Heinz-Georg Wetzstein

Molecular chemotaxonomy of Daldinia and other Xylariaceae

In a polyphasic classification approach, stromata and cultures of Daldinia and allied Xylariaceae from around the world were studied for: (1) morphology of teleomorphs and anamorphs; (2) metabolite patterns in stromata and cultures, employing analytical HPLC-UV-visible (diode array) detection and mass spectrometric detection; (3) amplified 18S rDNA restriction patterns (ARDRA); and (4) PCR amplified minisatellite regions. Comparison of type material, herbarium specimens, cultures, and freshly collected material revealed new evidence on their geographic distribution. Several Daldinia spp. were identified in Europe and other locations for the first time. The results point towards the existence of further undescribed species. Stromata of a given species never contained the same major metabolites as corresponding cultures. Most cultures of Daldinia spp. produced naphthalene and chromane derivatives, differing from allied genera by the absence of mellein. Stromata of Daldinia spp. did not produce mitorubrin, but generally contained binaphthyls. Metabolite profiles were correlated with colours of KOH-extractable stromatal pigments. The yellow azaphilones and benzophenones found in D. childiae were lacking in species with purple stromatal pigments. Cytochalasins were found in stromata of D. eschscholzii. Genetic fingerprints helped to distinguish morphologically closely related taxa. ARDRA gave specific results for species whose 18S rDNA contained insertions, while minisatellite PCR provided specific genetic fingerprints. A combination of both PCR based techniques provided a fair resolution of genetic subtypes, reflecting the intrageneric variance in Daldinia as established from morphological data and secondary metabolite profiles.

Hydroxylated Metabolites of 2,4-Dichlorophenol Imply a Fenton-Type Reaction in Gloeophyllum striatum

ABSTRACT While degrading 2,4-dichlorophenol, two strains ofGloeophyllum striatum, a basidiomycetous fungus causing brown rot decay of wood, simultaneously produced 4-chlorocatechol and 3,5-dichlorocatechol. These metabolites were identified by comparing high-performance liquid chromatography retention times and mass spectral data with those of chemically synthesized standards. Under similar conditions, 3-hydroxyphthalic hydrazide was generated from phthalic hydrazide, a reaction assumed to indicate hydroxyl radical formation. Accordingly, during chemical degradation of 2,4-dichlorophenol by Fentons reagent, identical metabolites were formed. Both activities, the conversion of 2,4-[U-14C]dichlorophenol into14CO2 and the generation of 3-hydroxyphthalic hydrazide, were strongly inhibited by the hydroxyl radical scavenger mannitol and in the absence of iron. These results provide new evidence in favor of a Fenton-type degradation mechanism operative inGloeophyllum.

Metabolite proving fungal cleavage of the aromatic core part of a fluoroquinolone antibiotic

Liquid cultures of the basidiomycetous fungus Gloeophyllum striatum were employed to study the biodegradation of pradofloxacin, a new veterinary fluoroquinolone antibiotic carrying a CN group at position C-8. After 16 days of incubation, metabolites were purified by micro-preparative high-performance liquid chromatography. Four metabolites could be identified by co-chromatography with chemically synthesized standards. The chemical structures of three compounds were resolved by 1H-nuclear magnetic resonance spectroscopy plus infrared spectroscopy in one case. All metabolites were confirmed by high resolution mass spectrometry-derived molecular formulae. They comprised compounds in which the carboxyl group or the fluorine atom had been exchanged for a hydroxyl group. Furthermore, replacement of the CN group and the intact amine moiety by a hydroxyl group as well as degradation of the amine substituent were observed. The chemical structure of a catechol-type fluoroquinolone metabolite (F-5) could be fully defined for the first time. The latter initiated a hypothetical degradation sequence providing a unique metabolite, F-13, which consisted of the cyclopropyl-substituted pyridone ring still carrying C-7 and C-8 of pradofloxacin, now linked by a double bond and substituted by a hydroxyl and the CN group, respectively. Most likely, all reactions were hydroxyl radical-driven. Metabolite F-13 proves fungal cleavage of the aromatic fluoroquinolone core for the first time. Hence, two decades after the emergence of the notion of the non-biodegradability of fluoroquinolones, fungal degradation of all key structural elements has been proven.

Biologische Abbaubarkeit der Gyrasehemmer: Chinolone in der Umwelt

FQs wie Enrofloxacin sind biologisch abbaubar. Entsprechende Potenziale finden sich bei Holz-, Dung- und Erdboden bewohnenden Pilzen, Hefen und einigen Bakterienarten, die vermutlich uber sehr unterschiedliche Abbaumechanismen verfugen. Bei Gloeophyllum striatum, einem Braunfaule verursachenden Basidiomyceten, liegt wahrscheinlich ein Hydroxylradikal-abhangiger Mechanismus vom Fenton-Typ vor. Die Metaboliten von Enrofloxacin haben entweder eine stark verringerte oder keine antibakterielle Aktivitat; im Gegensatz zum Wirkstoff sind einige chemisch labil. Die Eliminierung des einzigen naturfremden Strukturelementes, eines aromatisch gebundenen Fluors, erfolgte mit hoher Aktivitat. Trotz sehr niedriger 14CO2-Freisetzungsraten fur Markierungspositionen innerhalb des heterocyclischen Molekulkerns — bei wesentlich hoheren Raten fur den Aminteil von Enrofloxacin — durfte es in naturlichen Matrices wie Rinderdung und Ackerboden im Verlauf von Humifizierungsprozessen zu einer relativ schnellen Transformation und Inaktivierung von FQs kommen. Damit ist unwahrscheinlich, dass FQs in Mist und Ackerboden persistieren. Durch unspezifisches Binden an Dung und Ackerboden sowie Faeces und Restschlamme in Klaranlagen wird die Bioverfugbarkeit von FQs drastisch verringert, so dass selbst theoretische worst-case-Konzentrationen in Ackerboden weit unterhalb der Konzentrationen liegen, bei denen ein Selektionsdruck fur FQ-Resistenz auftreten konnte.