APPLIED MICROBIOLOGY, Apr. 1973, p. 650-658 Copyright 0 1973 American Society for Microbiology Vol. 25, No. 4 Printed in U.S.A. Distribution of Steroid 1-Dehydrogenation and Side-Chain Degradation Enzymes in the Spores of Fusarium solani: Causes of Metabolic Lag and Carbohydrate Independence ROSAIRE PLOURDE AND HAMDALLAH HAFEZ-ZEDAN Faculty of Pharmacy, University of Montreal, Montreal, Quebec, Canada Received for publication 1 January 1973 The spores of a strain of Fusarium solani 1-dehydrogenate ring A and cleave the 17#-acetyl side chain of 17a-hydroxypregn-4-ene-3,20-dione (17a-hydroxy- progesterone) to give 17a-hydroxypregna-1,4-diene-3,20-dione (the 1-dehydro analogue) and little androsta-1, 4-diene-3, 17-dione (androstadienedione). A 4-h lag period is observed in the course of metabolism, and there are no requirements for external additives. Exoenzymes or surface enzymes bound to the cell outside the plasma membrane, either in the periplasmic space or bound to the cell wall, cannot be detected. The spore activity is not destroyed by treatment with aqueous HCl (pH 1.50), indicating that the 1-dehydrogenation and side-chain degradation enzymes are located away from the surface of the spores. Phenethyl alcohol destroys the spore permeability barriers, and it is also likely that it exposes its enzymes to acid inactivation. The action of phenethyl alcohol is reversible at low concentrations and irreversible at high concentrations. This investigation shows that: (i) the spore 1-dehydrogenating and side-chain-degrad- ing enzymes appear to be bound to, or imbedded in, the plasma membrane; (ii) the lag period observed in the course of metabolism of the steroid by the spores might be required for enzyme activation or diffusion of the substrate through the cell wall; and (iii) the internal metabolites of the spores, that might be required for the conversion process, appear to be present in a nondiffusible form or bound to intrasporal macromolecules. The chemical activities of fungal spores were first discovered in 1958 by Gehrig and Knight (3). In recent years a number of investigations (R. Plourde, H. Hafez-Zedan, and J. P. Lem- oine, Abst. 39 Congr. Assoc. Can. Franc. Avanc. Sci., Sherbrooke, Quebec, p. 95, 1971; refer- ences 1, 6, 17, 20, 24, 25, 28, 29) established that nongerminating spores of fungi and ac- tinomycetes can accomplish a wide range of conversions of steroid molecules. The spores of certain microorganisms showed more selective enzymatic activities as compared to corre- sponding growing cultures. For instance, al- though spores and mycelium of Aspergillus ochraceus (28) are both highly active steroid lla-hydroxylators, the spores exhibit very little 6ft-hydroxylation activity as compared to the mycelium. In a previous communication (17), we reported that the growing mycelium of a strain of Fusarium solani 1-dehydrogenated ring A and degraded the side chain of 17a-hydroxy- progesterone; the spores of the same culture converted the same steroid into its 1-dehydro analogue (Fig. 1) without side-chain cleavage. Alternative explanations have been given for the cleavage of the side chain by the mycelium and not by the spores. (i) The spores may contain only 1-dehydrogenase and lack the side-chain-degrading enzymes. (ii) The main reaction in both mycelium and spore processes may be 1-dehydrogenation; further incubation may induce de novo protein synthesis of the side-chain-cleaving enzymes in the mycelium. This induction could not take place in spores due to absence of adequate nitrogen supple- ment. (iii) Both mycelium and spores contain both 1-dehydrogenase and side-chain-degrading enzymes. In spore-mediated reactions, the side- chain-degrading enzymes may be inactive due to a lack of certain cofactors which may be 650 on December 16, 2014 by guest http://aem.asm.org/ Downloaded from