Antifungal peptides homologous to the Penicillium chrysogenum antifungal protein (PAF) are widespread among Fusaria

Putative antifungal peptide encoding genes containing Penicillium chrysogenum antifungal protein (PAF) characteristic amino acid motifs were identified in 15 Fusarium isolates, representing 10 species. Based on the predicted sequences of mature peptides, discrepancy in one, two or three amino acids was observed between them. Phylogenetic investigations revealed that they show high amino acid sequence similarity to PAF and they belong to the group of fungal derived antifungal peptides with PAF-cluster. Ten from the 15 partially purified <10 kDa peptide fraction of Fusarium ferment broths showed antifungal activity. The presence of approximately 6.3 kDa molecular weight peptides was detected in all of the antifungally active ferment broths, and this peptide was isolated and purified from Fusarium polyphilaidicum. The minimal inhibitiory concentrations of F. polyphilaidicum antifungal protein (FPAP) were determined against different filamentous fungi, yeasts and bacteria. Filamentous fungal species were the most susceptible to FPAF, but some yeasts were also slightly sensitive.     

Ds-like restless deletion derivatives occur in Tolypocladium inflatum and two foreign hosts, Neurospora crassa and Penicillium chrysogenum

Single copies of the transposon Restless from Tolypocladium inflatum were introduced into Neurospora crassa and Penicillium chrysogenum. Excision of Restless from its donor site was investigated in N. crassa and in P. chrysogenum using direct selective conditions. In N. crassa, forward selection was also analyzed. Deleted Restless elements were frequently obtained in addition to the expected complete removal of Restless from its donor site. Similar deleted elements were also identified in T. inflatum employing a PCR amplification strategy. These deleted Restless copies strongly resemble maize Ds elements of various types, and direct repeated sequences of 3 to 16 bp were found to flank the truncated regions. In addition Ds1-like Restless elements were identified that carried foreign sequences between the inverted repeats. We discuss how Ds-like Restless elements might be generated by inaccurate excision from an active transposon copy.     

Penicillin Acylase Activity of Penicillium chrysogenum

The penicillin acylase activity of Penicillium chrysogenum was studied. Washed mycelial suspensions of a high penicillin-producing and a nonproducing strain were found to be similar in respect to relative acylase activity on benzylpenicillin, 2-pentenylpenicillin, heptylpenicillin, and phenoxymethylpenicillin. The relative rates for both strains, as determined by 6-aminopenicillanic acid formation, were approximately 1.0, 2.5, 3.5, and 6.0 on the penicillins in the order given. The high producing strain formed both 6-aminopenicillanic acid and "natural" penicillins in fermentations to which no side-chain precursor had been added. Therefore, its demonstrated ability to cleave the natural penicillins, 2-pentenylpenicillin and heptylpenicillin, suggests that at least some of the 6-aminopenicillanic acid produced during such fermentations arises from the hydrolysis of the natural penicillins. At pH 8.5, the mycelial acylase activity of the nonproducing strain was about three times that at pH 6.0; at 35 C, it was about 1.5 times as active as it was at 30 C. When tested on penicillin G or V, no differences in either total or specific penicillin acylase activity were observed among mycelia harvested from cultures of the nonproducer to which penicillin G, penicillin V, or no penicillin had been added. Acetone-dried mycelium from both strains displayed acylase activity, but considerably less than that shown by viable mycelium. Culture filtrates were essentially inactive, although a very low order of activity was detected when culture filtrate from the nonproducer was treated with acetone and the acetone-precipitated material was assayed in a minimal amount of buffer.     

Protein Kinase Activities in Neurospora crassa

Several protein kinase activities have been found in 105,000g supernatant of Neurospora crassa mycelia grown up to the logarithmic phase. By chromatography on DEAE-cellulose the following enzyme activities have been resolved: (i) a cyclic AMP-dependent protein kinase (peak I kinase) eluting at 0.20 m NaCl, more active with histone than with phosvitin (it was inhibited by both a thermolabile fraction having cyclic AMP-binding activity and a thermostable inhibitor isolated from 105,0005g mycelial supernates), (ii) a cyclic nucleotide-independent protein kinase (peak II kinase) eluting at 0.35 m NaCl, also more active with histone than with phosvitin (this kinase was not inhibited by the fraction having cyclic AMP-binding activity but it was sensitive to the thermostable inhibitor); and finally, (iii) a protein kinase eluting at 0.43 m NaCl (peak II kinase), with similar activity toward histone and phosvitin, insensitive to cyclic nucleotides and to fractions carrying cyclic AMP-binding capacity (this kinase activity also resulted insensitive to the thermostable inhibiting factor).     

