Molec. gen Genet. 151, 105-110 (1977) © by Springer-Verlag 1977
Some Genetical Aspects of Ornithine Metabolism in Aspergillus nidulans Herbert N. Arst, Jr. Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, England
Summary. 1. A possible minor route of ornithine catabolism in Aspergillus nidulans might begin with the ornithine decarboxylase reaction and end with the succinic semialdehyde dehydrogenase reaction. It is therefore of interest that the putative structural genes for these two enzymes, puA and ssuA, respectively, are tightly linked in linkage group II. However, this linkage is unlikely to have regulatory significance because ileA, the structural gene for threonine dehydratase, separates them. The gene order in this region is ssuA-ileA-puA-mauB-anB. (mauB- mutations result in loss of monoamine oxidase whilst anB- mutations lead to aneurin auxotrophy.) 2. An auxotrophy for ornithine or putrescine in A. nidulans occurs in double mutants lacking arginase and blocked before ornithine in the arginine biosynthetic pathway. Some residual ornithine synthesis in such double mutants can be catalysed by ornithine 6-transaminase, especially if it is synthesised constitutively.
mutation in puA can result in complete auxotrophy. The nutritional and developmental regulation of ornithine decarboxylase in A. nidulans has been studied by Stevens, McKinnon, and Winther (1976). Here I consider two genetical aspects of ornithine metabolism and its relationship to putrescine biosynthesis in A. nidulans." 1. Is the puA gene clustered with a functionally related gene? 2. Which metabolic pathways can supply ornithine for putrescine biosynthesis?
Introduction
Results and Discussion
One of the first indications that polyamines play an essential physiological role was the selection of a putrescine auxotroph of Aspergi[lus nidulans (Sneath, 1955). This auxotrophy results from loss of ornithine decarboxylase activity (Stevens, 1975) and is a consequence of a mutation in the puA gene in linkage group II (Clutterbuck, 1974). There is no biochemical evidence in A. nidulans for an alternative pathway ofputrescine biosynthesis from arginine via agmatine, involving arginine decarboxylase and agmatine ureohydrolase, such as exists in Escherichia coli (Stevens, 1975). This is consistent with the facts that no mutations to putrescine auxotrophy have been mapped to genes other than puA (Clutterbuck, 1974) and that
L Map Position ofpuA
Materials and Methods A list of A. nidulans markers mentioned in the text is given in Table 1. Other markers carried by strains are those in general use (Clutterbuck, 1974). I am very grateful to Drs P. Weglenski, J. Cybis, E. Bartnik, and M. Piotrowska of the Department of Genetics, University of Warsaw for supplying the agaA-90, arcA a47, ornC-31, and ornD-10 markers. Genetical methods were modified after Pontecorvo, Roper, Hemmons, Macdonald, and Bufton (1953). Growth testing of A. nidulans has been described previously (Arst and Cove, 1969, I973).
