Mucidin and strobilurin A are identical and inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain at the same site as myxothiazol

Mucidin and strobilurin A, antifungal antibiotics isolated from the basidiomycetes Oudemansiella mucida and Strobiluris tenacellus, respectively, inhibit electron-transfer reactions in the cytochrome bc1 complex of the mitochondrial respiratory chain. The two compounds have identical effects on oxidation-reduction reactions of the cytochromes b and c1 in isolated succinate-cytochrome c reductase. They inhibit reduction of cytochrome c1 by succinate but do not inhibit reduction of cytochrome b. When added in combination with antimycin, either inhibitor blocks reduction of both cytochromes b and c1. Mucidin and strobilurin A differ from antimycin in that they inhibit, rather than promote, oxidant-induced reduction of cytochrome b. They also differ from antimycin in that they do not block reduction of cytochrome b by succinate when cytochrome c1 is previously reduced by ascorbate and they do not inhibit oxidation of cytochrome b by fumarate. These effects of mucidin and strobilurin A are, however, qualitatively identical with those of myxothiazol, an antibiotic that inhibits respiration by binding to cytochrome b [Von Jagow, G., Ljungdahl, P. O., Graf, P., Ohnishi, T., & Trumpower, B. L. (1984) J. Biol. Chem. 259, 6319-6326]. Mucidin and strobilurin A have identical UV and mass spectra, and they elute together on high-pressure liquid chromatography. We thus conclude that these antibiotics, although isolated from different bacteria, are structurally identical. Our results indicate that strobilurin A and mucidin inhibit electron transport at the same site as myxothiazol and not at the antimycin site, as previously reported [Subik, J., Behren, M., & Musilek, V. (1974) Biochem. Biophys. Res. Commun. 57, 17-22].     

Phosphofrucktokinase and glucose catabolism of Mucor and Penicillium species.

The primary catabolic pathways in the fungi Penicillium notatum and P. duponti, and Mucor rouxii and M. miehei were examined by measuring the relative rate of 14CO2 production from different carbon atoms of specifically labelled glucose. It was found that these organisms dissimilate glucose predominantly via the Embden--Meyerhof pathway in conjunction with the tricarboxylic acid cycle and to a lesser extent by the pentose phosphate pathway. Phosphofructokinase (EC 2.7.1.11) activity could not be detected initially in Penicillium species because of the interference from mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and NADH oxidase (EC 1.6.99.3). A combination of differential centrifuging and a heat treatment of Penicillium cell-free extracts in the presence of fructose-6-phosphate removed the interfering enzymes. The kinetic characteristics of phosphofructokinase from P. notatum and M. rouxii are described. The enzyme presents highly cooperative kinetics for fructose-6-phosphate. The kinetics for ATP show no cooperativity and inhibition by excess ATP is observed. The addition of AMP activated the P. notatum enzyme, relieving ATP inhibition; slight inhibition by AMP was observed with the M. rouxii enzyme. In contrast M. rouxii pyruvate kinase (EC 2.7.1.40) is activated 50-fold by fructose-1,6-diphosphate whereas pyruvate kinase from P. notatum and P. duponti were unaffected by fructose-1,6-diphosphate.     

Why did the Fleming strain fail in penicillin industry?

Penicillin, discovered 75 years ago by Sir Alexander Fleming in Penicillium notatum, laid the foundations of modern antibiotic chemotherapy. Early work was carried out on the original Fleming strain, but it was later replaced by overproducing strains of Penicillium chrysogenum, which became the industrial penicillin producers. We show how a C(1357)-->T (A394V) change in the gene encoding PahA in P. chrysogenum may help to explain the drawback of P. notatum. PahA is a cytochrome P450 enzyme involved in the catabolism of phenylacetic acid (PA; a precursor of penicillin G). We expressed the pahA gene from P. notatum in P. chrysogenum obtaining transformants able to metabolize PA (P. chrysogenum does not), and observing penicillin production levels about fivefold lower than that of the parental strain. Our data thus show that a loss of function in P. chrysogenum PahA is directly related to penicillin overproduction, and support the historic choice of P. chrysogenum as the industrial producer of penicillin.     

