Altered Fatty Acid Distribution in Mutants of Neurospora crassa

Abstract

Morphological mutants of Neurospora with decreased levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide adenine dinucleotide (NADH) contained only 20% as much of a polyunsaturated fatty acid (linolenic acid) as the wild type in both the phospholipid and neutral lipid fractions. There was an excellent correlation between linolenic acid levels and morphological appearance as a function of total NADPH content, but no correlation with NADH content. The linolenic acid deficiency was balanced by a relative increase in the amounts of the less unsaturated fatty acids (oleic and linoleic acids), but the level of three other fatty acids did not appear to be changed. This accumulation of these two precursors suggests that the NADPH deficiency preferentially affected the final desaturation step, i.e., the conversion of linoleic to linolenic acid. The NADPH needed for this reaction in vivo was probably generated by the pentose phosphate shunt, since mutations affecting the shunt lead to the decreased levels of linolenic acid. It is not clear whether the changes in fatty acid distribution affect the morphogenesis of Neurospora, or if these changes are just part of the NADPH-deficiency syndrome.The primary biochemical deficiency of a morphological mutant of Neurospora crassa (col-2) was described (3) as an alteration in glucose-6phosphate dehydrogenase (EC 1.1.1.49), an enzyme which generates reduced nicotinamide adenine dinucleotide phosphate (NADPH). One pleiotropic effect produced by this mutational alteration was a decrease in the total content of NADPH as compared to that of the wild-type strain (2). The effects produced in turn by a decreased content of this cofactor could be numerous, but since it is generally thought that NADPH is primarily used for the synthesis of fatty acids and for the formation of unsaturated bonds in fatty acids, it was decided to investigate these possibilities first. NADPH is needed in those reactions catalyzed by mixed-function oxidases of the general reaction type: saturated fatty acid + 02 + NADPH yielding unsaturated fatty acid + nicotinamide adenine dinucleotide phosphate (NADP) + water. This paper reports the effects of an overall NADPH deficiency on the distribution of fatty acids in the phospholipid and neutral lipid fractions of Neurospora. MATERIALS AND METHODSCultures. RL3-8A (wild type), col-2, col-3, rg, col-2b2 inosSI, and col-2b2 inosRil (revertant) have all been described previously (3,4). Another morphological mutant (balloon) was obtained from the Fungal Genetics Stock Center, Dartmouth College, Hanover, N.H., as well as the nicotinic acid or tryptophan-requiring strain (nt). Double-mutant strains were constructed by the appropriate crosses.Growth conditions. All cultures were grown as liquid shake cultures in Vogel's minimal medium (10) containing 2% glucose at 23 C, unless otherwise stated. The col-2 nt strain received a supplement of 20 ug of nicotinic acid per ml. The growth and shearing (grinding in a sterile Waring Blendor) proced...

Purification and chemical characterization of the rodlet layer of Neurospora crassa conidia

“…Total yield based on summing maximum yield values is 792.5 mg g-1. lipids than to that of the neutral lipids (6). Extracting conidia with a lipid solvent caused no obvious change in rodlet structure (9).…”

Biochemical genetics of Neurospora crassa conidial germination.

“…Conidia do not contain many triglycerides (12, 146), whereas these lipids can account for more than 8% of the mycelial dry weight (17,143). The triglycerides are found predominantly in the older regions of the mycelia (240).…”

“…Perhaps the removal of cysteine would result in the synchronous formation of germ tubes but this has not been tested. Attempts to synchronize the appearance of germ tubes with heat shocks (13) or by flushing the culture with nitrogen (S. W. Bradford and B. I. Gibgot, Neurospora Newsletter 4: [17][18][19]1963) have not been very successful. For conidia from the wild-type strain, the requirements for the formation of germ tubes are simply a salt solution, a carbon source (132), and a supply of oxygen (82; Bradford and Gibgot, Neurospora Newsletter 4: [17][18][19]1963).…”

“…Another compound, benomyl Lmethyl-1-(butylcarbamoyl)-2-benzimidazole carbamatel, also inhibits hyphal elongation at concentrations that do not inhibit germ tube formation (K. S. Borck and H. D. Braymer, Neurospora Newsletter 20: [16][17]1973). Perhaps benomyl also inhibits the synthesis of chitin or some other cell wall component.…”

“…Two glucose transport systems, designated I and II (171), have been found in Neurospora mycelia (17,162,163,197). System I is constitutive and has a low affinity for glucose (162,171).…”