The principal pathway of L-ornithine catabolism in A. nidulans involves ornithine cS-transaminase-catalysed conversion of ornithine to L-glutamic 7-semialdehyde (Piotrowska, Sawicki, and Weglenski, 1969; Bartnik and Weglenski, 1974; Arst and MacDonald, 1975). A possible alternative pathway involving ornithine decarboxylase, the enzyme specific by puA, is shown in Figure 1. This pathway could not possibly account for more than a minor proportion of the ornithine catabolised because otaA mutations, leading to loss of ornithine 6-transaminase, largely eliminate ornithine catabolism (Piotrowska et al., 1969;
H.N. Arst, Jr. : Genetical Aspects of Ornithine Metabolism in Aspergillus
106 Table 1. List of Mutations Mentioned in Text Mutation
Relevant phenotype
References
Mutation
Relevant phenotype
References
agaA-90
loss of arginase
ornA-4
aneurin auxotrophy constitutive synthesis of ornithine 6-transaminase derepressed synthesis of some ammoniumrepressible enzymes and permeases loss of arginosuccinase
Arst and Cove (1973); Hynes (1975); (see also Arst and Scazzocchio (1975)) Brainbridge, Dalton, and Walpole (1966); Cybis, Piotrowska, and Weglenski (1972) Weglenski (1967); Cybis et al. (1972); Klimczuk and Weglenski (1974) Cybis et al. (1972)
ornC-31
blocked before ornithine in arginine biosynthetic pathway loss of N~-acetylornithine transaminase blocked before ornithine in arginine biosynthetic pathway blocked before ornithine in arginine biosynthetic pathway loss of ornithine &transaminase
Cybls et al. (1972)
anB-8 areAd-47
Bartnik and Weglenskl (1974) Forbes (1959) Bartnik and Weglenski (1974)
areA-102
argA-1
argB-2
argC-3 gatA-2
ileA-1 intA--lOl
intAC-2
mauB-4
loss of ornithine transcarbamylase
loss of arginosnccinate synthetase loss of v-aminon-butyrate (GABA) transaminase loss of threonine dehydratase uninducible synthesis of GABA transaminase constitutive synthesis of GABA transaminase loss of monoamine oxidase
puA
~ CO2
Arst (1976) ; H.A. Penfold and H.N. Arst (unpublished) MacDonald, Arst, and Cove (1974) Arst (1976) ; H.A. Penfold and H.N. Arst (unpublished) Arst (1976) ; H.A. Penfold and H.N. Arst (unpublished) Page (1971); Page and Cove (1972)
ornD-lO
otaA-2
proA-6
putrescine -
proB-9
puA-2 ssuA-1
uX-3 uY-5 uZ-4
- 7-amino-n-butyraldehyde --
~
7-amino-n-butyric acid
NH4+ ~-ketoglutarate
gatA
GABA transaminase
L-glutamate succinic semialdehyde dehydrogenase succinicsemialdehyde
succinicacid
ssuA Fig. 1. A possible minor route of ornithine catabolism
Arst (1976)
Cybis et al. (1972)
Cybis et al. (1972)
Piotrowska, Sawicki, and Weglenski (1969); Arst and MacDonald (1975) Weglenski (1966)
blocked before glutamic 7-semialdehyde on proline biosynthetic pathway blocked before Weglenski (1966) glutamic y-semialdehyde on proline biosynthetic pathway loss of ornithine Sneath (1955) ; decarboxylase Stevens (1975) loss of succinic semial- Arst (1976); dehyde dehydrogenase C.R. Bailey and H.N. Arst (unpublished) loss of urease Scazzocchio and Darlington (1968) loss of urease Scazzocchio and Darlington (1968) loss of urease Scazzocchio and Darlington (1968)
diamine oxidase
ornithine decarboxylase L-ornithine -
ornB-7
107
H.N. Arst, Jr. : Genetical Aspects of Ornithine Metabolism in Aspergillus Table 2. A s u m m a r y of data from crosses used to map the p u A region Relevant partial genotype
Selected recombinant class
Fraction carrying other markers
ssuA-1 x p u A - 2 m a u B - 4
ssuA + puA +
1/22 m a u B - 4
ssuA-1 p u A - 2 x anB-8
p u A + anB +
2/50 ssuA-1
ssuA-1 p u A - 2 anB-8 x ileA-1
i l e A + anB +
21/21 ssuA-1 21/21 p u A - 2
ssuA-1 p u A - 2 anB-8 x m a u B - 4
puA ÷ mauB +
0/I0 ssuA-1 10/10 anB-8
ileA-I anB-8 x p u A - 2 m a u B - 4
ileA + puA + p u A + anB +
0/5 m a u B - 4 5/5 anB-8 11/11 ileA-1 3/11 m a u B - 4
s s u A + ileA + ileA + p u A + ileA + anB +
11/11 p u A - 2 0/11 m a u B - 4 11/11 anB-8 22/22 ssuA-1 22/22 m a u B - 4 0/22 anB-8 87/88 ssuA-1 65/88 puA-2" 59/88 m a u B - 4 b
ssuA-1 p u A - 2 anB-8 x ileA-1 m a u B - 4
