A mutant strain No. 6, devoid of 5′-nucleotide-degrading ability, was derived from adenine auxotrophic strain No. 93 originating from Bacillussubtilis 209-4, and the accumulation of 5′-IMP under various conditions was examined. In No. 6, the early stage of growth was inhibited in the presence of more than 500μg/ml of adenine, whereas the duration of the maximum growth was maintained only shortly followed by a marked lysis in the presence of less than 180μg/ml of adenine. On the other hand, such phenomenon was not observed at all in the case of the adenineless mutant No. 93. Such a characteristic response to adenine in the mutant devoid of 5′-nucleotide-degrading ability seems to be related with activity of phosphatase (5′-nucleotidase or alkaline phosphatase) in cells. The mechanism of the accumulation of the purine and pyrimidine derivatives by No. 6 was also influenced by the adenine content in the medium. When 100μg/ml of adenine was added to the culture, nucleotides accumulated as the result of the degradation of cellular RNA. On the other hand, when more than 180μg/ml of the adenine was present, mostly inosine was accumulated in medium; this was considered to be synthesized de novo by viable cells.
Sixteen RD mutant strains of Saccharomyces carlsbergensis and Saccharomycescerevisiae, which were isolated by spontaneous mutation, acriflavine treatment, and ultraviolet irradiation, were studied for their ability to produce citramalic acid in a chemically defined medium. It was found that all those RD "petite" mutants which were clearly judged to be respiration-deficient in any of the four criteria, i.e. oxygen uptake, coloration of colonies on BCG agar, TTC reduction, and lactate utilization, did accumulate citramalate abundantly (13 to 46mg per 100ml of medium). None of the seven parent normal strains tested produced citramalic acid. Some variants isolated on BCG agar (verdant and variegated types), which were positive in reducing TTC, were not able to produce citramalic acid. RD mutant haploid strains of known cytochrome pattern (baker's yeast) were found to be divided into the citramalate formers and nonformers, but it was not possible to relate their citramalate-producing abilities to the kind of deficient cytochromes.
L-Proline production by an isoleucine auxotrophic mutant No. 14-5 (ATCC No. 15940), derived from glutamic acid-producing strain of Brevibacterium flavum 2247, was investigated. This strain produces a large amount of L-proline under vigorous aeration at 31° in a pH range from 7.0 to 7.5. Among various substances supplemented to the medium, the following substances are found to enhance L-proline production: (1) divalent cations, such as Fe++, Mn++, Zn++, and Mg++ (at rather high concentration); (2) organic acids related to the TCA cycle, and tartaric acid; (3) some organic nutrients, such as meat extract, "Ajieki" polypeptone, and yeast extract; (4) amino acids, such as L-histidine, L-tryptophan, L-tyrosine, L-phenylalanine, glycine, L-serine, L-cystine, L-cysteine, and L-ornithine. This microorganism is found to lack the threonine dehydratase activity, and a possible relation of the deficiency in threonine dehydratase activity in glutamic acid-producing bacteria to the abundant production of L-proline was discussed.
The genetic alteration in the mechanism of enzyme repression operating in the purine nucleotide synthesis of B. subtilis was investigated, using the mutants resistant to various concentrations of guanosine derived from strain G-6, a guanine-requiring mutant of GMP reductase-negative strain, TF-11. Strains 1-40 and 3-8, which were derived from G-6 as mutants resistant to 10μg/ml of guanosine, were derepressed for PRPP amidotransferase, IMP transformylase, and IMP dehydrogenase. Adenylosuccinate lyase and adenylosuccinate synthetase were still repressed in these strains. Strains 40-15 and 8-5, which were derived from 1-40 and 3-8, respectively, as mutants resistant to 100μg/ml of guanosine, could not be distinguished from their parent strains in the formation of the five enzymes tested. Strains 40-57, 4015-4, and 85-1, which were derived from 1-40, 40-15, and 8-5, respectively, as mutants resistant to 1, 000 μg/ml of guanosine, were derepressed for sAMP lyase. sAMP synthetase was still repressed. Strain GR-40, which was directly derived from TF-11 as a mutant resistant to 200μg/ml of guanosine, was derepressed for IMP transf ormylase and sAMP lyase. Based on these results, the genetic control of enzyme repression in purine nucleotide synthesis was discussed.
