The Nature of the Lesion in the Succinate-requiring Mutants of Neurospora crassa: Interaction between Carbohydrate and Nitrogen Metabolism

Abstract

SUMMARY: Mutants of Neurospora crmsa requiring dicarboxylic acids for an immediate growth response (SUC and at suc) oxidize acetate, and are inhibited by fluoroacetate with consequent citric acid accumulation to approximately the same extent as the wild-type. The concentration of nitrogen (as ammonium and nitrate salts) present in the conventional growth medium is inhibitory to the growth of these mutants and leads to an accumulation of acetylmethylcarbinol, pyruvic acid and a-ketoisovaleric acid. This inhibition is reduced and growth is stimulated by the addition of dicarboxylic acids or by diminution of the nitrogen present in 'minimal' medium. The addition of nitrogen salts to SUC mutants probably diverts dicarboxylic acids (already in short supply) from the catalysis of the oxidation of C, fragments via the tricarboxylic acid cycle to other reactions. This effect of nitrogen salts upsets the already precarious dicarboxylic acid balance of the suc mutants leading to a growth requirement and to the accumulation of intermediates.This investigation was begun because the existence of mutants of the ascomycete Neurospora crassa, which grow on minimal medium only after the addition of small quantities of dicarboxylic acids (succinate-requiring mutants), seemed difficult to reconcile with an apparent use of the tricarboxylic acid cycle for both synthesis and energy by Nearospora spp. (Lewis, 1948;Strauss, 1955a). Accepting the idea of a 'genetic block' in the cyclic metabolism of dicarboxylic acids by the succinate-requiring mutants seems to require the assumption that acetate is oxidized by some mechanism other than the tricarboxylic acid cycle (Krebs, Gurin & Eggleston, 1952) or that the 'genetic block' is not absolute. Since either of these explanations would be of interest, the properties of the succinate-requiring mutants have been studied in some detail.It now appears that the succinate-requiring mutants do use the tricarboxylic acid cycle, and that there is no enzymic block to the operation of the cycle. Dicarboxylic acids required to catalyse acetate oxidation are in short supply and, as a result, excess ammonium or nitrate nitrogen inhibits these mutants by withdrawing this limited supply of dicarboxylic acids for synthetic purposes. Inhibition by excess nitrogen is at least partly responsible for the failure of succinate-requiring mutants to grow normally on ' minimal medium' (Beadle & Tatum, 1945).It is the purpose of this paper to present the evidence for the interaction of carbohydrate and nitrogen metabolism in the succinate-requiring mutants, and to discuss briefly the implications of these findings.

AN ACETATE-REQUIRING MUTANT STRAIN OF <i>NEUROSPORA</i> <i>CRASSA</i> DUE TO A POSSIBLE PARACENTRIC INVERSION

“…Some characteristics of suc mutants have already been reported (Lewis 1948;Strauss 1956Strauss , 1957. Acetohydroxy acid synthetase activity of all the eleven suc strains has as high a derepressed level as that of strain 163 (Table 2).…”

Some Carbon-Dioxide Requiring Mutants of Neurospora crassa

“…The response to acetate is not difficult to understand because in many organisms acetate is known to be metabolized via the glyoxylate cycle, the net effect of which is to generate succinate from acetate. These 'carboxylic acid' mutants may be identical with the succinate mutants studied by Lewis (1948) and Strauss (1956, Some C0,-requiring mutants of N . crass 73 1957).…”

“…These ' succinate ' mutants did not require arginine or a purine or a pyrimidine, nor were they inhibited by these substances. In their nutritional requirements they resembled the ' succinate ' mutants studied by Lewis (1948) and Strauss (1956Strauss ( , 1957. They responded to several long-chain fatty acids; there is no record in the literature of the effect of these fatty acids on other SUCcinate mutants.…”

Cell Death and 'Unbalanced Growth' in Neurospora

“…Ethionine inhibits conidiation and this inhibition is overcome in a competitive fashion by methionine (Table 1). Ammonium ion inhibits conidiation by the at suc strain and this inhibition is overcome by succinate as is ammonium inhibition of the growth process (Strauss, 1956). Inhibition by ethionine is effective only when the ethionine is added within the first 9 hr.…”

“…In most of the previous investigations with Neurospora, conidia were collected from cultures grown on solid medium after growth periods ranging from 4 to 8 days (Strauss, 1956). Spores are formed throughout the growth period and what is actually obtained is a population of spores of different ages.…”