Selection for i l e A +, p u A +, and anB ~ was performed by omitting the necessary suppIement. Selection for s s u A + was performed by using a medium containing G A B A at 5 m M as sole nitrogen source or 50 m M as sole carbon and nitrogen source. Selection for m a u B + was performed by using a medium containing 10 m M e t h y l a m m o n i u m chloride as sole nitrogen source. The denominator o( each fraction is the total n u m b e r of recombinants of the selected class scored; the numerator is the number of those recombinants carrying the m u t a n t alleles listed above " All 23 ileA + p u A + anB + recombinants carry m a u B - 4 b All 29 i l e A + m a u B + anB + recombinants carry puA-2
Arst and MacDonald, 1975) whilst puA- mutations do not affect utilisation of ornithine as a carbon or nitrogen source. Moreover, although arginine and ornithine induce ornithine c~-transaminase (Weglenski, 1967; Bartnik, Weglenski, and Piotrowska, 1973; Bartnik and Weglenski, 1974; Stevens et al., 1976), they do not induce ornithine decarboxylase (Stevens et al., 1976). There is, nevertheless, evidence that at least putrescine catabolism does proceed through this pathway because both gatA- and intA- mutants grow more poorly than the wild type on 5 mM putrescine as nitrogen source, gatA- and intA- mutants are defective respectively in the putative structural gene and a positive regulatory gene for 7-amino-nbutyrate (GABA) transaminase (Arst, 1976; H.A. Penfold and H.N. Arst, unpublished). The ssuA gene, probably specifying succinic semialdehyde dehydrogenase, is tightly linked to puA in linkage group II (C.R. Bailey and H.N. Arst, unpublished). In view of the possible catabolic sequence depicted in Figure 1, it was of considerable interest to see if ssuA and puA might be contiguous and subject to a common form of control. However, several other genes having unrelated functions also map in this region (Clutterbuck, 1974): ileA, the structural gene for threonine dehydratase, the first enzyme of isoleucine biosynthesis (MacDonald, Arst, and Cove, 1974); mauB, a gene where mutation can lead to loss of monamine oxidase t (Page, 1971; Page and Cove, 1972); and anB, a gene where mutation can result in aneurin auxotrophy (Forbes, 1959). The only map i m a u B m u t a n t s utilise putrescine as a nitrogen source as well as wild type (Page, 1971). There is therefore no reason to suppose that m a u B is in any way involved with diamine oxidase.
order compatible with data in Table 2 is ssuA-ileApuA-mauB-anB. Thus the location of ileA between ssuA and puA precludes the possibility that the latter two genes share a cis-acting regulatory element (except in the improbable event that the element also participates in regulation of ileA). No attempt was made to determine actual map distances, but it should be noted that these five genes are very tightly linked to each other and any or all of them might be contiguous, the map distance between ssuA-1 and anB-8 is no more than 2 cM, and markers in genes listed as adjacent in the above series recombine so infrequently that in most cases data in Table 2 is based on the total progeny of several cleistothecia.
II. Sources of Ornithine for Putrescine Biosynthesis Even if A. nidulans is supplied with exogenous arginine, it must still be able to synthesise L-ornithine for putrescine biosynthesis. If metabolic blocks prevent ornithine formation, an auxotrophy for ornithine or putrescine (ornithine/putrescine) ought to be observable. Figure 2 summarises the relevant metabolic relationships. There are at least four possible metabolic routes by which A. nidulans might synthesise ornithine: 1. from L-glutamate via N-acetyl-L-glutamate and N%acetyl-L-ornithine on the arginine biosynthetic pathway. 2. By arginase-catalysed hydrolysis of arginine. 3. From L-glutamic 7-semialdehyde, an intermediate of proline biosynthesis and catabolism, by using the ornithine 6-transaminase reaction in the opposite direction (i.e., ornithine synthesis) from that necessary for its described physiological roles. 4.
