Crystalline structures of the fungus Mucor circinelloides were investigated substructurally and cytochemically by means of electron microscopy. The crystals, preferably localized within vacuoles and vesicles, are abundant in hyphae grown on n-alkanes or 1-decanol. Stripes and spot patterns with different dispositions are evident in the planes of sectioned crystals. Membraneous structures are in close spatial relation to crystalline inclusions.The crystals reveal NAD(P)H oxidase activity, as demonstrated by the cerium precipitation technique. The amount of reaction product as well as the quantity of reactive inclusions both depend on the carbon source of the nutrient medium. It was demonstrated that the formation of reaction product is insensitive to 1 mM KCN; it depends rather on both NADH and NADPH, with NADPH being the most efficient substrate. pchloromercuribenzoate in the incubation medium results in a drastic reduction in the amount of deposited precipitate ; this indicates that the observed cerium precipitation is attributed to enzyme activity. Catalase activity was fou&i to be restricted to microbodies.The accumulation of the reactive, crystalline condensed material on hydrocarbon-grown hyphae might account for a changed cellular metabolism.Hydrocarbon-grown microorganisms are characterized by different ultrastructural modifications. In mycelial fungi these are : intracellular substrate inclusions, a substantial enlargement of the vacuolar area (CUNDELL et al. 1976), small, spherical, electron-dense inclusions within the vacuoles, thin cell walls in both hyphae and spores, and the conspicuous appearance of microbodies (SMUCKER and COONEY 198 I), containing well developed crystalline cores (TURNER et al. 1980). In hyphae of hydrocarbon-grown Cladosporiurn resinae the occurrence of microbodies is accompanied by an. increased synthesis of catalase (SMUCKER and COONEY 1981).In the present study crystalline inclusions within the endomembrane system of filamentous hyphae of Mucor circinelloides, grown on various carbon sources, are described in detail. In view of the well known characteristic of oxidases and catalase to form highly regular crystalline structures, comparable to those found in Mucor circinelloides, histochemical investigations of NAD(P)H oxidase and catalase activity were performed.
Materials and methodsOrganism and culture conditions: The fungus Mucor circinelloides was isolated from a soil sample and described by BEMMANN (1981) with regard to its ability to utilize n-alkane. For inoculation, pieces of mycelium were obtained from 5-day old culture slants on malt extract agar medium. Filamentous hyphae were grown in standing culture at 28 "C in 200 ml ERLENMEYER flasks, containing 50 ml of a liquid basal medium, described by UDDER and KREGER-VAN RIJ (1952). The basal medium was supplemented with different carbon compounds as the sole source of carbon and energy: 1) Parex-Parafin I1 (mixture of n-alkanes with chain lengths of C,, to C,,, VEB Leuna-Werke), 2) I-decanol (FERAK, Berlin), 3) malt
