The effect of veratrole on carotenoid biosynthesis by Phycomyces blakesleeanus

A number of chemical compounds are known to affect the biosynthetic pathways of β-carotene. Both site-specific inhibitors as well as general stimulators of carotenogenesis have been described. It has been reported that veratrole enhances β-carotene synthesis when applied to agar cultures of Phycomyces blakesleeanus but we found no significant stimulation of β-carotene production in submerged culture. Moreover, veratrole in high concentrations (> 0.1% w/v), unlike diphenylamine, inhibits the formation of phytoene, resulting in an almost total block of carotenoid biosynthesis.     

Liposome-protoplast fusion in Phycomyces blakesleeanus

Abstract We describe here fusion between phospholipid vesicles (liposomes) and protoplasts to the fungus Phycomyces blakesleeanus . Both 6-carboxyfluorescein and the kanamycin resistance harboured by the plasmid have been transferred from liposomes to protoplasts of Phycomyces by the fusion technique.     

Carbamoyl-phosphate synthase in Phycomyces blakesleeanus

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.     

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.     

DNA sequence organization in Phycomyces blakesleeanus

Reassociation analysis shows that the genome of the Zygomycete Phycomyces blakesleeanus consists of repetitive and single copy sequences arranged in a long period interspersion pattern. The average lengths of the interspersed repetitive (16 kb) and single copy (25 kb) sequences appear to be very large. The sequence organization resembles that reported for the Oomycete Achlya (Hudspeth et al., 1977).     

Cell wall carbohydrates in Phycomyces blakesleeanus burgeff

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fucose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosugars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-¹⁴C]-glucose into wall materials.Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but not by 5-fluorouracil.     

A genetic map of Phycomyces blakesleeanus

Summary Complementation tests among Phycomyces auxotrophic strains revealed the existence of four genes with mutants requiring riboflavin, three genes with purine auxotrophs, two with nicotinic acid auxotrophs, and two with lysine auxotrophs. A total of 134 sexual crosses between strains carrying mutations affecting phototropism (madA-madE), carotenoid biosynthesis (carA), auxotrophy (ribA-ribD, purA-purC, lysA and lysB, nicA and nicB, and leuA) and resistance to 5-fluorouracil (furA and furB) were studied; mating type (sex) was also included as a marker. The results from random spore analysis, tetrad analysis, and gene-centromere distances shows that these markers are distributed into 11 linkage groups.     

Chitin synthetase mutants of Phycomyces blakesleeanus

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N′-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 μM nikkomycin, chsA mutants grew reasonably well in the presence of 50 μM nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased K_a for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.     

Olfactory responses of Phycomyces blakesleeanus.

Upon exposure to low levels of various volatile compounds such as n-heptanol, methanol, CHCl₃, mercaptoheptane, etc., the sporangiophore of $\text{Phycomyces}\text{blakesleeanus}$ responds with a transient and reproducible decrease in its elongation rate. All 22 volatile substances tested (except H₂O) elicited negative responses. The amplitude of the responses depends on the compound and its concentration. A characteristic concentration, required for 50% inhibition, correlates remarkable well with the human olfactory threshold (coefficient of correlation r = 0.89 (P < 0.001)). Perhaps some process in olfaction is common to this fungus and higher systems.     

Mutants of Phycomyces blakesleeanus Defective in Acetyl-CoA Synthetase

Nine mutants of the filamentous fungus Phycomyces blakesleeanus have been isolated on the basis of their resistance to fluoroacetate. None of the isolates uses acetate as the sole carbon source. Genetic complementation experiments revealed that all the mutants belong to the same complementation group. Biochemical analysis indicated that the acetate-induced acetyl-CoA synthetase activity is abolished in all nine mutants, thus suggesting that they are affected in the gene coding for acetyl-CoA synthetase (facA).     

Production of carotene stereoisomers by Phycomyces blakesleeanus

Summary -Carotene steroisomers, mainly all-trans and to a small extent 9-cis, may be produced by the fungus Phycomyces blakesleeanus under normal fermentation conditions. The amount of the 9-cis--carotene may comprise up to 15% of the total -carotene. Similarly, cis-lycopene or-phytoene stereoisomers may be obtained when the fungus is fermented in the presence of specific -carotene inhibitors such as nicotine or diphenylamine respectively. This is the first report on the occurrence of cis-stereoisomers of carotenes in mycelial fungi.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

Some properties of chitinase from Phycomyces blakesleeanus

The cytosol of the sporangiophore of $\text{Phycomyces}$$\text{blakesleeanus}$ has considerable chitinolytic activity. This activity is strongly dependent on the presence of a dialyzable activator. Maximal activity is achieved at pH 5.5; and ionic strength and Ca⁺⁺ or Mg⁺⁺ have little effect. Ungerminated spores do not contribute activity. The possibility is discussed that chitinase might be involved in the growth response system by transiently loosening the rigid framework of chitin at specific and defined points.