H.N. Arst, Jr. : Genetical Aspects of Ornithine Metabolism in Aspergillus
108 putrescine carbamyl phosphate
~"-....~ puA ornithlne decarboxylase
l argB "" L-ornittaine ~
ornB ornC ornD
/
/e/
~
arginosuccinate synthetase
t°rran~ts ch2bamylase
s / ~ia
Ncc ace!ylated intermediates
L arginosuccinate
argA L-arginine arginosuccinase
agaA
(
L-glutamate
otaA
argC
L-citrulline
arginase
urea
ornithine -transaminase
proHne oxidase "
~
L glutamic7semialdehyde
L-proline Al--pyrroline--5-carboxylate reductase
Fig. 2. Major pathways of ornithine metabolism
From arginine via citrulline by reversal of the last part of the arginine biosynthetic pathway. It is of further interest to test whether the intA gene, a positive regulatory gene interpretable as an integrator gene on the Britten and Davidson (1969) model (Arst, 1976), might have any particular association with putrescine biosynthesis. Two of the activities under intA control are acetamidase and a transaminase for 7amino-n-butyric acid (GABA) and other m-amino acids which can substitute in vivo for the biosynthetic enzyme which normally catalyses the conversion of N-acetyl-L-glutamic ?-semialdehyde to N%acetyl-Lornithine (Arst, 1976). As the acetamidase has acyl transferase activity (M.J. Hynes and P.J. Wright as cited in Hynes and Pateman, 1970), it might be postulated that intA controls an alternative pathway of ornithine biosynthesis or at least an alternative for the last two steps leading from N-acetyl-L@utamic ?-semialdehyde to ornithine. This could conceivably be a fifth source of ornithine for putrescine biosynthesis. Data in Table 3 show that the minimum number of mutations which result in an ornithine/putrescine auxotrophy is two: agaA-, resulting in loss of arginase (Bartnik and Weglenski, 1974) plus an orn- mutation, resulting in a block before ornithine in the arginine biosynthetic pathway. Mutations in the ornB gene show a slight locus-specific leakiness, presumably due to a low uninduced level of GABA transaminase catalysing some transamination of N-acetyl-Lglutamic 7-semialdehyde. Mutations in gatA, the putative structural gene for GABA transaminase, and
loss of function mutations ( i n t A ) in the positive regulatory gene intA eliminate this leakiness. Thus agaA- ornB- gatA- and agaA- ornB- intA- triple mutants are as stringently auxotrophic for ornithine/ putrescine as agaA- ornC double mutants. The ability of intAC-2 to suppress ornB lesions by leading to constitutivity for GABA transaminase (Arst, 1976; H.A. Penfold and H.N. Arst, unpublished) is also observable as a partial suppression of the ornithine/ putrescine auxotrophy in agaA- ornB- intAC-2 triple mutants. Similarly, fl-alanine and GABA, which probably activate the intA + product (Arst, 1976), partially supplement the ornithine/putrescine auxotrophy of agaA- ornB- double mutants. However, just as intAC-2 does not suppress and fl-alanine and GABA do not supplement ornC- ornithine/arginine auxotrophies (Arst, 1976), they have no effect on the stringency of the ornithine/putrescine requirement in strains carrying agaA- and ornC- mutations. However, some ornithine biosynthesis still occurs in agaA- orn- double mutants because they are definitely leakier thanpuA- strains (Table 3). This residual ornithine biosynthesis does not result from a reversal of the ornithine transcarbamylase reaction because inclusion of an argB- mutation, leading to loss of this enzyme (Weglenski, 1967; Cybis, Piotrowska, and Weglenski, 1972; Klimczuk and Weglenski, 1974), does not affect the leakiness. Most of the residual ornithine biosynthesis can be attributed to the ornithine ~5-transaminase reaction because its loss by an otaA- mutation in agaA- ornC- otaA- triple mutants leads to an auxotrophy almost as stringent as
H.N. Arst, Jr. : Genetical Aspects of Ornithine Metabolism in Aspergillus Table 3. Growth responses of strains of various genotypes with reference to ornithine/putrescine auxotrophy Relevant genotype
wild type puA-2 agaA-90 ornB-7 ornC-31 otaA-2 arcA
Some Genetical Aspects of Ornithine Metabolism in Aspergillus nidulans Herbert N. Arst, Jr. Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, England
Summary. 1. A possible minor route of ornithine catabolism in Aspergillus nidulans might begin with the ornithine decarboxylase reaction and end with the succinic semialdehyde dehydrogenase reaction. It is therefore of interest that the putative structural genes for these two enzymes, puA and ssuA, respectively, are tightly linked in linkage group II. However, this linkage is unlikely to have regulatory significance because ileA, the structural gene for threonine dehydratase, separates them. The gene order in this region is ssuA-ileA-puA-mauB-anB. (mauB- mutations result in loss of monoamine oxidase whilst anB- mutations lead to aneurin auxotrophy.) 2. An auxotrophy for ornithine or putrescine in A. nidulans occurs in double mutants lacking arginase and blocked before ornithine in the arginine biosynthetic pathway. Some residual ornithine synthesis in such double mutants can be catalysed by ornithine 6-transaminase, especially if it is synthesised constitutively.
