Folylpolyglutamate Deficiencies in Two Methionine-requiring Mutants of Neurospora crassa

Abstract

The growth of two Neurospora crassa methionine auxotrophs (met-6, 35809, FGSC 1330 and mac, 65109, FGSC 3609), that have lesions affecting folylpolyglutamate synthetase, was examined by culture in defined, liquid media. Both mutants responded to L-methionine supplements but additions of homocysteine, cysteine, serine, formate, thymine and adenine, supplied singly or in various combinations were not effective in stimulating growth. Mycelial yields after 22 hr growth in 0.5 mM L-methionine-supplemented media were comparable to those of a wild type strain (Lindegren A, FGSC 853) cultured in defined minimal medium. Pulse feeding radio-labelled cysteine, glycine, serine and formate showed that both mutants had less ability to generate methionine than the wild type. Standard microbiological assays, conjugase treatments and DEAE-cellulose chromatography showed that folylpolyglutamates were lacking in met-6 and mac but accounted for about 70% of the wild type folate pool. The polyglutamyl chain lengths of these folates were determined following tritium labelling, acid cleavage and Chromatographic separation as diazo dye derivatives. The wild type contained mainly tri- and hexaglutamyl folates. In met-6, mono- and diglutamates predominated but in mac only monoglutamates were found. Extracts of met-6: mac heterokaryons contained folates that were hexaglutamate derivatives. These findings provide additional evidence that folylpolyglutamates have an essential role in the methionine synthesis of N. crassa.