A new experimental approach to the study of the metabolism of steroids was launched in 1952, when the first microbial enzymes capable of carrying out highly specific and quantitative conversions of these substances were isolated in this laboratory.

Progressive subculture of a sample of California soil on a simple medium containing testosterone as the only source of organic carbon resulted in the isolation of a pure culture of an aerobic and highly motile bacterium (Pseudomonas testosteroni) (172). This organism grew rapidly on a medium containing inorganic nitrogen and other minerals supplemented with testosterone as the only organic matter. Colonies growing on Petri plates, in which testosterone was finely dispersed in the agar, became surrounded by clear halos, where the relatively insoluble steroid was utilized (Fig. 18).

Washed cell suspensions of P. testosteroni grown on steroid-containing media oxidized testosterone, progesterone, and related C19 and C21 steroids vigorously and without delay. In manometric experiments, rates of oxygen consumption up to 300 microliters per mg. bacterial dry weight per hour at 300 were observed. The total quantity of oxygen consumed in excess of the endogenous respiration was proportional to the amount of steroid added to the reaction vessel and amounted to between 40 and 60 per cent of that required for the complete oxidation of the steroid to carbon dioxide and water (172). It was concluded that under these conditions approximately one-half the steroid carbon skeleton was reutilized by the bacterium for synthetic processes. Addition of the uncoupling agent, 2,4-dinitrophenol, resulted in an increase in the total consumption of oxygen, approaching the theoretical limit for complete oxidation of the steroid.

Washed cell suspensions of this micro-organism grown in the absence of steroids did not immediately oxidize testosterone but only acquired this capacity upon prolonged incubation in the presence of the added hormone. Consequendy, the enzymatic machinery responsible for the oxidation of steroids is at least in part inducible (adaptive). Subsequent experiments have established that many of the enzymes concerned with steroid metabolism in P. testosteroni and in other micro-organisms are inducible (166).