Purification and properties of Penicillium glucose 6-phosphate dehydrogenase

1. Glucose 6-phosphate dehydrogenase was isolated and partially purified from a thermophilic fungus, Penicillium duponti, and a mesophilic fungus, Penicillium notatum. 2. The molecular weight of the P. duponti enzyme was found to be 120000+/-10000 by gelfiltration and sucrose-density-gradient-centrifugation techniques. No NADP(+)- or glucose 6-phosphate-induced change in molecular weight could be demonstrated. 3. Glucose 6-phosphate dehydrogenase from the thermophilic fungus was more heat-stable than that from the mesophile. Glucose 6-phosphate, but not NADP(+), protected the enzyme from both the thermophile and the mesophile from thermal inactivation. 4. The K(m) values determined for glucose 6-phosphate dehydrogenase from the thermophile P. duponti were 4.3x10(-5)m-NADP(+) and 1.6x10(-4)m-glucose 6-phosphate; for the enzyme from the mesophile P. notatum the values were 6.2x10(-5)m-NADP(+) and 2.5x10(-4)m-glucose 6-phosphate. 5. Inhibition by NADPH was competitive with respect to both NADP(+) and glucose 6-phosphate for both the P. duponti and P. notatum enzymes. The inhibition pattern indicated a rapid-equilibrium random mechanism, which may or may not involve a dead-end enzyme-NADP(+)-6-phosphogluconolactone complex; however, a compulsory-order mechanism that is consistent with all the results is proposed. 6. The activation energies for the P. duponti and P. notatum glucose 6-phosphate dehydrogenases were 40.2 and 41.4kJ.mol(-1) (9.6 and 9.9kcal.mol(-1)) respectively. 7. Palmitoyl-CoA inhibited P. duponti glucose 6-phosphate dehydrogenase and gave an inhibition constant of 5x10(-6)m. 8. Penicillium glucose 6-phosphate dehydrogenase had a high degree of substrate and coenzyme specificity.     

Oxidative ring fission of the naphthoquinones lapachol and dichloroallyl lawsone by Penicillium notatum

The naphthoquinones lapachol and dichloroallyl lawsone readily undergo oxidative ring fission when incubated with several fungi and streptomycetes. Penicillium notatum was employed to produce the ring fission product of dichloroallyl lawsone which was isolated and characterized by spectral analyses and chemical synthesis. The mechanism of oxidative ring fission of lapachol was studied by growing P. notatum cultures in an 18O2 atmosphere. Mass spectral analysis of the isolated and labeled metabolite indicates that ring fission occurs via a monooxygenase pathway most probably involving an epoxide intermediate.     

Pyranose Oxidase, a Major Source of H(2)O(2) during Wood Degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida

The production of the H(2)O(2)-generating enzyme pyranose oxidase (POD) (EC 1.1.3.10) (synonym, glucose 2-oxidase), two ligninolytic peroxidases, and laccase in wood decayed by three white rot fungi was investigated by correlated biochemical, immunological, and transmission electron microscopic techniques. Enzyme activities were assayed in extracts from decayed birch wood blocks obtained by a novel extraction procedure. With the coupled peroxidase-chromogen (3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolinone hydrazone hydrochloride) spectrophotometric assay, the highest POD activities were detected in wood blocks degraded for 4 months and were for Phanerochaete chrysosporium (149 mU g [dry weight] of decayed wood), Trametes versicolor (45 mU g), and Oudemansiella mucida (1.2 mU g), corresponding to wood dry weight losses of 74, 58, and 13%, respectively. Mn-dependent peroxidase activities in the same extracts were comparable to those of POD, while lignin peroxidase activity was below the detection limit for all fungi with the veratryl alcohol assay. Laccase activity was high with T. versicolor (422 mU g after 4 months), in trace levels with O. mucida, and undetectable in P. chrysosporium extracts. Evidence for C-2 specificity of POD was shown by thin-layer chromatography detection of 2-keto-d-glucose as the reaction product. By transmission electron microscopy-immunocytochemistry, POD was found to be preferentially localized in the hyphal periplasmic space of P. chrysosporium and O. mucida and associated with membranous materials in hyphae growing within the cell lumina or cell walls of partially and highly degraded birch fibers. An extracellular distribution of POD associated with slime coating wood cell walls was also noted. The periplasmic distribution in hyphae and extracellular location of POD are consistent with the reported ultrastructural distribution of H(2)O(2)-dependent Mn-dependent peroxidases. This fact and the dominant presence of POD and Mn-dependent peroxidase in extracts from degraded wood suggest a cooperative role of the two enzymes during white rot decay by the test fungi.     

