Eur. J. Biochem. 51, 573-577 (1975)

Carbon Catabolite Repression in Aspergillus nidulans Christopher BAILEY and Herbert N. ARST, Jr

Department of Genetics, University of Cambridge

(Received September 13, 1974)

Mutations leading to relief of carbon catabolite repression of the syntheses of a number of enzymes in Aspergillus nidulans can be selected in several different ways. They map at the creA locus in linkage group I and are recessive. Their phenotype is not due to defective carbon source uptake.

Although the mechanism of carbon catabolite repression in bacteria is now largely understood [l -41 (and references therein), relatively little is known about the way(s) in which glucose and certain other readily utilizable carbon sources prevent the syntheses of many enzymes involved in carbon metabolism in eukaryotes. Of considerable interest will be the way in which carbon catabolite repression is integrated with another general control mechanism, nitrogen metabolite repression, i.e. ammonium repres- sion of the syntheses of enzymes involved in nitrogen metabolism, for which the genetic basis has been at least partly established in Aspergillus nidulans [ 5 ] .

Here we report selection techniques for obtaining carbon catabolite derepressed mutants in A. nidulans and some preliminary characterization of these mu- tants. In particular, we identify a locus where mutations lead to derepression of many (but not all) carbon catabolite-repressible activities. These mutations bear some resemblance to those leading to carbon catabolite derepression in yeast [6 - 91.

MATERIALS AND METHODS

Wild-type and mutant strains used for enzyme assays carry a biotin auxotrophy (&A-l), and those used for uptake measurements carry a leucine auxo- trophy (IuA-1). The phenotypes of other relevant mutations are described in Table 1. Growth, harvesting

Enzymes. Alcohol dehydrogenase (EC 1 .I. 1.1) ; P-galac- tosidase(EC 3.2.1.23); D-quinatedehydrogenase(EC 1.1.1.24); NAD-linked glutamate dehydrogenase (EC 1.4. I .2) ; proline oxidase (EC 1.4.3.2); pyruvate dehydrogenase (EC 1.2.4.1); pyruvate carboxylase (EC 6.4.1.1) ; NADP-linked glutamate dehydrogenase (EC 1.4.1.4); galactose-I-phosphate uridyl transferase (EC 2.7.7.10).

of mycelium, and preparation of cell-free extracts are described elsewhere [lo]. Legends to Tables 2 and 3 give relevant details.

RESULTS AND DISCUSSION

Methods of Selection of Carbon Catabolite Derepressed Mutations

The selection of creA"-l has been described pre- viously [5]. The rationale can be summarized as follows : areA * strains are unable to utilize nitrogen sources other than ammonium on glucose minimal medium, although they can utilize acetamide, L-proline, L- alanine, L-histidine, L-threonine, L-arginine, L-orni- thine, L-glutamate, L-aspartate, and L-asparagine as sole carbon or sole carbon and nitrogen sources. Replacement of glucose by non-carbon catabolite- repressing carbon sources such as (at 1 %) L-arabinose, glycerol, ethanol, lactose or melibiose allows areA strains to utilize acetamide and proline but neither alanine, etc., nor non-carbon sources such as nitrate, formamide or hypoxanthine, as nitrogen sources. (This should not necessarily be taken to indicate two different mechanisms of carbon catabolite repression, however. Acetamidase and the proline catabolic enzymes might be simply less sensitive to carbon catabolite repression than the catabolic enzymes for alanine, etc.) creA d-l was therefore selected as a rever- sion mutation in an areA ' strain as mimicking replace- ment of glucose by non-carbon catdbolite-repressing carbon sources, i.e., as allowing utilization of acet- amide and proline but no other nitrogen sources on glucose-minimal medium. Characteristics in vivo of creA d-l are outlined in Table 1.

creA d-2, -3 , and -4 have an entirely different basis of selection. pdhA-1 strains lack pyruvate dehydro-

Eur. J. Biochem. 51 (1975)

574 Carbon Catabolite Repression in Aspergillus nidulans

Table 1. Phenotypic effects of creAd-1 in combination with other mutations CCR = carbon catabolite repression; PPP = pentose phosphate pathway

