Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.

Polyamine synthesis in Neurospora crassa ( Fig. 1) has been studied by several groups over the last decade. The sole origin of putrescine (1,4-diaminobutane) was identified as ornithine (8), and the enzyme responsible for putrescine formation, ornithine decarboxylase, was shown to be cytosolic (3,13,21). Two putrescinerequiring mutants of N. crassa were isolated by Deters et al. (Genetics 77:s16-s17, 1974), although there has been some doubt about their ornithine decarboxylase deficiency (14). The existence of spermine in N. crassa and other filamentous fungi has been debated recently (11,15), with general agreement presently that spermine does occur in low amounts in N. crassa (T. J. Paulus and R. H. Davis, Methods Enzymol., in press). A study of polyamine pool dynamics in N. crassa (16) suggests that polyamines are normally maintained in a narrow range of values (on a dry-weight basis) by a control mechanism for ornithine decarboxylase which responds with high efficiency to spermidine. It was also shown that little or no polyamine degradation takes place during exponential growth in N. crassa and that a substantial fraction of the polyamine pools may be bound (nondiffusible) within the cell (16,17).
In previous work and the work reported here, use has been made of the arginaseless (aga) mutation. Because strains carrying an aga mutation cannot form ornithine from arginine ( Fig. 1) and because arginine feedback inhibits ornithine biosynthesis, aga mutants grown with arginine develop severe ornithine and polyamine depletion. The resulting nutritional needs are met by addition of ornithine or polyamines (8).
In this report, we wish to extend and clarify our knowledge of polyamine metabolism in N. crassa. We confirm the previous work on genetic control of ornithine decarboxylase and show that ornithine decarboxylase is highly derepressed in the aga strain when depleted of ornithine. Finally, we explore the origin of cadaverine (1,5-diaminopentane), an analog of putrescine found in the ornithine-starved aga mutant, and its role in supporting the slow and indefinite growth of this strain despite the lack of the normal polyamines. MATERIALS AND METHODS Strains, media, and growth. The strains of N. crassa used were IC-1 and IC-2 (wild types) and strains carrying the aga, various spe-1, and inl (inositol) mutations (Table 1). In this paper, we rename the put-i The media used were Vogel medium N corn meal agar without dextrose and suppli appropriate for crosses, and sorbose platin testing media for genetic analysis (7).
Growth was done in 10-ml Vogel mediu flasks for stationary cultures, in 1,000exponential cultures for enzyme and poly determination, and in 25-ml solidified med ml Erlenmeyer flasks to grow conidia used and for mutational work (7). Media were s ed as noted in the text.
Genetic methods. Mutagenesis of N. cra of both wild-type and polyamine-depleted was done. Polyamine-depleted aga conidi; pared by growing cultures in 1 mM ail medium and limiting (0.2 mM) putrescine. sis of conidia with UV light to 20 to 80! followed by the fltration-enrichment tec spermidine-requiring mutants, was done t methods (7). Crosses were made by standa (7); most media in which ascospores were I supplemented with 500 SLg of spermidine counteract the very poor germination of spores.

S-ADSoOL
Complementation tests were performed by combin-M"ETh"NINE ing drops of conidia in 1 ml of liquid minimal medium DC-SAN in culture tubes (13 by 100 mm) and incubating them for up to 10 days at room temperature.
Ornitine decarboxylse extrctin and asay. Assays were performed on extracts of cells grown in exponential cultures. Mycelia collected by ifitration were ground with sand in a cold mortar in 0.05 M K+and sperm-phosphate.buffer (pH 7.1) containing 1 mM EDTA.