Toxigenic Aspergilli and Penicillia Isolated from Aged, Cured Meats

Eighty-nine cultures of Aspergillus and 54 cultures of Penicillium isolated from aged, cured meats were tested for toxicity to chicken embryos. Two of 22 isolates of A. ruber, 5 of 28 A. repens, 2 of 12 A. sydowi, 1 of 12 A. restrictus, 2 of 7 A. amstelodami, 1 of 2 A. chevalieri, and an A. fumigatus isolate exhibited toxicity. Similarly, 2 of 15 isolates of P. expansum, 1 of 3 P. notatum, 1 of 2 P. brevi-compactum, and 1 of 8 Penicillium spp. were found to be the most toxic. Among these fungi, the chloroform extract from the growth of an A. sydowi isolate showed the greatest toxicity. There was no direct or indirect evidence that aged, cured meats contain toxic metabolites.     

Screening of microorganisms for improvement of beta-galactosidase production.

The strain of Penicillium notatum 1 most effective for producing beta-galactosidase (see lactase 3.2.1.23), was selected out of 110 moulds belonging to 15 different species, by the test-tube microculture method. The dynamics of beta-galactosidase synthesis was investigated in P. notatum 1 during its culture by submerged method.     

MYCOLOGICAL SYNTHESIS OF FAT FROM WHEY. II. COMPARATIVE STUDIES WITH SHAKEN AND STATIONARY CULTURES USING SELECTED MOULDS

SUMMARY: In detailed studies of the growth of Aspergillus ustus, Penicillium oxalicum, P. frequentans and P. notatum in whey with fat production in view, the first two showed the highest lactose utilization and felt weights in shaken cultures while the last two gave as good or better felt yields in stationary cultures.
When A. ustus was grown in shaken culture in whey with and without the addition of 1·14 g/1. of ammonium nitrate, the extra nitrogen led to the production of larger amounts of fat, but P. frequentans did not form additional fat in these circumstances. A. ustus was the best mould; in whey plus ammonium nitrate it utilized up to 96% of the lactose and formed, per litre of whey, about 17 g of mycelial felt containing 13% of protein and 28% of fat.     

MYCOLOGICAL SYNTHESIS OF FAT FROM WHEY. I. PRELIMINARY STUDIES WITH STATIONARY CULTURES

SUMMARY: Forty mould species, 30 of which were from the genera Aspergillus, Penicillium, Fusarium and Mucor , were examined for their ability to utilize the lactose in whey and to produce fat in the mycelium in stationary cultures.
In terms of lactose consumed and weight of mycelial felt produced the most promising species were A. ustus, P. frequentans, P. oxalicum and P. notatum. The first three of these warrant further study as fat producers.     

甘草药材上的污染真菌类群及其产毒素特性

对药材市场上霉变甘草样品的污染真菌进行分析,共得到4属7种真菌,包括Penicillium、Aspergillus、Fusarium、Mucor属,其中Penicillium polonicum、Aspergillus parasiticus以及P.crustosum是优势真菌。采用高效液相色谱-质谱联用技术对优势菌菌株产黄曲霉毒素及赭曲霉毒素A的特性进行检测。结果表明A.parasiticus主要产生黄曲霉毒素(AFG2、AFG1、AFB2、AFB1)和赭曲霉毒素A(OTA);而Penicillium polonicum主要产生赭曲霉毒素A(OTA)。     

The protective role of pigments against UV rays in fungi isolated from the mesosphere

Five among six species of microorganisms isolated from the mesosphere contained pigments which made them more resistant to the action of UV as compared to pigmentless microorganisms in the atmosphere of Earth. UV irradiation in the atmosphere is supposed to select resistant pigmented forms, so that they predominate in the mesosphere. To confirm this assumption, mutants of Aspergillus niger, Penicillium notatum and Circinella muscae were sported by irradiating them four times and then subjecting to stepwise selection. These mutants either synthesized pigments at a very low rate or did not produce them at all. No significant differences were found by studying the biomass, mycelium and sporeforming organs of the parent cultures and their mutants. However, their resistance to UV was not the same. Addition of the pigment apsergillin, isolated from the conidia of Aspergillus niger, to a suspension of the pigmentless (mutant) conidia of Penicillium notatum, the spores of Circinella muscae, and the vegetative cells of Micrococcus albus, before their irradiation with UV, considerably increased their resistance to this factor.     