Mutation tested and phenotype Phenotype of double mutant with creAd-I Inference

1. areA'-1 creAd-I suppressesareA'-I on L-proline and creAd-I relieves CCR of enzymes of proline Lack N!&+-repressible acetamide as nitrogen sources degradation and acetamidase enzymes [5]

2. pdhA-I creAd-I allowspdhd-I to be supplemented by Lack pyruvate dehydrogenase ethanol, ethylammonium and acetamide in convertingethanol, ethylammo- [11,121 presence Of D-glucose, sucrose, D-XylOSe, etc.

3. PYCA-3 creAd-I allows pycA-3 to be partially supple- creAd-I relieves CCR of activities converting Lack pyruvate carboxylase mented by (+)-tartrate, ethanol, acetamide,

L-proline, L-ornithine, etc., in the presence of' L-ornithine, etc., to oxaloacetate or concentrations of D-glucose, etc., which prevent supplementation of pycA-3 single mutants creAd-I partially suppresses gdhA-10 [5]

creAd-1 relieves CCR of enzymes

nium, and acetamide to acetyl-CoA

(+)-tartrate, ethanol, acetamide, L-proline,

L-aspartate 1231

4. gdhA-10 creA d-l relieves CCR of enzyme(s) respon- sible for ammonium assimilation in absence of NADP-linked glutamate dehydrogenase

creAd-I prevents D-glucose, etc., from creAd-I relieves CCR of enzymes converting protecting pppA-I strains against L-arabinose, L-arabinose, D-xylose, and D-glucuronate to D-XylOSe, and D-glucuronate toxicity, but PPP intermediates, but does not affect control

of PPP enzymes themselves ppp A-I strains

Lack NADP-linked glutamate dehydrogenase [24,25]

Unable to utilize NO; and NO, as N and L-arabinose, D-xylose, and D-glucuronate as does not affect NO; and NO; toxicity to C due to altered levels of' pentose phosphate pathway (PPP) enzymes leading to an accumulation of sedoheptu- lose-7-phosphate [26]

creAd-1 prevents D-glucose, etc., from creA d-l relieves CCR of activities responsible Unable to utilize D-fructose protectingfrucd-I strains against D-fructose for accumulation of a toxic fructose 121 1 toxicity derivative infrucA-1 strains

creA d-3 partially prevents D-glucose, etc., creA d-l relieves CCR of activities converting Unable to utilize D-sorbitol; from protecting sbA-3 strains against D-sorbitol to a toxic derivative probably lack a dehydrogen- D-sorbitol toxicity ase (Table 3) [20,21]

creA d-l partially prevents D-glucose, etc., Lack galactose-I-phosphate from protecting galD-5 strains against and/or galactose permease uridyl transferase [27] D-galactose toxicity

5. pppA-1

6. frucA-I

7. sbA-3

8. gdD-5 creA d-l relieves CCR of galactokinase

genase and consequently require acetate [ 1 1,121. Ethanol, ethylammonium and acetamide can supple- ment pdhA-I strains when serving as sole carbon sources or in the presence of non-carbon catabolite- repressing carbon sources but not in the presence of strongly repressing carbon sources such as (1 %) D-glucose, D-xylose, or sucrose [5]. Alcohol dehydro- genase [13,14], monoamine oxidase [13,14] and acetamidase [15- 171 which are necessary for the conversion to acetate of ethanol, ethylammonium and acetamide, respectively, are subject to carbon catabolite repression in A . niduluns. Thus creAd-2, -3 and -4 were selected, after N-methyl-N-nitro-N-nitro- soguanidine mutagenesis [18,19] of a strain of geno- type biA-I udE-10 pdhA-1 (biotin-requiring7 adenine-

requiring, lacking pyruvate dehydrogenase), as allow- ing 1 % (v/v) ethanol to supplement the acetate auxotrophy in the presence of 1 % D-glucose plus 1 % sucrose. The majority of mutations obtained by this selection technique are probably defective in sugar uptake because, unlike creA mutations, they confer resistance to the toxic sugars L-sorbose and 2-deoxy- D-glucose [20]. Regulation of glucose transport differs from that of sucrose in A . nidulans [I 1,121, suggesting that different permeases are involved. Inclusion of both was an unsuccessful attempt to avoid uptake mutations. The selection of other putative carbon ca- tabolite-derepressed mutants by an entirely analogous method as enabling supplementation of strains lack- ing pyruvate carboxylase (pycA) under conditions of