(utations (in Slurries were centrifuged at 18,500 x g, and the De btansamisupernatants were desalted on Sephadex G-25 col-We-deficient. umns equilibrated with the extraction buffer. Extracts S-adenosylwere frozen (-70°C) with little loss of activity. Enxylase (EC zyme assays were performed as described previously press) in reaction mixtures (0.3 ml) containing 100 mM K+ phosphate (pH 7.1), 1 mM EDTA, 1.7 mM ,Bmercaptoethanol, 50 ,M pyridoxal phosphate, 2 mM L-ormithine, and sufficient L-[1-14C]ornithine to bring pall spe-l to the specific radioactivity to 200 to 700 cpm/nmol. polyamine Incubations were carried out at 37°C for 10 to 90 min; specific activity is expressed as nanomoles per hour for growth, per millig of protein. Lysine decarboxylase activilemented as ty was measured in the above reaction mixture, in ig and spot-which L-[U-14C]lysine (5,800 cpm/nmole) was used in place of ornithine. Due account was taken of the fact im in 50-ml that only one of the six lysine C atoms is released upon ml aerated decarboxylation. ,amine pool anlyamne deterinatis ad n. Polyium in 125amine pools were determined in perchloric acid exlfor inocula tracts of mycelia by the double-isotope derivative upplementassay described previously (16; Paulus and Davis, in press). The method depends upon dilution of added issa conidia 'C-labeled polyamine by polyamines in the extract. aga strains The quantitation of polyamines recovered from silica a were pre-gel, thin-layer chromatograms is done by use of ginine agar [3H]dansyl-chloride of known specific activity. Dan-Mutagenesyl-polyamines dissolved in benzene were spotted on % survival, Sil G plates (20 by 20 cm). For one-dimensional :hnique for separations, the solvent used was ethyl acetate-cyclo-3y standard hexane (2:3, vol/vol) (16). For two-dimensional separd methods rations, the first dimension was run with the solvent plated were above and the second dimension was run with chloroper ml to form-n-butyl-alcohol-dioxane (48:1:1) (1). Two-di-Speasco-mensional separations are essential for dependable resolution of the dansyl derivatives of putrescine,  cadaverine, spermidine, and aminopropylcadaverine ( Fig. 2). No attempt was made to measure the amount of aminopropylcadaverine, but both this compound and cadaverine were further identified by rechromatographing their dansyl derivatives eluted from twodimensional plates in additional one-dimensional plates with standards.
Materials. All chemicals were of reagent grade. Most biochemicals were obtained from Sigma Chemical Co. Triton X-100 was purchased from Research Products International, and Polygram Sil G thin-layer chromatograms were obtained from Brinkman Instru-ments, Inc. Isotopes were purchased from Amersham-Searle (L-1l-14C]ornithine) and New England Nuclear Corp. (polyamines and dansyl-chloride). Aminopropylcadaverine was a gift from David R. Morris, University of Washington, Seattle.

RESULTS
Mutants requiring polyamines. In the course of numerous attempts, only one polyamine-requiring strain was isolated from conidia of the aga strain 1C-3, as described in Materials and Methods. It grew very slightly in minimal medium but grew well in medium supplemented with putrescine, spermidine, or spermine. By a cross to the wild type, strains 1C-6 and 1C-7, carrying the spe mutation TP-138 in otherwise wild-type backgrounds, were isolated for study.
The TP-138 mutation, like other polyaminerequiring mutations (14), caused poor ascospore germination even in supplemented media, particularly in allelic crosses. Despite this, crosses of strain IC-6 with wild type gave no indication that the putrescine-requiring phenotype was other than monogenic (Table 2). A cross of strains IC-6 or IC-7 with strains carrying previously isolated putrescine auxotrophic mutations (IC-4 or IC-5) yielded few spores, but none were prototrophic ( Table 2). This is significant in view of the high degree of selection for prototrophs in germination. A cross of strain IC-6 to the inlbearing IC-8 strain demonstrated linkage of TP-138 and inl (ca. 5 centimorgans [cM], based on Spe+ progeny). This was confirmed in a second cross, spe, aga x inl (Table 2), in which the map distance was approximately 4 cM. These map distances are somewhat shorter than those de-  (8). Growth of wild type was not influenced by the presence or absence of polyamines; only the data for spermidine are given. Strain IC-6 grown in minimal medium yielded a trace of growth at 25C and none at 35°C. Strain IC4 did not grow at all in minimal medium.
termined by McDougall et al. (14) (ca. 10 cM) and by us ( Table 2) with spe-l allele 462JM. A test of complementation among three spe-l strains of the same mating type and carrying different alleles (TP-138, 462JM, and 521KW) was negative. We conclude on the basis of intercrosses (Table 2), complementation, and linkage data that the three mutations are allelic. The locus is hereby named spe-l with the consent of K. J. McDougall. The symbol, derived from the end product(s) of the pathway, supplants put-1, which is based on the intermediate.
All three spe-l mutants grew well with 1.0 mM putrescine, spermidine, or spermine at 25°C (Fig. 3). The same was true at 35°C for strains caiyming the earlier mutations, 462JM (IC-4) and 521KW (IC-5). Strains carrying the TP-138 mutation (e.g., IC-6), however, grew much more poorly at 35°C even on 1 mM (Fig. 3) or 5 mM polyamiines. This character cosegregated in small progenies with TP-138 ( Table 2), but its genetic and physiological basis cannot be inferred until more spe-l mutants are isolated from the parent strain.