Comparative study of cultures of four species of the genusOudemansiella

Thirty stationary and submerged cultures of species of the genusOudemansiella Speg.,viz. two narrow-region species (O. brunneomarginata, O. canarii) and two primarily wideregion species (O.mucida, O. radicata) were compared. Stationary cultures have identical macroscopic and microscopic features (they produce crust, teleomorph, large thick-walled basidiospores with an apiculum and an uneven surface, dikaryotic hyphae have clamp connections). “Coils” were detected in the mycelium of two species by SEM. Submerged cultures produce antibacterial and particularly antifungal antibiotics, although with various intensity.O. brunneomarginata was first studied in pure culture and its microstructures were first studied by SEM. It could be demonstrated that this species is related with other representatives of the genusOudemansiella. In the present taxonomy of basidiomycetes it is advisable to extend and deepen data concerning a single genusOudemansiella with a series of interspecies taxons, without impairing “borders” of the genus.     

Organophosphonate Utilization by the Wild-Type Strain of Penicillium notatum

We studied the biodegradation of compounds containing phosphorus-to-carbon bonds by using a wild-type strain of Penicillium notatum. The substrate specificity of this strain was studied, and we found that it is able to utilize structurally diverse organophosphonates as sole sources of phosphorus. This ability seems to be inducible, as indicated by the presence of a lag phase during growth. A popular herbicide, glyphosate, inhibited fungal growth, but it was also degraded by the fungus if it was applied in sublethal doses. This indicates that P. notatum may play an important role in biodegradation of organophosphonates. The strain which we used did not metabolize any of the phosphonates which we tested when they were used as sole carbon or nitrogen sources.     

Enhancement of anthocyanin production in callus cultures ofDaucus canota L. under the influence of fungal elicitors

Aqueous extracts of fungal biomass ofAspergillus niger, A. flavus, Penicillium notatum, Fusarium oxysporum and the filtrates of their culture media were analysed for elicitation capability to enhance anthocyanin production in callus cultures ofDaucus carota. The mycelial extract ofA. flavus at the 2.5% level gave maximum elicitation, which resulted in a two-fold increase in anthocyanin with maximal productivity of 23.7% on a dry weight basis. Whereas the media filtrates ofA. flavus induced a 1.25-fold increase in anthocyanin production with a yield of 20.6% on a dry weight basis,P. notatum andF. oxysporum were not as effective asA. flavus orA. niger. The contact time and the concentration required for maximal elicitation of anthocyanin differed with the type of elicitor used. Qualitative analysis of anthocyanins revealed the presence of cyanidin glycosides in control and elicited cultures.     

Sesquiterpene aryl ester natural products in North American Armillaria species

In the first comprehensive study on the secondary metabolism of all ten North American Armillaria species, we analyzed their capacity to produce sesquiterpene aryl ester natural products. All species were identified as producers. Species-specific trends were observed for some species, e.g., for Armillaria ostoyae and A. gemina (low diversity of aryl esters) or A. tabescens (very low number of chlorinated aryl esters). The crude extracts were assayed for antibiotic activity against wood-rotting basidiomycetes and ubiquitous soil fungi. Inhibitory effects were strongly dependent on the target organism, with basidiomycetes being more susceptible than other fungi. Tapinella panuoides and Omphalotus illudens were inhibited by extracts of all Armillaria species whereas the same extracts were completely inactive against Trichoderma harzianum, widely inactive against Mucor racemosus, and only moderately active against Aspergillus flavus and Penicillium oxalicum. Activity tests with seven purified aryl esters suggest that lipophilicity and the position of the double bond in the tricyclic sesquiterpene moiety impact upon bioactivity while the substitution pattern of the aromatic ring does not. Arnamial was the most active compound and showed a minimum inhibitory concentration of <5 μg/ml for all four basidiomycetes and Penicillium oxalicum, but did not inhibit Trichoderma harzianum and Mucor racemosus.     

