An apparatus which allows the continuous deproteinization of nucleic acid solutions, reducing manual work, for methods in which the deproteinizing reagent is constituted by a liquid phase not mixing with the aqueous phase containing nucleic acids, is described. An example of the application on DNA from Bacillus subtilis is reported and the results regarding the extraction yield, percentage protein content, sedimentation coefficient, and the transforming activity are compared with those obtained by the original Marmur method.
DNA of B. subtilis, extracted according to Marmur but deproteinized in the apparatus described in the preceding paper of this series, has a higher transforming efficiency and a higher percentage protein content than DNA obtained exclusively by the Marmur method. Experiments were carried out in order to establish whether the higher transforming ability was due to the higher residual content of proteins. Results obtained by the action of Pronase on both samples of DNA support this hypothesis.
Bacterial cells adsorbed on an anion-exchange resin, Dowex 1, were incubated in a growth medium. Cell concentration in the liquid part of culture increased gradually at first (first step) and then rapidly (second step). With an incomplete medium or a medium which contains chloramphenicol, secondary rapid increase of the cells was not observed. The rate of secondary increase was much greater than the specific growth rate of free cells. These observations show that the first step is attributable to desorption of adsorbed cells from the resin without growth, while the second step is to detachment of adsorbed cells through growth and the rate of secondary increase reflects the growth rate of adsorbed cells. As the initial density of adsorbed cells increased, length of the first step decreased and the minimum length of which was 1 to 2hr. Relationship between the relative growth rate and pH shows that the optimum pH for growth of adsorbed cells is about one unit higher than that of free cells. It was observed that resin has many physiological influences not only on adsorbed cells growing on its surface but also on the cells detached from it as a result of growth, the most remarkable influence being shown in the case of enzyme induction.
The behavior of adsorbed cells on the surface of a resin was followed by electron microscopy by the carbon replica method. Cell division was not observed among adsorbed cells within 1 to 2hr after incubation in a growth medium, and cell density on the surface of the resin decreased slightly. After 2 to 6hr of incubation, adsorbed cells divided and the density increased at first but decreased later. The change in cell density on the resin surface was explained in relation to the change of cell concentration in the liquid part of culture reported in the previous report (1).
Twenty strains of methanol strongly assimilating yeasts were isolated from rotten tomato and a flower of azalea through investigations on the single- cell protein production and on the microbial utilization of C1 compound. Taxonomic studies indicated that these yeasts were limited to certain species of Candida and Torulopsis, including two new species; C. methanolica OKI et KOUNO sp. nov. and T. methanolovescens OKI et KOUNO sp. nov. One hundred and ninety-one strains of yeasts obtained from type culture collections did not exhibit methanol assimilability at all.
The cells of Escherichia coli at the logarithmic phase of growth lost their colony-forming ability when the cells were suspended in Tris buffer containing 0.15M sodium chloride. The colony-forming ability was recovered rapidly when sodium chloride-treated cells were incubated in Tris buffer containing magnesium ion immediately after the treatment. The magnesium- mediated recovery was not inhibited by either chloramphenicol, 2, 4-dinitrophenol, or sodium azide. Moreover, a higher recovery was observed by incubation at 3° rather than at 30°. A significant release of UV-absorbing materials occurred during incubation in Tris buffer containing sodium chloride. This release was almost completely suppressed when magnesium ion was present in the incubation medium.