Onithe decarboxylase activity. Ornithine decarboxylase activity was measured in extracts of wild-type, an aga-bearing strain, and strains carrying the three spe-l mutations grown exponentially under various nutritional conditions (Table 3). Wild-type N. crassa had an activity of about 20 U/mg of protein, a value that did not change significantly when the strain was grown in putrescine or arginine. This is in contrast to the finding of Sikora and McDougall (19) that ornithine decarboxylase activity of wild type was augmented fourfold in arginine-grown cultures.
The arginaseless strain grown in minimal medium had normal ornithine decarboxylase activity. When grown in the presence of arginine, it became ornithineand polyamine-starved (see above) and grew more slowly. As expected on the basis of previous data (16,19), the strain had about 60-fold normal enzyme activity (Table 3),  in keeping with the postulated negativ of the enzyme by polyamines (16).
The three putrescine-requiring strain er grown on limiting or unlimiting pi displayed no detectable ornithine decal activity in assays that would have meat than 1% wild-type activity (Table 3). N spe-l extracts altered the ornithine de ase activity of wild-type or aga extra mixed with them.
A test of the extracts for lysine decai showed easily detectable activity in the grown aga strain, IC-3. Wild-type ext little activity, and spe-) extracts had no 3). The proportional variation of 1l ornithine decarboxylase activities sug the activities are properties of the samt particularly in view of their simultaneo D.25 mutation. When the extract of the argininer grown aga strain was used, the Km value for ornithine was estimated to be 0.3 mM. The Km D.20 z for lysine was too high to be measured (Fig. 4). g -At 5 mM substrate (saturating with respect to IV <> ornithine), lysine was decarboxylated at 1.2% 0^the rate of ornithine (Fig. 4). Lysine ( The spe-l mutants grew somewhat initially in minimal medium, but the amount of polyamines ctordnIaCe) found in such cultures can be accounted for by miino acid carry-over from the inoculum. Growth of spe-l mutants on 1 mM putrescine restored their pools to the normal range. Growth on spermidine did not lead to the appearance of putrescine (Davis re control and Paulus, in press). Strains given exogenous spermidine did not contain much more spermi-IS, wheth-dine than normal, as though polyamine uptake utrescine, was inefficient or polyamine capacity was fixed. rboxylase Of primary interest here are the polyamine sured less pools of the aga strain during nutritional maone of the nipulations (see above). The polyamine pools of carboxyl-strain IC-3 (aga) were compared after growth in lcts when minimal medium or in arginine-supplemented medium. The data (Table 4) show that arginine rboxylase caused a complete loss of the ornithine and arginine-putrescine pools, and the small amount of spertracts had midine present could be accounted for by the me (Table spermidine of the inoculum. (Pools of wild-type ysine and cultures on arginine-supplemented medium are gests that quite normal [Davis and Paulus, in press].) Howe enzyme, ever, in contrast to the spe-l cultures, cadaverus loss by ine (0.6 nmol/mg [dry weight]) and a detectable c Pools of wild type (in nanomoles per milligram) are, generally: ornithine, 30; putrescine, 1.1; and spermidine, 16.2 (Davis and Paulus, in press). d Carry-over from the inoculum accounts for spermidine in polyamine-starved cultures. amount of aminopropylcadaverine appeared in strains carrying aga. The amounts of these compounds were greater after lysine was added to such cultures. These observations suggest that the lack of ornithine and the extreme derepression of ornithine decarboxylase led to decarboxylation of lysine (endogenous or exogenous). The fact that starving spe-) cultures did not show any trace of cadaverine or its aminopropyl derivative suggests that cadaverine synthesis requires ornithine decarboxylase.
It now might be asked whether cadaverine serves as a polyamine substitute in the growth of N. crassa. Indeed, cadaverine did stimulate growth of spe-l mutants (Fig. 3). This was confirmed (Table 4) by the reduction of doubling time for exponential cultures. It is very likely that the indefinite growth of arginine-grown aga cultures (8) can be attributed to the endogenous synthesis of cadaverine, because they grew at a rate similar to that of spe-l cultures supplemented with added cadaverine (Table 4). It is not known whether the analog(s) is less effective than its normal counterpart(s) on a molar basis, because no cultures are available with intracellular levels of analogs equal to those of normal polyamines.