mutation in puA can result in complete auxotrophy. The nutritional and developmental regulation of ornithine decarboxylase in A. nidulans has been studied by Stevens, McKinnon, and Winther (1976). Here I consider two genetical aspects of ornithine metabolism and its relationship to putrescine biosynthesis in A. nidulans." 1. Is the puA gene clustered with a functionally related gene? 2. Which metabolic pathways can supply ornithine for putrescine biosynthesis?
Introduction
Results and Discussion
One of the first indications that polyamines play an essential physiological role was the selection of a putrescine auxotroph of Aspergi[lus nidulans (Sneath, 1955). This auxotrophy results from loss of ornithine decarboxylase activity (Stevens, 1975) and is a consequence of a mutation in the puA gene in linkage group II (Clutterbuck, 1974). There is no biochemical evidence in A. nidulans for an alternative pathway ofputrescine biosynthesis from arginine via agmatine, involving arginine decarboxylase and agmatine ureohydrolase, such as exists in Escherichia coli (Stevens, 1975). This is consistent with the facts that no mutations to putrescine auxotrophy have been mapped to genes other than puA (Clutterbuck, 1974) and that
L Map Position ofpuA
Materials and Methods A list of A. nidulans markers mentioned in the text is given in Table 1. Other markers carried by strains are those in general use (Clutterbuck, 1974). I am very grateful to Drs P. Weglenski, J. Cybis, E. Bartnik, and M. Piotrowska of the Department of Genetics, University of Warsaw for supplying the agaA-90, arcA a47, ornC-31, and ornD-10 markers. Genetical methods were modified after Pontecorvo, Roper, Hemmons, Macdonald, and Bufton (1953). Growth testing of A. nidulans has been described previously (Arst and Cove, 1969, I973).
