Both ATP deaminase from Microsporum audouini and acid ADP-deaminating enzyme from Aspergillus melleus were found to catalyze the dechlorination of 6-chloropurine ribotide more rapidly than that of 6-chloropurine riboside. Specific AMP deaminase from rabbit muscle was also confirmed to hydrolyze 6-chloropurine ribotide to IMP and chloride ion. The Km values of ATP deaminase and acid ADP-deaminating enzyme for 6-chloropurine ribotide were 1.2×10-3M and 1.1×10-3M, respectively, while the Vmax of these two deaminases for dechlorinating activity was about 1/4-1/3 of that for deamination of 5′-AMP. The deamination of ATP deaminase and acid ADP-deaminating enzyme were competitively inhibited by 6-chloropurine ribotide, dechlorination of which, in turn, was competitively inhibited by pyrophosphate and it was suggested that both the deamination and the dechlorination occurred at the same active center on deaminase. The fact that the dechlorinating activity of ATP deaminase for 6-chloropurine ribotide was greater than that for 6-chloropurine riboside and pyrophosphate inhibition of the dechlorination reaction brought about a more evaluation of the schematic model of ATP deaminase previously proposed by the authors.
Following the previous study on the dynamical behavior of a chemostatic culture of Azotobacter vinelandii, the microbial response to a delta type of pulse effected in a medium via glucose was studied experimentally in this account. The glucose concentration in the medium and concentrations of cellular material and its constituents-DNA, RNA, protein, polysaccharide, and so forth-were fully analysed in unbalanced growth which immediately followed the delta pulse imposed. The fraction of RNA in the cells was confirmed to increase sigmoidally, while the protein fraction decreased exponentially as an approximation, other cellular components remaining approximately unchanged in the unbalanced growth. A plausible definition of unbalanced end balanced growth of microbial cells was made possible thus far. Employing the RNA fraction as parameter, a set of equations was proposed to represent the dynamical behavior of the bacterium.
1. Cell wall-cell membrane fractions were prepared from the cells of Brevibacterium flavum No. 2247 (ATCC No. 14067) and their fatty acid compositions were examined. Cultures from which cells were collected were as follows: Biotin-deficient culture of glucose medium, biotin-excess culture of glucose medium, biotin-excess and penicillin-added culture of glucose medium, biotin-excess and stearic acid ester (Tween 60)-added culture of glucose medium, biotin-deficient culture of acetate medium, and biotin-excess culture of acetate medium. 2. Myristic, palmitic, stearic, octadecenoic, and octadecadienoic acids were detected in all the samples. In addition to these acids, cell wall-cell membrane fraction of acetate-grown cells contained unidentified fatty acid. The content of these fatty acids decreased in the order of palmitic acid, octadecenoic acid, stearic acid, octadecadienoic acid, and myristic acid. This order was observed without exception in every sample. Content of each of these fatty acids in cell wall-cell membrane fractions of the cells possessing an ability to produce glutamate was generally low in comparison with that of the respective fatty acids in cell wall-cell membrane fraction of cells lacking the ability to produce glutamate.
Mycelial mats of Aspergillus niger 1617 grown on cellophane membrane placed on agar media were examined for activities of enzymes related to phosphorus metabolism during the course of cultural development. Most enzymes tested, phosphomonoesterase, pyrophosphatase, ribonuclease, and deoxyribonuclease, showed two pH optima at acid and neutral to slightly alkaline regions except phosphodiesterase, which had an optimum at pH 5.8. In general, enzymes showing higher pH optima were active in younger mycelia and those of lower pH optima were active in aged ones. Ribonucleases of T1-type (optimum pH 6.0) together with T2-type (optimum pH 4.5) were found in younger mycelia, while that of T2-type was most active in aged mycelia. The latter enzyme was considered to be responsible for the ribosome degradation.