Protein kinase activities in Neurospora crassa

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified from human erythrocytes using a simple chromatographic procedure. Purified enzyme was obtained from individuals who were homozygous for the principal isozyme (ADA 1) as well as from individuals who were heterogyzous for the major variant (ADA 2-1). Although ADA 1 and ADA 2-1 are electrophoretically distinguishable, they have many common physical and catalytic properties. No significant differences between the two isozymic forms were found in measurements of molecular weight, catalytic activity in the presence of various substrates and inhibitors, pH optimum, turnover number, and stability in conditions of both high and low pH. ADA 2-1 was, however, substantially less stable than ADA 1 with respect to thermal denaturation. These studies support the idea that adenosine deaminase activity in erythrocytes is lower in those individuals who possess the variant form of the enzyme.     

Calcium as a branching signal in Neurospora crassa

The divalent cation ionophore A23187 was found to induce apical branching in Neurospora crassa. Optimal effects were obtained by treatment with 0.1 mM ionophore for 30 min. Branching first became manifest during or shortly after treatment; successive rounds of branching could be observed at later times. Calcium starvation of the mycelium markedly reduced its subsequent response to the ionophore, whereas starvation for other divalent cations had no detectable effect. The branching response was markedly reduced in the presence of 10 to 30 mM cyclic AMP or derivatives thereof.     

Production of Calcium Gluconate by Penicillium chrysogenum in Submerged Culture

The waste mycelium of Penicillium chrysogenum HA-10 (obtained at the end of penicillin fermentation), or a 24-hr-old freshly grown vegetative inoculum of this organism, was found to utilize glucose for the production of calcium gluconate by submerged fermentation in shake flasks. After 72 to 96 hr of fermentation at 24 C, 90 to 95% of the reducing sugar from the 15% glucose medium was converted to calcium gluconate. Reuse of the mycelium for successive experiments reduced the fermentation period to 72 hr or less because of an enhancement of glucose utilization. Ten successive batches of 15% glucose medium were fermented by the reuse method. Fermentation media containing up to 30% glucose could be used, provided boric acid was added to prevent the precipitation of calcium gluconate formed. We found that 30% hydrol (a by-product of glucose manufacture containing 50 to 55% reducing sugar), when used in place of glucose in the fermentation medium, inhibited the rate of glucose utilization. However, this effect was partially reversed by pretreatment of hydrol with 2 to 4% activated charcoal before addition to the fermentation medium.     

Protein import into mitochondria of Neurospora crassa

Biogenesis of mitochondria requires import of several hundreds of different nuclear-encoded preproteins needed for mitochondrial structure and function. Import and sorting of these preproteins is a multistep process facilitated by complex proteinaceous machineries located in the mitochondrial outer and inner membranes. The translocase of the mitochondrial outer membrane, the TOM complex, comprises receptors which specifically recognize mitochondrial preproteins and a protein conducting channel formed by TOM40. The TOM complex is able to insert resident proteins into the outer membrane and to translocate proteins into the intermembrane space. For import of inner membrane or matrix proteins, the TOM complex cooperates with translocases of the inner membrane, the TIM complexes. During the past 30 years, intense research on fungi enabled the identification and mechanistic characterization of a number of different proteins involved in protein translocation. This review focuses on the contributions of the filamentous fungus Neurospora crassa to our current understanding of mitochondrial protein import, with special emphasis on the structure and function of the TOM complex.     

Protein changes during the asexual cycle of Neurospora crassa.

A method for synchronizing conidiation and isolating large numbers of cells at discrete stages of conidia development is described. Using two-dimensional gel electrophoresis, we analyzed the protein profiles of mycelia, aerial hyphae, and conidia and observed that the concentration of 14 polypeptides increase and 38 decrease during the asexual cycle. Twelve polypeptides were present in extracts of aerial hyphae or conidia, but not mycelia, suggesting that they may be conidiation specific. The protein profiles of mutants defective in conidiation were also analyzed. Differences were detected in the two-dimensional profiles of protein extracts from fluffy and wild-type aerial hyphae. Polyadenylated RNA isolated from wild-type mycelia and conidiating cultures was translated in vitro in a rabbit reticulocyte lysate. Differences were detected in the polypeptide products specified by the two RNA populations, suggesting that changes in steady-state levels of polyadenylated RNAs also occur during conidiation.     

Effect of growth temperature on lipid fatty acids of four fungi (Aspergillus niger,Neurospora crassa,Penicillium chrysogenum,andTrichoderma reesei)

The effect of growth temperature on the lipid fatty acid composition was studied over a temperature range from 35 to 10° C with 5° C intervals in four exponentially growing fungi: Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Trichoderma reesei. Fatty acid unsaturation increased in A. niger, P. chrysogenum, and T. reesei when the temperature was lowered to 20–15, 20, and 26–20° C, respectively. In A. niger and T. reesei, this was due to the increase in linolenic acid content. In P. chrysogenum, the linolenic acid content increased concomitantly with a more pronounced decrease in the less-unsaturated fatty acid, oleic acid, and in palmitic and linoleic acids; consequently, the fatty acid content decreased as the temperature was lowered to 20° C. In T. reesei, when the growth temperature was reduced below 26–20° C, fatty acid unsaturation decreased since the mycelial linolenic acid content decreased. In A. niger and P. chrysogenum, the mycelial fatty acid content increased greatly at temperatures below 20–15° C. In contrast, in N. crassa, fatty acid unsaturation was nearly temperature-independent, although palmitic and linoleic acid contents clearly decreased when the temperature was lowered between 26 and 20° C; concomitantly, the growth rate decreased. Therefore, large differences in the effects of growth temperature on mycelial fatty acids were observed among various fungal species. However, the similarities found may indicate common regulatory mechanisms causing the responses. Received: 1 March 1995 / Accepted: 8 May 1995     

