Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optinum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.Large amounts of the disaccharide trehalose are present in the spores and cysts of a variety of organisms, including actinomycetes, fungi, nematodes, and brine shrimp (2, 4, 13). In these organisms trehalose is degraded very slowly during periods of dormancy but is rapidly metabolized following the onset of vegetative growth (1,9,14,20,22).Spores of Streptomyces griseus contain large amounts of trehalose (13). Extracts of the spores also contain a high specific activity of the enzyme trehalase (14). Nongerminating spores metabolize their endogenous trehalose reserves very slowly. Upon transfer to conditions allowing spore germination, trehalose is rapidly metabolized, while the level of trehalase activity remains constant (14).In this paper we report the results of an investigation of the properties of trehalase and the localization of trehalase activity and trehalose in spores of S. griseus. The goal of this study was to determine the basis for the apparent coexistence of trehalase and trehalose in dormant spores and the rapid utilization of trehalose following the initiation of germination.MATERIALS AND METHODS Growth conditions. S. griseus NRRL B-2682 was maintained as described previously (13). Spores were harvested from solid DMC medium after 7 days of incubation at 30°C unless indicated otherwise. DMC medium was modified to obtain lysozyme-sensitive spores by adding glycine to a final concentration of 0.35% and by adjusting the pH to 6.6 prior to sterilization. Spores were germinated in the complex germination medium described previously (13). Mycelia were grown in liquid DMC medium containing 0.15 M KCl. Protoplast regeneration medium consisted of 25 mM morpholinopropanesulfonic acid (MOPS) (pH 7.0), 5 mM (NH4)...