The effect of nystatin on the acid protease formation by Aspergillus niger as induced by sulfur deficiency was studied. Nystatin at the concentration of 10μg/ml was found to inhibit the growth and the induced protease formation. The leakage of free amino acids from mycelia was also noted at this concentration. When nystatin was added to S-deficient cultures at different intervals in the induction period, a complete inhibition of protease formation was observed when added within the first 2.5hr. Later additions caused less effective inhibition. After the protease induction in sulfur-deficient media, mycelia were either treated or not treated with nystatin and they were further cultured for protease formation on S-deficient media containing 14C-amino acid mixture. It was found that the nystatin-treated mycelium produced protease of higher specific radioactivity than the non-treated mycelium. This suggests that a fungal mycelium which has already established the protease-forming system can produce protease at the expense of exogenous nutrients, irrespective of the loss of the pool amino acid from the mycelium.
The duration of nuclear cycle and activities of RNA and protein syntheses along the length of growing hyphae of Aspergillus oryzae were estimated by the techniques of autoradiography. The labeling of DNA was effected by incubating the hyphae in the presence of adenine-3H followed by the removal of the labeled RNA with ribonucleases I and T1. For the estimation of the duration of the nuclear cycle, two methods were presented. Estimations of activities of RNA and protein syntheses were carried out by the short term labeling with adenine-3H and L-leucine-3H, respectively. The statistic analysis of the data on the labeling of nuclear regions showed that, in short hyphae of several hundreds microns in length, the generation time of the end nuclei was 1.5-2 times longer than that of the head nuclei. Activities of RNA and protein synthesis, so far observed by autoradiography, were apparently less active in the apical region than in regions behind the apex. It is discussed that the latter observation may not necessarily gives the real picture, since the presence of the nucleotide and amino acid pool highly concentrated in the apical region possibly causes the dilution of radioactive precursors taken up by hyphae.
The rate of oxidation of several substrates by cell-free extracts increased during the aeration of anaerobically grown Escherichia coli. Succinate oxidation by extracts from aerated cells was not suppressed by the addition of extracts from anaerobic cells. Dialysis of extracts from aerated or aerobiccells resulted in marked decrease of the oxidative activity, but the activity was restored to the original level by the addition of boiled extracts from anaerobic cells. The oxidative activity of dialyzed extracts from anaerobic cells showed little improvement by the addition of boiled extracts from aerated or aerobic cells. Uptake of fumarate by aerated cells occurred even when the oxidation of the substrate was completely inhibited by cyclohexanol, provided that glucose was present in the reaction mixture. No such uptake occurred in anaerobic cells or in aerobic cells grown in glucose-containing media. From these results, it was inferred that the development of respiration by aeration of anaerobic cells was caused by the increase of permeability to substrates and by the formation of high molecular substances (possibly enzyme proteins), but not by the increase of coenzyme content or by the disappearance of respiratory inhibitors.
The minimum inhibitory concentrations (MIC) of chloramphenicol, tetracycline and penicillin for cholera vibrios were found to be 1.5μg/ml, 2.5μg/ml and 30μg/ml, respectively. No strains were found completely or partially resistant to either chloramphenicol or tetracycline at their MIC but a number of strains were partially resistant to these antibiotics at lower concentrations. The incidence of partially resistant strains to tetracycline was higher and it was found increasing year to year. There was no significant difference between V. eltor and V. cholerae in their sensitivity to other two of these antibiotics. The action of chloramphenicol, tetracycline and penicillin was studied in growth kinetic experiments using normal vibrio cells as well as protoplasts. All the three antibiotics caused a sharp fall in viable count of V. cholerae as well as V. eltor during the first 4hr of incubation. Penicillin-treated vibrios of both types then resumed growth and in 24hr multiplied to the same extent as control cultures. V. eltor cells treated with chloramphenicol or tetracycline resumed very slow growth after 4hr and this continued up to 24hr whereas the vibriocidal action of the two antibiotics on V. cholerae cells continued to be exerted for up to 24hr. Similar concentrations of the three antibiotics totally inhibited the growth of protoplasts of both V. eltor and V. cholerae. Unlike in the case of normal cells, the inhibition of growth of protoplasts by penicillin was found to be irreversible. These findings have been discussed in relation to likely sites of action of the antibiotics on the vibrio cells.