The principal pathway of L-ornithine catabolism in A. nidulans involves ornithine cS-transaminase-catalysed conversion of ornithine to L-glutamic 7-semialdehyde (Piotrowska, Sawicki, and Weglenski, 1969; Bartnik and Weglenski, 1974; Arst and MacDonald, 1975). A possible alternative pathway involving ornithine decarboxylase, the enzyme specific by puA, is shown in Figure 1. This pathway could not possibly account for more than a minor proportion of the ornithine catabolised because otaA mutations, leading to loss of ornithine 6-transaminase, largely eliminate ornithine catabolism (Piotrowska et al., 1969;
H.N. Arst, Jr. : Genetical Aspects of Ornithine Metabolism in Aspergillus
106 Table 1. List of Mutations Mentioned in Text Mutation
Relevant phenotype
References
Mutation
Relevant phenotype
References
agaA-90
loss of arginase
ornA-4
aneurin auxotrophy constitutive synthesis of ornithine 6-transaminase derepressed synthesis of some ammoniumrepressible enzymes and permeases loss of arginosuccinase
Arst and Cove (1973); Hynes (1975); (see also Arst and Scazzocchio (1975)) Brainbridge, Dalton, and Walpole (1966); Cybis, Piotrowska, and Weglenski (1972) Weglenski (1967); Cybis et al. (1972); Klimczuk and Weglenski (1974) Cybis et al. (1972)
ornC-31
blocked before ornithine in arginine biosynthetic pathway loss of N~-acetylornithine transaminase blocked before ornithine in arginine biosynthetic pathway blocked before ornithine in arginine biosynthetic pathway loss of ornithine &transaminase
Cybls et al. (1972)
anB-8 areAd-47
Bartnik and Weglenskl (1974) Forbes (1959) Bartnik and Weglenski (1974)
areA-102
argA-1
argB-2
argC-3 gatA-2
ileA-1 intA--lOl
intAC-2
mauB-4
loss of ornithine transcarbamylase
loss of arginosnccinate synthetase loss of v-aminon-butyrate (GABA) transaminase loss of threonine dehydratase uninducible synthesis of GABA transaminase constitutive synthesis of GABA transaminase loss of monoamine oxidase
puA
~ CO2
Arst (1976) ; H.A. Penfold and H.N. Arst (unpublished) MacDonald, Arst, and Cove (1974) Arst (1976) ; H.A. Penfold and H.N. Arst (unpublished) Arst (1976) ; H.A. Penfold and H.N. Arst (unpublished) Page (1971); Page and Cove (1972)
ornD-lO
otaA-2
proA-6
putrescine -
proB-9
puA-2 ssuA-1
uX-3 uY-5 uZ-4
- 7-amino-n-butyraldehyde --
~
7-amino-n-butyric acid
NH4+ ~-ketoglutarate
gatA
GABA transaminase
L-glutamate succinic semialdehyde dehydrogenase succinicsemialdehyde
succinicacid
ssuA Fig. 1. A possible minor route of ornithine catabolism
Arst (1976)
Cybis et al. (1972)
Cybis et al. (1972)
Piotrowska, Sawicki, and Weglenski (1969); Arst and MacDonald (1975) Weglenski (1966)
blocked before glutamic 7-semialdehyde on proline biosynthetic pathway blocked before Weglenski (1966) glutamic y-semialdehyde on proline biosynthetic pathway loss of ornithine Sneath (1955) ; decarboxylase Stevens (1975) loss of succinic semial- Arst (1976); dehyde dehydrogenase C.R. Bailey and H.N. Arst (unpublished) loss of urease Scazzocchio and Darlington (1968) loss of urease Scazzocchio and Darlington (1968) loss of urease Scazzocchio and Darlington (1968)
diamine oxidase
ornithine decarboxylase L-ornithine -
ornB-7
107
H.N. Arst, Jr. : Genetical Aspects of Ornithine Metabolism in Aspergillus Table 2. A s u m m a r y of data from crosses used to map the p u A region Relevant partial genotype
Selected recombinant class
Fraction carrying other markers
ssuA-1 x p u A - 2 m a u B - 4
ssuA + puA +
1/22 m a u B - 4
ssuA-1 p u A - 2 x anB-8
p u A + anB +
2/50 ssuA-1
ssuA-1 p u A - 2 anB-8 x ileA-1
i l e A + anB +
21/21 ssuA-1 21/21 p u A - 2
ssuA-1 p u A - 2 anB-8 x m a u B - 4
puA ÷ mauB +
0/I0 ssuA-1 10/10 anB-8
ileA-I anB-8 x p u A - 2 m a u B - 4
ileA + puA + p u A + anB +
0/5 m a u B - 4 5/5 anB-8 11/11 ileA-1 3/11 m a u B - 4
s s u A + ileA + ileA + p u A + ileA + anB +
11/11 p u A - 2 0/11 m a u B - 4 11/11 anB-8 22/22 ssuA-1 22/22 m a u B - 4 0/22 anB-8 87/88 ssuA-1 65/88 puA-2" 59/88 m a u B - 4 b
ssuA-1 p u A - 2 anB-8 x ileA-1 m a u B - 4