Rhizosphere and rhizoplane fungi of Triticum vulgare cultivated in Saudi Arabia

131 species in addition to 7 varieties which belong to 40 genera were collected from the rhizosphere (36 genera and 120 species + 7 varieties) and rhizoplane (27 genera and 56 species + 2 varieties) of Triticum vulgare. More than 22 species and 3 varieties were not encountered previously from soils or other sources in Saudi Arabia.In the rhizosphere, Aspergillus and Penicillium contributed the broadest spectrum of species (26 species + 5 varieties; and 24 species, respectively). The most frequent fungi were Aspergillus niger, A. clavatus, A. flavus, A. terreus, A. carneus, Penicillium citrinum, P. notatum, P. chrysogenum and Fusarium solani. In the rhizoplane which is a more selective substratum for fungi than the rhizosphere, the picture of dominance became different and the most common fungi were Fusarium solani, F. oxysporum, F. moniliforme, Drechslera spicifera, Cephalosporium roseo-griseum, Stemphylium botryosum, Acremonium strictum and Rhizoctonia solani.     

Gene organization and plasticity of the β-lactam genes in different filamentous fungi

The genes pcbAB, pcbC and penDE encoding enzymes that catalyze the three steps of the penicillin biosynthesis have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are located in a cluster in Penicillium chrysogenum, Penicillium notatum, Aspergillus nidulans and Penicillium nalgiovense. The three genes are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of P. notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. The cluster of the penicillin biosynthetic genes is amplified in strains with high level of antibiotic production. About five to six copies of the cluster are present in the AS-P-78 strain and 11 to 14 copies in the E1 strain (an industrial isolate), whereas only one copy is present in the wild type (NRRL 1951) strain and in the low producer Wis 54-1255 strain. The amplified region in strains AS-P-78 and E1 is arranged in tandem repeats of 106.5 or 57.6-kb units, respectively. In Acremonium chrysogenum the genes involved in cephalosporin biosynthesis are separated in at least two clusters. The pcbAB and pcbC genes are linked in the so-called early cluster of genes involved in the cephalosporin biosynthesis. The late cluster, which includes the cefEF and cefG genes, is involved in the last steps of cephalosporin biosynthesis. The early cluster was located in chromosome VII (4.6 Mb) in the C10 strain and the late cluster in chromosome I (2.2 Mb). Both clusters are present in a single copy in the A. chrysogenum genome, in the wild-type and in the high cephalosporin-producing C10 strains.     

Selection of Trichoderma strains capable of increasing laccase production by Pleurotus ostreatus and Agaricus bisporus in dual cultures

Aims:  To select Trichoderma strains for enhanced laccase production in Pleurotus ostreatus or Agaricus bisporus cultures.
Methods and Results:  Laccase production by P. ostreatus and A. bisporus was evaluated in liquid (axenic) and solid (dual cultures) malt extract medium. Oxidation of ABTS, DMP and syringaldazine was evaluated in order to assess the potential of Trichoderma strains to enhance laccase production by basidiomycetes. Selected Pleurotus–Trichoderma interactions yielded higher increases in laccase volumetric activity and an additional laccase isoform was produced. By contrast, Agaricus–Trichoderma interactions lead to smaller increases on laccase volumetric activity, probably as result of repression (or degradation) towards one of the laccases isoforms.
Conclusions:  The strains of P. ostreatus and A. bisporus assessed in this work showed good potential as laccase producers. The Trichoderma -mediated biological stimulation of laccase production by P. ostreatus and A. bisporus is relevant in order to develop highly productive processes.
Significance and Impact of the Study:  Extracellular laccases from basidiomycetes are produced only in small amounts. It is therefore important to increase process productivity for potential industrial applications. The results from this study enable the selection Trichoderma strains capable of increasing laccase production by P. ostreatus or A. bisporus in dual cultures.