Metabolism of ricinoleate by Neurospora crassa

Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and of 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate of 14 M–3 mM [1-¹⁴C]ricinoleate. The fate of the [¹⁴C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.     

Metabolism of pyrimidine deoxyribonucleosides in Neurospora crassa.

The experiments in this report involve the following series of reactions which were previously demonstrated with purified enzyme preparations from Neurospora crassa: thymidine a yields thymine ribonucleoside b yields thymine c yields 5-hydroxymethyluracil d yields 5-formyluracil e yields uracil-5-carboxylic acid f yields uracil. The evidence for some of the reactions occurring in vivo has been incomplete and for others totally lacking. In this paper intact cells of Neurospora are shown to be capable of converting the substrates of each of the reactions to the corresponding products. Studies are described which were carried out in vivo and in vitro with the pyrimidineless strains pyr-4,uc-1,uc-2 and pyr-4,uc-1,uc-3, developed by Williams and Mitchell. The results reported in the present paper indicate that (reaction a) and the uc-3 mutation affects thymine 7-hydroxylase (reactions c,d, and e). Evidence is presented for the 2'-hydroxylase reaction being the major, if not only, way by which Neurospora can initiate the conversion of thymidine to the pyrimidines of nucleic acids and for the 2'-hydroxylation of thymidine and deoxyuridine being catalyzed by the same enzyme. Deoxycytidine was shown not to be hydroxylated in intact cells but instead deaminated to deoxyuridine, which in turn was converted to uridine. Further studies with the uc-3-carrying strain showed that an enzyme other than thymine 7-hydroxylase can also convert 5-formyluracil to uracil-5-carboxylic acid.     

Nitrogen regulation of amino acid utilization by Neurospora crassa.

The production of an extracellular deaminase activity involved with the utilization of amino acids as sole sources of nitrogen is under the control of the nit-2 locus of Neurospora crassa. This locus is the sole major nitrogen regulatory locus described for N. crassa and is believed to encode a positive effector required for induction of activities involved with the utilization of alternate nitrogen sources. Production of deaminase activity requires the lifting of nitrogen metabolite repression, the presence of a functional nit-2 gene product, and specific induction by amino acids. Additional parameters of enzyme production are described.     

Glutamine Metabolism and Cycling in Neurospora crassa

[This corrects the article on p. toc in vol. 54.].     

Unsaturated fatty acid mutants of Neurospora crassa.

Unsaturated fatty acid (ufa) auxotrophs of Neurospora crassa were obtained by treatment of conidia with N-methyl-N'-nitro-N-nitrosoguanidine followed by isolation on media containing polyunsaturated fatty acids suspended in Tergitol NP-40. The 24 mutants for which reisolates were obtained from crosses with wild type were assigned to two complementation classes, ufa-1 and ufa-2, located on linkage group V. Unsaturated fatty acids with varying degrees of unsaturation, chain length, and double-bond position as well as different steric configurations were tested for growth requirements.     

Nitrogen regulation of acid phosphatase in Neurospora crassa.

Neurospora crassa possesses a repressible acid phosphatase with phosphodiesterase activity which appears to permit it to utilize ribonucleic acid as a phosphorus and as a nitrogen source. This acid phosphatase, which is specified by the pho-3 locus, is derepressed approximately eightfold during nitrogen limitation and to an even greater extent during phosphorus limitation, but is unaffected by sulfur limitation. Derepression of the enzyme did not occur when adenosine 5'-monophosphate was the sole phosphorus or nitrogen source. Synthesis of the acid phosphatase is not under the control of the nit-2 locus, which regulates the expression of a large number of other nitrogen catabolic enzymes. The structural gene of the acid phosphatase appears to be a member of both the phosphorus and nitrogen regulatory circuits.     

Circadian Rhythms of Nucleic Acid Metabolism in Neurospora crassa

Wild-type, band, and fluffy strains of Neurospora crassa exhibit circadian rhythms of ribonucleic acid and deoxyribonucleic acid content in the growth-front hyphae of cultures grown on a solid medium. There is also a rhythm of (3)H-uridine incorporation into the nucleic acids of the band strain. Maximum incorporation precedes the peaks of nucleic acid content which occur during conidiation. As cultures age, ribonucleic acid content decreases rapidly and deoxyribonucleic acid content decreases gradually in standing, shake, and bubble cultures. A reduction of ribonuclease activity with age is also noted in standing and shake cultures. The nucleic acid content, nuclease activity, and changes associated with age vary with the culture conditions.     

Cryobiology of Neurospora crassa. II. Alteration in freeze response of Neurospora crassa conidia by additives

Neurospora crassa conidia in aqueous suspensions were frozen and thawed in the presence of various agents. Colony counts with these treatments were compared with those of the following (a) unfrozen, agent-treated, (b) unfrozen water suspended, and (c) frozen, water suspended. It was found that dimethyl sulfoxide (0.5–20%) resulted in total protection against freeze damage. Glycerol and calcium chloride decreased survival as much as 90% with fast freeze. The latter agents have properties which should decrease the rate of outflow of cellular water during temperature lowering. The results are consistent with the proposal that intracellular ice crystal growth to membrane rupturing dimensions is the damaging freeze mechanism under these conditions.     

Regulation of biosynthesis of l-amino acid oxidase by Neurospora crassa

A temperature gradient device has been used to produce pH-temperature gradient plates. The response of Salmonella typhimurium and Bacillus cereus to continuous gradients of pH and temperature has been mapped. A third variable, sodium chloride, was investigated by incorporating it at different concentrations in a series of gradient plates. Both organisms exhibited an optimum temperature and pH range for maximum salt tolerance.     

Cellulase of Neurospora crassa.

Mycelia and ungerminated conidia of Neurospora crassa were found to secrete extracellular endocellulase (EC 3.2.1.4). A simple induction system of potassium phosphate buffer (ph 6.0) plus inducer relied on the internal metabolic reserves of conicia or mycelia to provide energy and substrates for protein synthesis. Buffer concentration for optimum enzyme production was 100 mM, but at higher buffer concentrations enzyme production was inhibited. Cellobiose was clearly the best inducer, with an optimum effect from 0.05 to 1 mM. In deionized water, cellulase remained mostly associated with the cell, but a variety of salts stimulated the release of cellulase into the medium.