Induction of Neurospora crassa Laccase with Protein Synthesis Inhibitors

Rapidly growing Neurospora crassa does not produce laccase (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2). Low concentrations of cycloheximide induce the production of this enzyme, most of which is secreted into the media. In general, limited inhibition of protein synthesis seems to derepress laccase synthesis since actinomycin D and, to a limited extent, puromycin also induce laccase production. Similarities in the conditions of laccase and tyrosinase induction, plus investigations with two tyrosinase regulatory mutants, suggest that the production of these two phenol oxidases is controlled by the same mechanism. As shown by polyacrylamide gel electrophoresis, most of the 10 to 12 proteins normally present in the medium virtually disappear during cycloheximide treatment. In contrast, the amounts of two proteins that are present in only very minor quantities, if at all, in normal culture filtrates increase dramatically. One of these proteins co-migrates with laccase, whereas the other has not been identified.     

Protein chemistry of the Neurospora crassa plasma membrane H+-ATPase

A highly effective procedure for fragmenting the Neurospora crassa plasma membrane H+-ATPase and purifying the resulting peptides is described. The enzyme is cleaved with trypsin to form a limit digest containing both hydrophobic and hydrophilic peptides, and the hydrophobic and hydrophilic peptides are then separated by extraction with an aqueous ammonium bicarbonate solution. The hydrophilic peptides are fractionated by Sephadex G-25 column chromatography into three pools, and the individual peptides in each pool are purified by high-performance liquid chromatography. The hydrophobic peptides are dissolved in neat trifluoroacetic acid (TFA), diluted with chloroform-methanol (1:1), and the hydrophobic peptide solution thus obtained is then fractionated by Sephadex LH-60 column chromatography in chloroform-methanol (1:1) containing 0.1% TFA. The recoveries in all of the above procedures are greater than 90%. The N-terminal amino acid sequences of three of the hydrophobic H+-ATPase peptides purified by this methodology have been determined, which establishes the position of these peptides in the 100,000 Da polypeptide chain by reference to the published gene sequence, and documents the sequencability of the hydrophobic peptides purified in this way. This methodology should facilitate the identification of a variety of amino acid residues important for the structure and function of the H+-ATPase molecule. Moreover, the overall strategy for working with the protein chemistry of the H+-ATPase should be applicable to other amphiphilic integral membrane proteins as well.     

Rhythms of Enzyme Activity Associated with Circadian Conidiation in Neurospora crassa

The mycelial growth front of the band strain of Neurospora grown on a solid surface exhibits a circadian rhythm of conidiation. Enzyme assays on extracts from that mycelium have shown that the activities of 6 of 13 enzymes (nicotinamide adenine dinucleotide nucleosidase, isocitrate lyase, citrate synthase, glyceraldehydephosphate dehydrogenase, phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase) and soluble-protein content oscillate with the visible morphological change. The rhythmic enzymes associated with the Krebs and glyoxylate cycles are more active during conidiogenesis, whereas the activities of the rhythmic enzymes of glycolysis and the hexose monophosphate shunt are reduced during that phase. The absence of enzyme oscillations in wild-type and fluffy strains which do not form conidia under the conditions employed suggests that the enzyme fluctuations are associated with conidiogenesis itself. Oscillations of enzyme activity as a function of time are restricted to the growth front. A permanent record of rhythmicity associated with conidial and nonconidial regions does, however, exist in the mycelial mat behind the growth front. The activities of three enzymes (nicotinamide adenine dinucleotide nucleosidase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase) are not directly influenced by CO(2) concentration, but are correlated with the prescence or absence of conidiation which is controlled by CO(2) concentration. In contrast, citrate synthase and malate dehydrogenase activities are correlated with changes in CO(2) concentration.     

The Structure of Neurospora crassa Glycogen

The mycelia of a wild type strain of Neurospora crassa (6068, IFO) contain a polysaccharide which is stained reddish brown by iodine. The polysaccharide purified by repeated precipitation with ethanol is made up of d-glucose and has a molecular weight of about (more than) 2 × 10⁷, 101 S on ultracentrifugation analysis, an average chain length of 10, β-amylolysis limit of 33.6%, and α-amylolysis limit of 58.3%. The highly branched structure, therefore, resembles to that of a typical glycogen. The properties of the glycogen from N. crassa are discussed in comparison with the commercial glycogens from shellfish and rabbit liver.