The principle underlying the sensitivity to antibacterial agents of wild type Escherichia coli P and its γ-irradiation resistant mutants was studied. These strains were classified as typical rough (R) strains, belonging to classes Ra (P and γ), and Rc (6γ and 12γ). The galactose-deficient (Rc) strains, 6γ and 12γ, leaked large amounts of alkaline phosphatase, acid phosphatase, 2, 3-cyclic phosphodiesterase, and β-galactosidase into the culture media during active growth, indicating defects in both the outer and inner membranes. Unlike strains P and γ, strains 6γ and 12γ were hypersensitive to Actinomycin-D, Bacitracin, Erythromycin, Kanamycin, Novobiocin, penicillin G, Rifamycin, Vancomycin, sodium deoxycholate (DOC), sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), lysozyme, and Methylene Blue. Strains P, 6γ, and 12γ showed similar sensitivity to phages T2, T3, T4, T5, T6, and T7, and differed from strain γ, which was sensitive only to phages T2 and T4. Strains P and 6γ were sensitive to colicins E1, E2, E3, G, H, and K, whereas strain γ was resistant to colicin H, and 12γ was tolerant to colicins H and K. On the basis of these observations it is suggested that the leakage of periplasmic enzymes from and the sensitivity of these mutant strains to antibacterial agents are associated with both defects in the cell wall and plasma membrane.
Developmental process of the carpophore of Polyporellus brumalis (Pers. ex Fr.) Karst. was histochemically observed. Locality of RNA, succinic dehydrogenase, cytochrome oxidase, esterase, peroxidase, and phosphatases was investigated. Acid phosphatase was stained intensively throughout the development of the carpophore but alkaline phosphatase was not detected. Acid phosphatase, esterase, peroxidase, cytochrome oxidase, and succinic dehydrogenase were detected in growth zones of this fungal carpophore, such as the apex of stipe, margin of pileus, and hymenial layers.
Preparation of cell-free progesterone hydroxylases from Aspergillus niger 12Y was achieved by extraction of the mycelium with acetate, citrate-phosphate, and phosphate buffer solutions at different pH values. All buffer solutions afforded cell-free preparations containing 11α-hydroxylase, 11β-hydroxylase, and 21-hydroxylase. The results indicated that 11α-hydroxylase and 21-hydroxylase comprised the major and minor components of the enzyme preparations, respectively. The extractability and/or the activity of the hydroxylases decreased and increased with the increase of the pH of acetate and citrate-phosphate buffers used in the mycelium extraction, respectively. Citrate-phosphate buffer provided enzyme preparations which were more active than those of acetate buffer. The latter buffer had a harmful effect on both the isolated hydroxylases and those remaining in the mycelium debris. On the other hand, grinding the mycelium with phosphate buffer at pH 6.24 provided the most active enzyme preparation. Homogenization of the mycelium with the last-mentioned buffer solution had an adverse effect on the activity of the resulting cell-free preparation. Addition of EDTA to the phosphate buffer of pH 6.24 resulted in enzyme preparations possessing weak 11α-hydroxylase and 21-hydroxylase activities and almost no 11β-hydroxylase activity.
Ammonium sulphate was unsuitable for salting out active fractions of progesterone hydroxylases from the cell-free preparation of Aspergillusniger 12Y while ethanol provided precipitates possessing weak activities. Acetone afforded precipitates possessing moderate activities and the precipitate obtained by treatment with 6 volumes of acetone showed only 11β-hydroxylase activity. Centrifugation of a buffered cell-free preparation at different velocities provided fractions rich in 11α-hydroxylase and others rich in 11β-hydroxylase. The supernatant fluid remaining after centrifugation at 18, 000rpm and the sediment obtained at 7, 000rpm showed maximal activities of 11α-hydroxylase and 11β-hydroxylase, respectively. Paper electrophoresis of the cell-free preparation of progesterone hydroxylases was studied using 26 different buffer solutions. Successful separation of 11α-hydroxylase and 11β-hydroxylase was achieved with acetate buffer of pH 3.42 (0.02M and 0.2M) and pH 4.05 (0.02M), as well as with phosphate buffer of pH 5.29 (0.0006M) and pH 8.0 (0.0001M). Acetate buffer of low pH had an inhibitory effect on the electrophoresed 11β-hydroxylase. However, both 11α-hydroxylase and 11β-hydroxylase became inactive when electrophoresed with acetate buffer of pH 5.89. In no case 21-hydroxylase appeared on the electropherograms, probably due to its presence as a minor component of the enzyme sample.
The intracellular localization of acid and alkaline phosphatases in Aspergillus oryzae A 1-5 was studied cytochemically with light and electron microscopy. Two different acid phosphatases were present, one (acid phosphatase I) having a pH optimum of 4.0 and the other (acid phosphatase II) of 5.5. They were shown to be localized at different sites in the fungal mycelium under this experimental condition; acid phosphatase I was demonstrated to be present at the surface of cell wall, whereas acid phosphatase II both at the surface of cell wall and at the cytoplasmic membrane. It was demonstrated cytochemically that excess amount of inorganic phosphate in growth medium promoted the formation of acid phosphatase I but repressed that of acid phosphatase II. Alkaline phosphatase was found to be localized in nucleoli and unknown electron-dense bodies in the cytoplasm. It was also seen at the surface of cell wall and at the cytoplasmic membrane.
Phospholipid compositions were radiochemically studied on nocardioform and coryneform bacteria. Phosphatidylethanolamine (PE) was present in the strains of the genera Corynebacterium, Nocardia, and Mycobacterium. Cardiolipin and phosphatidylglycerol were found in all the genera tested. Sugar-containing phospholipids such as phosphatidyl-inositolmannosides and phosphatidylinositol were present in about half of the genera tested. The genera Corynebacterium was divided into two sub-groups by the phospholipid composition. Corynebacterium strains having a high GC-content exhibited the presence of a large amount of PE and those having a low GC-content a trace amount of PE. Nocardia and Mycobacterium were different from Actinomadura and Oerskovia in the point of phospholipid profile. The phospholipid composition of Nocardia and Mycobacterium was similar to that of the high GC-content group of Corynebacterium. From these findings, the phospholipid composition is considered to be useful for differentiation of coryneform and nocardioform bacteria.