The present observation indicates that, besides the nitrogenous compounds, other biologically active cell constituents like vitamin B12 and auxins may also appreciably contribute to the fertilizing action of the nitrogen-fixing blue-green algae. Of the various intracellular amino acids of Cylindrospermum muscicola, cystine, tyrosine and phenylalanine seem to be available to the rice plants. The alga can synthesize vitamin B12 active compounds, the yield of which was about 1.2 to 1.5μg/g dry weight of the alga. The synthesis of these compounds was promoted by the addition of cobalt salt. The alga also contains interconvertible auxin-like substances which stimulate the root growth of rice seedlings.
DNA base composition and degree of homology relative to Streptomycesgriseus DNA of various morphologically distinct actinomycetes were studied in order to find out any genotypic difference that may exist between their DNA's, for a more rational approach to the taxonomy and phylogeny of the actinomycetes. The present results indicated that the average percentage of GC, analyzed by the paper chromatographic method, was in the range of 67.5-74.5 and, if Nocardia and its relatives be excepted, generally between 70 and 74; the difference was gradual and failed to delineate any organisms. However, organisms having the nocardial type of cell wall, i.e., Nocardia and Micropolyspora, occupied the lower region, followed by Streptosporangium and Microellobosporia. Furthermore, the DNA homology test by the DNA-agar method showed that, under the experimental conditions used, no conspicuous difference applicable to the classification was recognizable inter- and intragenerically, including even strains of Mycobacterium and Thermoactinomyces, as compared with the marked difference of E. coli used as a control. Using these data, as well as the previously reported cell-wall composition (1), a possible phylogenetic position of various mesophilic actinomycetes is presented.
With a chemostatic culture of Azotobacter vinelandii, the values of yield factor for glucose were measured; the measurement was repeated after steady state in each value of dilution rate could be realized. In parallel with the measurement of yield factor, the bacterial cells sampled in each run of steady state were processed properly to determine the nucleic materials such as DNA, RNA and specific substances, the latter of which could be dissolved in a cold perchloric acid solution. The glucose (limiting substrate) consumption by the cells was assumed to be a sum of that primarily due to cellular growth and that principally due to cellular maintenance. This assumption was justified experimentally. The efficiency of glucose utilization for cellular growth, YG, and the specific rate of glucose consumption for cellular maintenance, m, were 0.172 and 3.90 (hr-1), respectively. Although the contents of DNA and the acid-soluble substances remained fairly unchanged without regard to the values of dilution rate, a logarithmic relationship was confirmed between the RNA content, NR, and the value of dilution rate, D, or the specific growth-rate, μX, of the bacterium.
Following the previous work on nucleic approach to a chemostatic culture of Azotobacter vinelandii (1), unsteady state relevant to a continuous cultivation of the same bacterium was studied especially from a macro-molecular and engineering viewpoint. Prior to the nucleic approach to this subject, unsteady state realized when batch cultivation is transferred to continuous run by initiating the flow of fresh medium into a single vessel was studied both by experimentation and by calculation using an Analog computer. So far as a marked difference, if any, between experiment and calculation-the latter of which was conducted from a material balance using the value of yield factor secured previously under steady-state condition-does not become problematical, the length of time required before steady state in continuous culture was achieved after initiating the continuous operation could be assessed by the computation. The nucleic analyses of cells experiencing unsteady state of different nature which was realized by changing the value of dilution rate stepwise from one level to another revealed that the response of cells to a new environment was rather rapid. Another steady and balanced state of cells was reached, being quite independent of the time when a new level of steady state in terms of cell and substrate concentrations became apparent in an engineering sense of the term. This finding suggests a need of introducing two significant concepts of "biological" and "mechanical" time-lag in handling a transient state due to be associated with a chemostatic culture of micro-organisms.
Escherichia coli K-12 E64 was grown in a synthetic medium containing succinate or glucose as the carbon source and changes in the cell age distribution pattern occurring in the aged culture was examined employing biochemical and cytological methods including autoradiography. Following the log phase, the growth proceeded linearly for a short period and then the stationary phase ensued. In the linear phase, cells gradually ceased to synthesize DNA leaving protein and RNA synthetic activities still active, although the rate was fairly reduced. These processes in the linear phase were accompanied with a decrease in DNA content per cell to a certain low level and the decrease in nuclear number toward one and two nuclei per cell. In the late linear phase the cultures seemed to be synchronized at least partially at the post-division stage. It was indeed demonstrated that when the aged cultures were diluted with fresh media, a characteristic synchronous cell division occurred after about one generation time.
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