Eur. J. Biochem. 51 (1975)

C. Bailey and H. N. Arst, Jr 575

Table 2. Enzyme activities in wild-type and creAd-l strains Derepressing conditions: 24 h growth on 0.1 % D-glucose (NAD-linked glutamate dehydrogenase, proline oxidase) or 27 h growth on 0.1 % D-fructose (other enzymes) as C source. Repressing conditions: 21 h growth on 1 % D-glucose. N source: 10 mM NH,f (NAD-linked glutamate dehydrogenase) or 100 mg/l uric acid (proline oxidase) or 10 mM NO; (other enzymes). Alcohol dehydro- genase induced by 1 % (v/v) ethanol at 0 h and assayed after [13]. 8-Galactosidase induced by 1 % lactose at 0 h and assayed after [28]. D-Quinate dehydrogenase induced by 0.3 % D-quinic acid at 0 hand assayed after [29]. NAD-linked glutamate dehydrogenase (GDH) induced by 5 mM L-proline added 5 h before harvesting and assayed after [24]. Proline oxidase induced similarly and assayed after MacDonald (unpublished) : assay mixture contained 50 mM L-proline, 833 pM 2-(4’-iodophenyl)-3-(4”-nitrophenyl)- 5-phenyltetrazolium chloride, 133 pg/ml phenazine methosulphate, 15 pM FAD, 100 mM Tris-HCI, pH 8.5, and cell-free extract; activity measured at 30 “C spectrophotometrically at 500 nm against a blank containing all components except proline. Specific activities: nmol product x min-’ x mg soluble protein-’ in biuret reaction [30]

Strain Growth Induction Specific activities _ _ ~ - conditions

alcohol 8-galacto- quinate NAD- proline dehydrogenase sidase dehydrogenase linked GDH oxidase

Wild derepressing uninduced 13 0 0 330 26 63 5

derepressing induced 118 6.3 117 874 89 type repressing uninduced - - -

repressing induced 2 0.3 22 107 59

creA d-l derepressing uninduced 12 0 0 307 18 63 5

repressing induced 84 0.2 15 218 85

repressing uninduced - - -

derepressing induced 126 6.2 135 932 73

Table 3. Sorbitol and mannitol dehydrogenase activities in wild-type and mutant strains Derepressing conditions: 27 h growth on 2% (v/v) glycerol as C source. Repressing conditions: 21 h growth on 1 % D-glucose. N source: 10 mM NO;. Induced conditions: 1 % D-sorbitol at 0 h. D-Sorbitol dehydrogenase assayed after [31]. Mannitol used as alternative substrate at 50 mM. Specific activities expressed as in Table 2

Growth Induction Substrate Specific activities conditions ~~

wild type creAd-1 sbA-3 creA d-l, sbA-3

Derepressing uninduced sorbitol 28 25 20 20 mannitol 106 104 72 92

Derepressing induced sorbitol 264 262 40 46 mannitol 137 158 197 195

Repressing induced sorbitol 178 267 23 23 mannitol 84 146 95 170

carbon catabolite repression is now in progress. (See properties of creAd-1 pycA-3 double mutants in Table 1 . )

The ability of creAd-1 to allow supplementation of pdhA-1 by ethanol, ethylammonium and acetamide under conditions of carbon catabolite repression and of creAd-2, -3 and -4 to allow areA‘ strains to utilize acetamide has been shown by constructing the corre- sponding double mutants. All four mutants are tightly linked (< 0.5 % recombination) and lead to compact colony morphology. However, in double mutants with areA‘-1, creAd-2 and -4 allow less utilization of acetamide than creAd-1 and -3 and allow virtually no utilization of L-proline (whereas creA d-l and -3 allow considerable proline utilization). In

contrast, creAd-1 and -3 have a less pronounced morphological effect than creA d-2 or -4.

