1973, 1974) showed t h a t the (camA) markers, like the also extranuclear (oliA-1) and (cs-67), rapidly assort to yield resistant and sensitive heterokaryons or sectors of heterokaryons. This accounts for the difference between progeny of heterokaryons I, I I and I I I in Table 1. Sensitive and resistant heterokaryons retain their respective phenotypes upon repeated subculture. Occasionally, areas of intermediate resistance occur on the chloramphenieol-containing replica plate. Conidiospores from these areas give rise to sensitive, resistant, and mosaic (mixed sensitive and resistant) colonies. However, the heteroplasmio state is only transient, and subculture of hyphae or conidiospores quickly leads to pure breeding strains of both resistant and sensitive types. Table 1. Progeny analysis of three independent heterokaryons formed between a (camA) and a sensitive tester strain Nuclear genotype

Number of progeny from heterokaryon having extranuelear genotype I

(camA-112) yA-2 pyroA-4 cnxC-3 15 biA-1 luA-1 cnxH-5 28

II

III

(cam+) (camA-li2) (cam+) (camA-112) (cam+) 16 11

43 30

1 0

2 1

39 33

Three independent heterokaryons were formed between a ehloramphenicol resistant strain yA-2 pyroA-4 cnxC-3 (camA-ll2) and the sensitive tester strain biA-I luA-1 cnxH-5.

Cleistothecia from sexual crosses between (camA) and wild type strains, whether crossed or whether selfed from either nuclear genotype parent, contained either all resistant or all sensitive ascospores. Similar b~haviour has been described for (oliA-1) (Rowlands and Turner, 1973) and (cs-67) (Waldron and Roberts, 1973). Presumably, the cytoplasmic genotype of the region in which fertilisation occurs determines the genotype of the ascospores independently of the nuclear genotype of the zygote giving rise to the eleistothecium. Further confirmation of the cytoplasmic inheritance of (camA-112) and (camA-141) was obtained b y showing t h a t they do not segwegate with any of the eight nuclear linkage groups. Both resistant and sensitive diploid strains were obtained from heterokaryons between (camA) strains and chloramphenicolsensitive " m a s t e r strains" (McCully and Forbes, 1965), which carry at least one marker in each of the eight nuclear linkage groups. All haploids obtained upon haploidisation of these diploids retained the chloramphenicol phenotype of the

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parental diploid. Presumably t h e cytoplasmic genotype of a diploid strain merely reflects the cytoplasmic genotype of the region in which the nuclear fusion event occurred. (camA-112) and (camA-141) are stable and do not affect growth on chloramphenieol-free media. They do not affect sensitivity to cyeloheximide, which inhibits cytoplasmic protein synthesis in A. nidulans (Turner, 1973), or neomycin, which inhibits both mitochondrial and cytoplasmic protein synthesis in Saccharomyces cerevisiae (Davey et al., 1970). Growth of wild type A. nidulans is not affected by erythromycin, spiramycin, oleandomycin, lincomycin, or tetracycline, all of which resemble chloramphenicol in inhibiting mitochondrial protein synthesis in yeast (Lamb et al., 1968). Experiments in collaboration with Drs. G. Turner and R. T. Rowlands (unpublished) show that (camA-112) recombines readily with (oliA-1) and (cs-67). Double and triple mutants of extranuclear genotypes (camA-112 oliA- 1), (camA- 112 cs-67) and (camA-112 oliA-1 cs-67) have been obtained without difficulty and no interactions between (camA-112) and (oliA-1) or (cs-67) have been detected. No chloramphenicol-scnsitive progeny have been obtained among a large number of eonidial isolates of heterokaryons between a (camA-112) strain and a (camA- 141) strain. This tight linkage probably indicates allelism since the (camA) markers apparently do not interfere with extranuclear recombination between (oliA-1) and (cs-67). We cannot, however, eliminate the possibility that (camA-112) and (camA.141) are not independent mutations but resulted from a single spontaneous event prior to mutagenesis.

2. Nuclear Mutations Seventeen nuclear mutations were tested for possible atlelism on the basis of tight linkage. They fell into three distinct groups within which no ehloramphenicol-sensitive recombinants were obtained among at least one hundred progeny from a sexual cross. Haploidisation analysis (McCully and Forbes, 1965) located camB (allele numbers 105, 138 and 139) to linkage group II, camC (allele numbers 108 and 129) to linkage group I, and camD (allele numbers 26, 27, 30, 31, 32, 35, 36, 37, 39, 47, 101 and 147) to linkage group V. camB-105, camC-108 and camD-27 were all shown to be partially dominant in diploids. No pleiotropic effects of camB or camC mutations were detected. However, eleven of the twelve cared mutations (careD-26, -27, -30, -31, -35, -36, -37, -39, -47, -101 and -147) lead pleiotropieally to hypersensitivity to cycloheximide toxicity and eight of these (careD-26, -27, -30, -31, -35, -36, -37, and -47) also lead to hypersensitivity to salicylhydroxamic acid (SHAM) toxicity. The existence of three distinct mutant cared phenotypes has been confirmed by extensive progeny analysis upon outcrossing strains carrying careD-27, careD-32, and camD-39, representing the three phenotypes. The location of camD in linkage group V shows that cared mutations cannot be allelic to as-25 (Waldron and Roberts, 1974a, b), which also confers hypersensitivity to cycloheximide but has been located to linkage group IV or VI. Similarly, careD mutations cannot be alleles at the ActA (Wart and Roper, 1965) or arpA (Waldron and Roberts, 1974a, b) loci, where mutations to cyclohcximide

