Acid ribonuclease (RNase) was extracted from mycelia of Aspergillusniger 1617 and separated into two fractions designated acid RNases I (EC 220.127.116.11) and II (EC 18.104.22.168). Each fraction was partially purified and its enzymatic properties were examined. The molecular weight of acid RNase I was approximately 45, 000, and that of II was 13, 000. The optimal pH of acid RNase I was 3.5 and that of II, 4.5. Acid RNase I was inhibited markedly by Fe3+, Cu2+, Hg2+ and 5′-ATP, whereas II was inhibited markedly by Fe3+. Acid RNase I had no base specificity producing 3′-monophosphates of the four nucleosides and guanosine-2′, 3′-cyclic monophosphate. Acid RNase II was a guanine-specific RNase. Changes in the activity of acid RNases I and II were studied during culture on agar medium. The activity of acid RNases I and II increased and reached their maxima during the late phase of active growth. After their maxima, the activity of acid RNase I remained unchanged, while that of II decreased and finally disappeared.
A superoxide dismutase (EC 22.214.171.124) was purified 113-fold with a yield of 38% from the crude extract of Rhodococcus bronchialis (Gordonabronchialis). The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis, analytical ultracentrifugation and immunological procedures. The molecular weights of the enzyme and its subunit were estimated to be approximately 80, 000 and 21, 000, by sedimentation equilibrium analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The amino acid composition was similar to that of the enzyme from Mycobacterium smegmatis. Metal analysis showed that the enzyme contained approximately 2.2 and 0.9 atoms of manganese and iron per mol, respectively. Correlation of enzymic activity, and manganese and iron contents in fractions of gel filtration on a Sephadex G-100 column indicated that both the metals were tightly bound to the enzyme.
Pure culture isolation of aerobic chemoheterotrophic spirilla from mud and sand samples collected from various freshwater and seawater areas in Japan was accomplished by applying the following three methods in the sequence shown: (i) boiled shellfish infusion method for enrichment of spirilla; (ii) glass capillary method for selection of spirilla; and (iii) ordinary streak plate method for pure culture isolation of spirilla. The aerobic spirilla grew well in all enrichment cultures in the 20 experiments performed. Crude cultures containing abundant spirilla were obtained from all enrichment cultures by use of glass capillaries. Pure culture isolation of spirilla from the crude cultures was reliably achieved by the ordinary streak plate method.
The stainability of ascospores and vegetative cells of Saccharomycescerevisiae to acid-fast staining, using hot Ziehl's carbolic fuchsin solution, 5% sulfuric acid, and diluted Löffler's methylene blue, was examined. Resting spores and growing haploid cells (a type strain 24428 and α type 3626) retained much fuchsin dye in the cells. Only mature spores of diploid G2-2 resisted methylene blue staining. The stainability of Mycobacterium phlei IFO 3158 also examined. The kinetics of germinationn were examined. The loss of the stainability with acid-fuchsin and of the resistance to methylene blue was used as a criterion of germination. The ascospores germinated anaerobically as well as aerobically. Especially in the early stage of germination, there was found no difference in the germination rates under both conditions. Ultrastructure of germinating ascospores cultured in aerobic and anaerobic conditions was examined by ultrathin sectioning and electron microscopy. At the first stage of germination, the ascospores swelled in aerobic as well as anaerobic cultures. The outer spore coat and the outer zone of inner spore wall disappeared during the germination process, the inner zone of the spore wall then giving rise to a germinated spore cell wall (=extruded germ tube wall). The vacuole became granular. The mitochondria showed no change in shape and number in aerobic cultures, but seemed to swell and disintegrate in the later stages of anaerobic germination.
The envelope system as seen in the thin section of the cell of Lactobacilluscasei ATCC 27092 was of the multi-layered type. The transverse section of the isolated cell walls had a homogeneous appearance with a width of about 20nm. The cell wall preparations were considered to correspond to the surface layer in the thin section of whole cells. Electron microscopic examination of the mixtures of PL-1 phages and cell walls after fixation with osmic acid revealed that all the phages were adsorbed to cell walls in a tail-first orientation and that the adsorbed phages were intact. When the adsorption mixtures were not fixed with osmic acid, the adsorbed phages were eluted as infective virions by centrifuging and resuspending in fresh medium. In a control where the phages were mixed with the cell walls isolated from a phage-resistant strain named K-12, the phages showed no tendency to bind to the cell walls. Thin section of osmic acid-treated mixtures of phages and cell walls confirmed that the phages bound by their tail tip to the one side of cell wall preparations.