Evidence that creAd-l Relieves Catabolite Repression

Data in Tables 2 and 3 show that creAd-1 leads to marked derepression of alcohol dehydrogenase, sorbi- to1 dehydrogenase, and proline oxidase and to slight derepression of NAD-linked glutamate dehydrogenase whilst not affecting induction of these enzymes. creA d-l does not affect carbon catabolite repression of quinate dehydrogenase or P-galactosidase. Thus, in both A . nidulans and Saccharomyces carlsbergensis [8], mutations relieving carbon catabolite repression

Eur. J. Biochem. 51 (1975)

576 Carbon Catabolite Repression in Aspergillus nidulans

5 t 4

I

0 0 . L 3 0) x

Q 7 c

$ 2 s 5

1

0 0 5 10 15 20 25

Time (rnin)

Fig. 1. Glucose uptake in wild-type and creAd-1 strains. The method has been previously described [32]. The solid growth medium contained 1 % L-arabinose + 0.1 "/, D-glUCOSe (induc- ed) or 1 % L-arabinose (uninduced) as C source(s). Incorpora- tion of ~-[4,5-~H]leucine served as a measure of growth [32]. The liquid uptake medium contained 1 % L-arabinose and 0.1 % ~-[U-'~C]glucose (700 mCi/mol). w.t. = wild type

affect the syntheses of some enzymes much more strongly than others, but in neither organism has it been established whether these differential effects are locus-specific or allele-specific. The yeast mutations also lead to morphological abnormalities [6 - 91.

When most of the sorbitol dehydrogenase activity is eliminated by the sbA-3 mutation [20,21], a sorbitol- inducible, carbon catabolite-repressible mannitol de- hydrogenase activity is observed (Table 3). Although the relationship of this activity to sorbitol dehydro- genase is unclear, creAd-1 does relieve its carbon catabolite-repressibility .

Uptake of ~-[ '~C]Glucose is unaffected by creAd-1

One possible way in which a mutation might relieve carbon catabolite repression would be by reducing uptake of carbon catabolite-repressing com- pounds. However, data in Fig. 1 show that creAd-1 has no effect on glucose uptake. Fig. 1 also shows that glucose uptake is not inducible in A. nidulans. 0.1 % D-glucose in the growth medium used prior to uptake studies reduces glucose uptake (perhaps the effect of preloading with unlabelled glucose).

Mapping and Dominance Relationships

Using standard procedures [22], creA d-l has been located to linkage group I. Preliminary meiotic analysis has shown that it recombines freely with the sugar uptake mutation sorA-2 [20], also in linkage group I.

However, creAd-1 is tightly linked (2 - 3 % recombina- tion) to galD-5. creAd-1 is recessive in diploids of partial genotype creAd-l/creA +, areA'-l/areA'-l with respect to acetamide and proline utilization and morphological effect. This recessivity has been con- firmed in diploids of partial genotype creA d-l /creA+, areA +/areA + where creAd-1 is recessive with respect to both morphology and the resistance it confers to inhibitors such as Cs', methylammonium, and thio- urea on acetamide and proline as nitrogen sources [ 5 ] .

We are grateful to Dr Mary Page, who participated in the initial stages of this work, and to Drs D. J . Cove, C. Scaz- zocchio, D. W. MacDonald and R. W. F. LePage for useful discussions. We thank the Science Research Council for sup- port, through a research studentship (C.R.B.) and a grant to Dr D. J. Cove (H.N.A.).

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C. Bailey and H. N. Arst, Jr, Department of Genetics, University of Cambridge, Milton Road, Cambridge, Great Britain CB4 IXH

Note Added in Proof (January 28, 1975). It has recently been shown that creAd-I also leads to carbon catabolite derepression of a proline permease [Arst, H. N., Jr & Mac- Donald, D. W., Nature, in press (1975)l.

Eur. J. Biochem. 51 (1975)