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resistance have been described, since these are in linkage groups I I I and either VI or VII, respectively. Although the pleiotropie effects of careD mutations are potentially revealing, the role of the careD gene remains obscure. In Saccharomyces cerevisiae highly pleiotropic nuclear mutations have been described and proposed to affect components of the mitochondria] inner membrane (Rank and Bech-Hansen, 1973; Rank, 1974). However, camD mutations would appear to differ from the yeast mutations in that they can affect growth responses to a substance whose toxicity presumably does not require transport into mitochondria, cycloheximide. Cycloheximide toxicity is almost certainly correlated with its ability to inhibit cytoplasmic protein synthesis in A. nidulans (Cybis and Weglenski, 1972; Turner, 1973; Waldron and Roberts, 1974b). Thus if camD mutations affect membrane components, these would probably have to be components of the cytoplasmic rather than mitochondrial membrane. The involvement of growth responses to SHAM is perhaps slightly less obscure. Aspergillus nidulans, like Neurosporct crassa (Lambowitz and Slayman, 1971; Lambowitz, Slayman, Slayman and Bonner, 1972; Lambowitz, Smith and Slayman, 1972a, b; Jagow et al., 1973; Edwards et al., 1974), possesses an alternate respiratory pathway which is insensitive to cyanide and antimycin A but is subject to inhibition by salicylhydroxamate (unpublished results). As with N. crassa (Lambowitz and Slayman, 1971; Lambowitz, Smith and Slayman, 1972b; Jagow and Klingenberg, 1972; Edwards et al., 1974), growth in the presence of chloramphenicol increases the capacity for SHAM-sensitive respiration relative to cyanide-sensitive respiration. I t is therefore relevant whether in vivo sensitivity to SHAM might be correlated with utilisation of the alternate oxidase system. Although our first experiment indicated markedly higher SHAM-sensitive respiratory activity in a careD-27 strain than in the wild type, two subsequent experiments failed to confirm the difference, possibly owing to poorer quality of the mitochondrial preparations. Further work is necessary to clarify the situation. Hypersensitivity to SHAM toxicity to growth is a feature of the cni-1 mutation of N. crassa, correlating with high SHAM-sensitive respiration of isolated mitoehondria (Edwards and Kwiecinski, 1973). The hypersensitivity of certain careD mutants to cycloheximide, although more marked than their hypersensitivity to SHAM, probably does not warrant extensive discussion at this point, except to note that there is evidence that in N. crassa synthesis of the components of the alternate oxidase system involves cytoplasmic rather than mitochondrial ribosomes (Edwards et al., 1974). None of the careD mutants is hypersensitive to anisomycin toxicity. Although anisomycin, another inhibitor of eukaryotic protein synthesis (Grollman and Huang, 1973), is effective in A. nidulans (Arst and Scazzocchio, 1972), there is no evidence whether this is the basis of its inhibition of growth. Acknowledgements. I.A.U.N.G. gratefully acknowledges a postgraduate studentship from the University of Sri Lanka (Vidyalankara Campus). C.S. and H.N.A. thank the Science Research Council for support through a grant to Dr. D. J. Cove. During the final part of this work, It. N. A. was supported by a Smithson Research Fellowship (Royal Society). We thank Mr. D. A. Webb and Miss B. Pritchard for valuable technical assistance.

