Respiration of the Mycelia and Mitochondria of the Filamentous Watermold, Allomyces Macrogynus

Abstract

The immediate purpose of this investigation of respiration in the watermold Allomyces macrogynus was to provide a basis for the ultimate explanation of the effect of various nutritional conditions on the utilization of mannose and fructose by this mold (28,29,37). Its primary significance, however, is the isolation from one filamentous fungus of mitochondria which oxidize the various intermediates of the Krebs cycle. Only mitochondria from y-east, among the fungi, have been shown to contain an organized system associated with mitochondria for carrying out these reactions (24, 32). The reader is referred to Bonner (4) for a comprehensive review of pathways of carbohydrate metabolism in the fungi and the related actinomycetes.MATERIALS AN-D METHODS GROWTH AND PREPARATION OF THE FUNGUS: The organism used was the Burma 1 Da strain of Allomyces macrogynus (10). It was grown in liquid, agitated culture in 50 ml of medium in 125-ml Erlenmeyer flasks as described by AMachlis (27). The complete medium, as used in this study, is the same as the minimal medium of Machlis (27) except for the addition of 10i4 M L-glutamic acid. Low-sugar medium differs only in that the concentration of glucose is 0.05 % instead of 0.5 %. Each flask of medium was inoculated with 0.5 or 1.0 ml of mitospores and then incubated in the dark (except when room lights were turned on for observations) on a rotary shaker at 250 C for 48 to 96 hours. The mitospore inoculum was prepared as previously described (26).For the manometric determinations, the flasks of spherical plants were emptied over a wire screen or nylon cloth, the occasional large plaints removed, and the remaining retained plants rinsed with three or four washes of 0.04 M potassium phosphate buffer at pH 7.0. The rinsed plants from the requisite number of flasks were then composited, susl)ended in the solution to be used in the vessels and transferred to the Warburg vessels with a calibrated pipette having an opening approximately 5 mm in diameter.PREPARATION OF 'MITOCHONIDRIA: Initial trials to obtain mitochondria were unsuccessful using homogenizers of the Ten-Broeck and Potter-Elvehjem type, carborundum of several mesh sizes, and washed sand.At the suggestion of Dr. V. WV. Cochrane, ground Pyrex glass was tried and mesh sizes 20 through 50 were found to work well. The screened glass was washed with acid followed by distilled water until all acid was removed. It was then oven-dried. For a typical experiment 40 flasks of plants grown in complete medium for 80 to 96 hours were used. They were removed from the growth medium and washed on a nylon cloth over a Biuchner funnel with approximately 500 ml of cold 0.04 MI potassium phosphate buffer (pH 7.0 or 7.4). Excess buffer was pressed from the plants which were then ground in a cold room with 50 to 60 gm of ground glass and a final total of 150 to 180 ml of a solution containing 0.5 M sucrose, 0.1 M potassium phosphate, and 0.001 M potassium ethylenediamine tetraacetate at a pH o 7.0 or 7.4. Both pH 7.0 and 7.4 gave identical results.The...

Fine Structure of Fungi as Revealed by Electron Microscopy

“…Early electron micrographs of thin sections of vegetative cells of Saccharomyces cerevisiae (Bartholomew & Mittwer, 1952; Bartholomew & Levin, 1955; Agar & Douglas, 1955a, b, 1957 were of generally poor definition, but nevertheless yielded information which has been confirmed and extended by later workers (Hashimoto, Conti & Naylor, 1959; Yotsuyanagi, 1959Yotsuyanagi, , 1960; Mundkur, 1960, 1 9 6 1 4 b; Vitols, North & Linnane, 1961a; Koehler, 1962; Koehler, Birnbaum & Hayes, 1961; Marquardt, 1962b).…”

Influence of Growth Conditions, Metabolic Inhibitors, and Phenolic Compounds on the ATP Pool in Fomes annosus

“…Vitols and Linnane 1961). Besides yeasts, very few examples of successful P/O estimations with fungi have been reported (Bonner andMachlis 1957, Grabbe et al 1968).…”

The Breakdown of Carbohydrate by Schizophyllum Commune Fr.

“…COCHRANE, 1958), but relatively little work has been done on the isolation of functionally active subcellular particles from fungi. (BONNER and MACHLIS, 1957). In this report some observations on subcellular particles from Schizophyllum commune are given.…”