Selection for i l e A +, p u A +, and anB ~ was performed by omitting the necessary suppIement. Selection for s s u A + was performed by using a medium containing G A B A at 5 m M as sole nitrogen source or 50 m M as sole carbon and nitrogen source. Selection for m a u B + was performed by using a medium containing 10 m M e t h y l a m m o n i u m chloride as sole nitrogen source. The denominator o( each fraction is the total n u m b e r of recombinants of the selected class scored; the numerator is the number of those recombinants carrying the m u t a n t alleles listed above " All 23 ileA + p u A + anB + recombinants carry m a u B - 4 b All 29 i l e A + m a u B + anB + recombinants carry puA-2
Arst and MacDonald, 1975) whilst puA- mutations do not affect utilisation of ornithine as a carbon or nitrogen source. Moreover, although arginine and ornithine induce ornithine c~-transaminase (Weglenski, 1967; Bartnik, Weglenski, and Piotrowska, 1973; Bartnik and Weglenski, 1974; Stevens et al., 1976), they do not induce ornithine decarboxylase (Stevens et al., 1976). There is, nevertheless, evidence that at least putrescine catabolism does proceed through this pathway because both gatA- and intA- mutants grow more poorly than the wild type on 5 mM putrescine as nitrogen source, gatA- and intA- mutants are defective respectively in the putative structural gene and a positive regulatory gene for 7-amino-nbutyrate (GABA) transaminase (Arst, 1976; H.A. Penfold and H.N. Arst, unpublished). The ssuA gene, probably specifying succinic semialdehyde dehydrogenase, is tightly linked to puA in linkage group II (C.R. Bailey and H.N. Arst, unpublished). In view of the possible catabolic sequence depicted in Figure 1, it was of considerable interest to see if ssuA and puA might be contiguous and subject to a common form of control. However, several other genes having unrelated functions also map in this region (Clutterbuck, 1974): ileA, the structural gene for threonine dehydratase, the first enzyme of isoleucine biosynthesis (MacDonald, Arst, and Cove, 1974); mauB, a gene where mutation can lead to loss of monamine oxidase t (Page, 1971; Page and Cove, 1972); and anB, a gene where mutation can result in aneurin auxotrophy (Forbes, 1959). The only map i m a u B m u t a n t s utilise putrescine as a nitrogen source as well as wild type (Page, 1971). There is therefore no reason to suppose that m a u B is in any way involved with diamine oxidase.
order compatible with data in Table 2 is ssuA-ileApuA-mauB-anB. Thus the location of ileA between ssuA and puA precludes the possibility that the latter two genes share a cis-acting regulatory element (except in the improbable event that the element also participates in regulation of ileA). No attempt was made to determine actual map distances, but it should be noted that these five genes are very tightly linked to each other and any or all of them might be contiguous, the map distance between ssuA-1 and anB-8 is no more than 2 cM, and markers in genes listed as adjacent in the above series recombine so infrequently that in most cases data in Table 2 is based on the total progeny of several cleistothecia.
II. Sources of Ornithine for Putrescine Biosynthesis Even if A. nidulans is supplied with exogenous arginine, it must still be able to synthesise L-ornithine for putrescine biosynthesis. If metabolic blocks prevent ornithine formation, an auxotrophy for ornithine or putrescine (ornithine/putrescine) ought to be observable. Figure 2 summarises the relevant metabolic relationships. There are at least four possible metabolic routes by which A. nidulans might synthesise ornithine: 1. from L-glutamate via N-acetyl-L-glutamate and N%acetyl-L-ornithine on the arginine biosynthetic pathway. 2. By arginase-catalysed hydrolysis of arginine. 3. From L-glutamic 7-semialdehyde, an intermediate of proline biosynthesis and catabolism, by using the ornithine 6-transaminase reaction in the opposite direction (i.e., ornithine synthesis) from that necessary for its described physiological roles. 4.
