Autolysis of Thermoactinomyces vulgaris spores lacking carbon dioxide during germination

Abstract

Ultrathin sections of early germinating endospores of !Phermoactinomyces vulgaris were studied by electron microscope. Only spores aerated with a n air-COB mixture (5% C02) grow out, while spores aerated with air (0.03% CO,) lyse by the 25th min of inoculation. The lysis is due to progressive, unlimited degradation of the spore integuments and a lack of cell wall formation around the spore protoplast.The requirement of CO, for outgrowth could not be replaced by oxaloacetate. CO, seems to be needed to energize the dormant cytoplasmic membrane of the spore t o render i t capable of initiating active transport processes and of synthesizing the germ cell wall.Studying the germination behaviour of Thermoactinomyces vulgaris spores we found that aeration inhibited outgrowth. The initiation of germination, i.e. swelling and fall of optical density was not affected. The inhibitory effect of aeration was compensated by mixing 2-5% CO, to the flowing air during the first 15 min, or by using very dense spore suspensions. Measuring the respiratory activity of germinating spores we found that CO, was produced immediately after swelling. At first CO, was produced with low, but then with steadily increasing rate. This CO, accumulated in the unaerated cultures, but was eliminated in the aerated ones (KRETSCHMER and JACOB 1978).Since inhibition became irreversible already by the gth min after swelling, aeration is thought to damage the spores severely. I n order to elucidate the kind of damage and to discover the function of CO, in supporting outgrowth, the fine structure of spores germinated during aeration (0.03% CO,) was conipared to that of spores aerated with an air-CO, mixture (5% 0,).Spores of Thermoactinomyces vulgaris strain 4 were used. The procedures for harvesting spores from agar medium, for activation, storage and germination were described by STROHBACH and KRETSCHMER (1977). The spores were inoculated into liquid medium (salt-sucrose-casamino acids medium, 50 "C) a t a density of about 1.5. l o 7 . ml-I. One culture was aerated with prewarmed air (aerated spores) and the other with an air-5% CO, mixture (C0,-spores) by bubbling each with 20 1 h-1 gas per 7 0 ml medium. 0.02 M phosphate buffer was added to both cultures to maintain the p H a t about 7.0. After 25 min the spores were fixed with OsO, and embedded according t o KELLENBERGER et aZ. (1958). Ultrathin sections of the spores were studied with a n electronmicroscope Type SEM 3-2 (VEB Werk fur Fernsehelektronik, Berlin-Oberschoneweide) operating at 80 kV.Concerning fine structure as well as sporulation and germination behaviour the endospores of T . vulgaris are similar to those of bacilli (CROSS and ATTWELL 1975, KALAKOUTSKII and AGRE 1976, ENSIGN 1978. The spore integuments consist of an electron dense outer coat, a niultilayered inner coat and the inner pale cortex (Fig. 1). As is known from spores of bacilli and T. sacchari (FITZ-JAMES and YOUNG 1969, 28 S. KRETSCHMER and H.-E. JACOB c : W encloses the spore protoplast.d, e: The spore protoplast surroun...