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Lambowitz, A.M., Slayman, C.W.: Cyanide-resistant respiration in Neurospora erassa. J. Bact. 108, 1087-1096 (1971) Lambowitz, A. M., Slayman, C. W., Slayman, C. L., Bonner, W. D., Jr.: The electron transport components of wild type and poky strains of Neurospora erassa. J. biol. Chem. 247, 1536-1545 (1972) Lambowitz, A.M., Smith, E.W., Slayman, C.W.: Electron transport in Neurospora mitochondri~. Studies on wild type and poky. J. biol. Chem. 247, 4850-4858 (1972a) Lambowitz, A. M., Smith, E. W., Slayman, C. W. : Oxidative phosphorylation in Neurospora mitochondria. Studies on wild type, poky, and ehloramphenieol-inducedwild type. J. biol. Chem, 247, 4859-4865 (1972b) MeCully, K.S., Forbes, E.: The use of p-fluorophcnylalanine with "master strains" of AspergilIus nidulans for assigning genes to linkage groups. Genet. Res. 6, 352-359 (1965) ~olloy, P. L., Howell, N., Plummet, D.T., Linnane, A.W., Lukins, H.B.: Mitochondrial mutants of the yeast Saccharomyees eerevisiae showing resistance ~n vitro to ehloramphenicol inhibition of mitochondrial protein synthesis. Biochem. biophys. Res. Commun. 52, 9-14 (1973) l~ank, G. H. : Pleiotropy of cytoplasmically and nuclearly inherited resistance to inhibitors of mitochondrial function in Saccharomyces cerevisiae. Canad. J. Microbiol. 29, 9-i2 (i974) Rank, G. H., Bech-Hansen, N. T. : Single nuclear gene inherited cross resistance and collateral sensitivity to 17 inhibitors of mitochondrial function in S. eerevisiae. Molec. gen. Genet. 126, 93-102 (1973) Rever, B. M. : Biochemical and genetical studies of inorganic nitrogen metabolism in Aspergillus nidulans. Ph.D. Thesis, University of Cambridge (1965) Roberts, C. T., Jr., Orias, E. : Cytoplasmic inheritance of chloramphenicol resistance in Tetrahymena. Genetics 73, 259-272 (1973) Roodyn, D. B., Wilkie, D. : The biogenesis of mitochondria. London: Methuen & Co. Ltd. 1968 Rowlands, R. T., Turner, G. : Nuclear and extranuclear inherit~,nce of oligomycin resistance in Aspergillus nidulans. 1V[olec.gem Genet. 126, 201-216 (1973) l%owlands, R. T., Turner, G. : l~ccombination between the extranuclear genes conferring oligomycin resistance and cold sensitivity in Aspergillus nidulans. Molec. gen. Genet. 133, 15116i (1974) Schlanger, G., Sager, i%.: Localisation of five antibiotic resistance at the subunit level in chloroplast ribosomes of Chlamydomonas. Proc. nat. Acad. Sci. (Wash.) 71, 1715-1719 (1974) Trembath, M. K., Bunn, C. L., Lukins, H. B., Linnane, A. W. : Biogenesis of mitochondria 27. Genetic and biochemical characterisation of cytoplasmic and nuclear mutations to spiramycin resistance in Saecharomyces cerevisiae. Molee. gem Genet. 121, 35-48 (1973) Turner, G.: Cycloheximide-resistant synthesis of mitochondrial-membrane components in Aspergillus nidulans. Europ. J. Bioehem. 40, 201-206 (1973) Waldron, C., Roberts, C. F. : Cytoplasmic inheritance of a cold-sensitive mutant in AspergilIus nidulans. J. gen. Microbiol. 78, 379-381 (1973) Waldron, C., Roberts, C. F. : Cold-sensitive mutants in Aspergitlus nidulans. I. Isolation and general eharacterisation. Molec. gen. Genet. 134, 99-113 (1974a) Waldron, C., Roberts, C.F.: Cold-sensitive mutants in Aspergillus nidulans. II. Mutations affecting ribosome production. Molee. gen. Genet. 134, 115-132 (197~b) Warr, J. R., Roper, J. A. : Resistance to various inhibitors in Aspergillus nidulans. J. gen. Microbiol. 40, 273-281 (1965)

Note added in proo], eareD.27, camD-37, camC-108, and particularly eamB-105 confer a low level of resistance in vivo to pyrrolnitrin [M. P. Landini-Ricc5 and Arst, unpublished]. In Neurospora crassa mitochondria, pyrrolnitrin is an uncoupler, which, at higher concentrations, also inhibits electron transport [A. M. Lambowitz and C. W. Slayman, J. Bact. 112, 1020-1022 (1972)]. Other nuclear cam mutations have not been tested, but the extranuclear (camA) mutations do not affect pyrrolnitrin sensitivity. As both pyrrolnitrin and chloramphenicol contain substituted nitrobenzene moieties, this cross resistance might implicate the three nuclear cam loci in membrane functions associated with transport.

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As in Saccharomyces cerevisiae [W. E. Lancashire and D. E. Griffiths, Europ. J. Biochem. 51, 377-392 (1975)], a class of nuclear triethyltin resistance mutations in A. nidulans also confer a limited degree of cross resistance to chloramphenicol. These partially dominant mutations also confer cross resistance to trimethyltin and low level cross resistance to pyrrolnitrin and ethidium bromide but do not affect sensitivity to cycloheximide. C o m m u n i c a t e d b y W. Gajewski I. A. U. N. Gunatilleke Department of Botany Vidyalankara Campus University of Sri Lanka Kelaniya Sri Lanka

C. Scazzocchio Department of Biology University of Essex Wivenhoe Park Colchester C04 3S Q England

H. N. Arst, Jr. (/or reprints) Dept. of Genetics University of Cambridge Milton Road Cambridge CB4 1XH England