DISCUSSION
Our major findings can be summarized as follows. (i) There is a gene controlling ornithine decarboxylase in N. crassa on the right arm of linkage group V, confirming the original work of McDougall. It is not known whether the gene is the structural gene for the enzyme. (ii) Polyamine-deprived mycelia have greatly enhanced (60to 100-fold) ornithine decarboxylase activity, confirming expectations of earlier work (16). (iii) Cadaverine and its aminopropyl derivative appear only in cells having high ornithine decarboxylase activity and a lack of ornithine. Ornithine decarboxylase appears to be the sole enzyme of lysine decarboxylation in the preparations we have used.
There are several discrepancies between our results and those of McDougall (14,19). First, as noted above, the map distance (4 to 5 cM) between our new mutation, TP-138, and inl was less than the distance between 462JM and inl found by McDougall. We have confirmed the higher recombination rate between mutation 462JM and inl and consider that this may reflect pecularities of the chromosome segments carrying the 462JM and TP-138 alleles. Precedents for such differences are frequent in N. crassa (4). The fact that the TP-138 and 462JM neither complement nor recombine defines them as alleles of the same locus, spe-1.
We have been unable to confirm the fourfold augmentation of ornithine decarboxylase in argi-nine-supplemented wild-type cultures. More important, we have been unable to confirm the rather high ornithine decarboxylase activities reported by McDougall (14) for his mutants (carrying alleles 462JM and 521KW). We find that they conform to his original report (Genetics 77:s16-s17, 1974) in having less than 0.5% wild-type activity. The discrepancy may be related to our finding that strains carrying allele 462JM quite often revert to a Spe+ phenotype; mass transfers in nonselective media may allow revertants to contaminate an initially Spe-culture.
Yeast mutants lacking enzymes of polyamine synthesis have been isolated by others (5,6,22). The spe-10 locus (originally designated spe-1) controls ornithine decarboxylase, but its action does not appear to be straightforward (6). Yeast polyamine mutants share with those of N. crassa the ability to grow considerably in unsupplemented media until the exhaustion of internal polyamines (22). Yeast mutants, moreover, are exquisitely sensitive to the addition of polyamines to the medium. As little as 10-10 M of any polyamine elicits a growth response, and 10-6 M spermidine suffices for optimal growth (6). In contrast, N. crassa mutants grow optimally only with 5 x 10-4 M or more polyamine. The difference between species probably reflects differences in polyamine uptake capability rather than differences in demand or catabolism (16).
The synthesis of cadaverine and aminopropylcadaverine upon starvation of mutant cells for the normal polyamines was first described by Dion and Cohen (9) working with Escherichia coli. Using cells having only the ornithine decarboxylase route of polyamine synthesis, they imposed ornithine deprivation by feedback inhibiting ornithine formation with addition of arginine to the medium. (E. coli cannot make ornithine from arginine.) The derivation of cadaverine and aminopropylcadaverine from lysine was proven, and both lysine and cadaverine in the medium stimulated the growth of ornithine-deprived cells (9). Studies of spermidine analogs on growth and DNA replication of polyamine-requiring E. coli showed that aminopropylcadaverine and aniinopropyl-1,6-diaminohexane were stimulatory (10,12). The synthesis of cadaverine in E. coli is due largely to activity of a lysine decarboxylase (20). Strains lacking this enzyme make only traces of the compound. (It is not excluded that the ornithine decarboxylases of E. coli can weakly catalyze lysine decarboxylation.) Whereas E. coli strains unable to make any polyamine grow slowly, the fungal mutants eventually do not grow at all. In both N.
crassa and E. coli (20), however, cadaverine stimulates growth slightly when added to the medium. It is probable that the indefinite growth of the arginaseless strain of N. crassa when grown on arginine is due in large measure to its ability to synthesize cadaverine endogenously.
In animal cells, cadaverine occasionally appears in certain tissues. In a study of lysine decarboxylase activity in vitro, Pegg and McGill (18) could attribute all activity to a nonspecific action of ornithine decarboxylase with which the lysine decarboxylase activity copurified. (Results from cultured mammalian cells which suggested the presence of a specific lysine decarboxylase [1] were later shown to be attributable to mycoplasmas in the culture [2].) Thus, N. crassa fits the animal pattern for cadaverine synthesis, inasmuch as ornithine decarboxylasedeficient mutants cannot decarboxylate lysine.