H.N. Arst, Jr. : Genetical Aspects of Ornithine Metabolism in Aspergillus
108 putrescine carbamyl phosphate
~"-....~ puA ornithlne decarboxylase
l argB "" L-ornittaine ~
ornB ornC ornD
/
/e/
~
arginosuccinate synthetase
t°rran~ts ch2bamylase
s / ~ia
Ncc ace!ylated intermediates
L arginosuccinate
argA L-arginine arginosuccinase
agaA
(
L-glutamate
otaA
argC
L-citrulline
arginase
urea
ornithine -transaminase
proHne oxidase "
~
L glutamic7semialdehyde
L-proline Al--pyrroline--5-carboxylate reductase
Fig. 2. Major pathways of ornithine metabolism
From arginine via citrulline by reversal of the last part of the arginine biosynthetic pathway. It is of further interest to test whether the intA gene, a positive regulatory gene interpretable as an integrator gene on the Britten and Davidson (1969) model (Arst, 1976), might have any particular association with putrescine biosynthesis. Two of the activities under intA control are acetamidase and a transaminase for 7amino-n-butyric acid (GABA) and other m-amino acids which can substitute in vivo for the biosynthetic enzyme which normally catalyses the conversion of N-acetyl-L-glutamic ?-semialdehyde to N%acetyl-Lornithine (Arst, 1976). As the acetamidase has acyl transferase activity (M.J. Hynes and P.J. Wright as cited in Hynes and Pateman, 1970), it might be postulated that intA controls an alternative pathway of ornithine biosynthesis or at least an alternative for the last two steps leading from N-acetyl-L@utamic ?-semialdehyde to ornithine. This could conceivably be a fifth source of ornithine for putrescine biosynthesis. Data in Table 3 show that the minimum number of mutations which result in an ornithine/putrescine auxotrophy is two: agaA-, resulting in loss of arginase (Bartnik and Weglenski, 1974) plus an orn- mutation, resulting in a block before ornithine in the arginine biosynthetic pathway. Mutations in the ornB gene show a slight locus-specific leakiness, presumably due to a low uninduced level of GABA transaminase catalysing some transamination of N-acetyl-Lglutamic 7-semialdehyde. Mutations in gatA, the putative structural gene for GABA transaminase, and
loss of function mutations ( i n t A ) in the positive regulatory gene intA eliminate this leakiness. Thus agaA- ornB- gatA- and agaA- ornB- intA- triple mutants are as stringently auxotrophic for ornithine/ putrescine as agaA- ornC double mutants. The ability of intAC-2 to suppress ornB lesions by leading to constitutivity for GABA transaminase (Arst, 1976; H.A. Penfold and H.N. Arst, unpublished) is also observable as a partial suppression of the ornithine/ putrescine auxotrophy in agaA- ornB- intAC-2 triple mutants. Similarly, fl-alanine and GABA, which probably activate the intA + product (Arst, 1976), partially supplement the ornithine/putrescine auxotrophy of agaA- ornB- double mutants. However, just as intAC-2 does not suppress and fl-alanine and GABA do not supplement ornC- ornithine/arginine auxotrophies (Arst, 1976), they have no effect on the stringency of the ornithine/putrescine requirement in strains carrying agaA- and ornC- mutations. However, some ornithine biosynthesis still occurs in agaA- orn- double mutants because they are definitely leakier thanpuA- strains (Table 3). This residual ornithine biosynthesis does not result from a reversal of the ornithine transcarbamylase reaction because inclusion of an argB- mutation, leading to loss of this enzyme (Weglenski, 1967; Cybis, Piotrowska, and Weglenski, 1972; Klimczuk and Weglenski, 1974), does not affect the leakiness. Most of the residual ornithine biosynthesis can be attributed to the ornithine ~5-transaminase reaction because its loss by an otaA- mutation in agaA- ornC- otaA- triple mutants leads to an auxotrophy almost as stringent as
H.N. Arst, Jr. : Genetical Aspects of Ornithine Metabolism in Aspergillus Table 3. Growth responses of strains of various genotypes with reference to ornithine/putrescine auxotrophy Relevant genotype
wild type puA-2 agaA-90 ornB-7 ornC-31 otaA-2 arcA
