M. Kohoutová

Mechanism of binding of the competence substance to the cell wall receptor sites. Inhibition of induction of competence by the competence substance in the presence of phytohemagglutinins and some amino sugars.

The induction of competence by the competence substance is strongly inhibited in the presence of phytohemagglutinins. A great inhibition effect is also found when the competence substance is preincubated with some amino sugars. Both the phytohemagglutinins and the competence substance exhibit a similar binding affinity to cross-linked dextran (Sephadex) and to the cell surfaces, as both are inhibited by some sugars in their interaction with the sensitive cells. It is proposed that they are bound to similar or identical cell receptor sites. These seem to be some specific sugar molecules forming a part of cell wall structures of the transformable and to competence inducible bacterial strains.

Role of deoxyribonuclease in genetic transformation. I. Deoxyribonuclease activity in variously transformable strains.

The deoxyribonuclease activity was investigated in cell-free filtrates of cultures of strains of different transformability. The highly transformable stable strains display a relatively low, probably optimal, deoxyribonuclease activity. Transformable but unstable strains produce a very high deoxyribonuclease activity in comparison with the aforementioned ones. In some nontransformable strains no deoxyribonuclease activity could be detected. The role of deoxyribonuclease in the competence of recipient cells s discussed.

Contribution on the biochemical investigation of external factors required for type transformation in thePneumococcus

Summary(1)It was found that different anti-R-Pneumococcus sera display a great variability in DNase activity contained therein. There are sera in which a high DNase activity remains even after inactivation at 65° C for 30 min. At dilutions which are commonly used in the experiments a certain residual activity is always found.(2)The presence of suitable concentrations of salts with univalent cations in the transformation medium inhibits the DNase activity in it, thus regularizing the results obtained during type transformation in the Pneumococcus. This explains the results of our earlier work concerning the significance of these salts in the transformation reaction even if their role is manifold.(3)By preliminary estimation of DNase activity in sera or serous fluids, globulin and albumin preparations, it can readily be shown whether these sera are or are not suitable for supporting the transformation reaction. Antisera with a high residual activity of DNase do not support the transformation reaction unless DNase had been suitably inhibited.(4)High DNase content in serum may by inhibited by higher temperature or occasionally by increased salt concentration. By using a mixture of citrate, magnesium ions and a small amount of pancreatic DNase activated for 15 min. at 30° C and then inactivated for 15 min. at 60° C, together with DNA, a positive result may be achieved during transformation using some antisera with a high DNAse activity which will not allow such a reaction to proceed without the agents added.(5)The transformation reaction always takes place in the presence of a small amount of DNase which is blocked by different factors in the system. The possible active role of DNase in the transformation reaction is discussed. The author is indebted to Academician I. Málek for the interest with which he followed the entire work described. Technical assistance of PhMr. P. Křečková and Mr. J. Kubíček is acknowledged. Our thanks are due to Dr. Hilgert of the Institute of Experimental Biology and Genetics for a sample of DNA from chick erythrocytes.Abstract(1)Мв установили, что раэличные анти-R пневмококковые сыворотки отличаются эначителыюй иэменчивостъю активности содержащейся в них деэоксирибонуклеаэ ы(ДН аэа). Это сывороеки, в которых высокая степенъ активности ДНϕэы сохраняется и после инактивирования при 65° C в течение 30 мин. В раэведениях, применяемых обычно для опытов, всегда находится иэвестная остаточная активностъ ДНаэы.(2)Присутствие в трансϕормационной среде соответствующих концентраций солей с одновалентными катионами подавляет активностъ ДНаэы, чем определяются реэулътаты трансϕормации типа пневмококка. Этим объясняются реэулътаты наших предшествовавших работ по иэучению эначения этих солей для реакдии трансϕормадии, хотя ролъ этих солеи, по-видимому, является более многосторонней.(3)Путем предварителъного определения активности ДНаэы в сыворотках или в сероэных жидкостях, глобулино вых и алъбуминовых препаратах можно быстро установитъ, пригодны ли они для поддержания реакции трансϕорма ции - или нет. Антисыворотки с высокой остаточной активностъю ДНϕэы не способствуют реакции трансϕормации, если не имело места предварителъное соответственное угнетение ДНаэы.(4)Угнетение ДНаэы в сыворотках воэможно с помощъю повышения температуры или иногда с помощъю более высоких концентрации солеи. лсполъэуя смесъ цитратов, ионы Mg++и неболъшое количество панкреатическои ДНϕэы, активированной 15 мин. при 30° C и после этого инактивированной 15 мин. при 60° С вместе с ДНК, можно добитъся положителъных реэулътатов трансϕормации с применением некоторых антисывороток с высокой активностъю ДНаэы там, шже беэ этои смеси это невоэможно.(5)Реакция трансϕормации протекает всегда в присутствии неболъшого количества ДНаэы, которая в системе блокируется раэличными ϕакторами. — Обсуждается воэможностъ активной роли ДНаэы в реакции трансϕормации.

Regulation by the precompetent cell culture of the appearance of spontaneous and induced competence

Biochemical and biophysical changes in the precompetent cell culture, rather than merely the cell size, regulate the appearance of competence. The more physiologically mature the incompetent cell culture is, the less competence substance is required for maximal induction of competence. The kinetics of induction of competence as the function of the physiological state of the incompetent culture and as the function of the concentration of the competence substance seems to support the idea that the competent cell is a temporary spheroplast.

Genetic transformation and transfection ofBacillus subtilis spheroplasts

Genetic transformation and transfection of lysozyme-treatedBacillus subtilis spheroplasts 168M ind occurs only if they are stabilized with 0.5m phosphate buffer and not if they are stabilized with 0.5m sucrose. Spheroplasts prepared from maximally competent cells give maximum transformation and transfection results. The results indicate that the DNA receptors must also be intact in the spheroplasts.

A special deoxyribonuclease activity accompanying competence-inducing activity

A deoxyribonuclease activity accompanies the competence substance isolated from transformableDiplococcus pneumoniae even in well purified fractions. The deoxyribonuclease seems to exhibit a rather different kind of activity from the one found as a major nuclease in a partially purified competence substance. The products of interaction between the enzyme and double-stranded DNA would indicate that the enzyme might act as an “unwindase” on the double-stranded DNA.

The substance with competence inducing activity interacts with DNA and produces a hyperchromic increase of optical density.

The active substance inducing competence in non-competent cells ofDiplococcus pneumoniae was isolated by ammonium sulphate precipitation from the sterile filtrate of a culture 40–60 min after the peak of competence had passed. After being dialyzed and dried from the frozen state the substance was further purified by chromatography on Sephadex G100 or DEAE cellulose column. In all cases the fractions containing the competence inducing activity contained also the transforming DNA-inactivating activity. A hyperchromic increase of optical density was found on incubating the active substance with various DNAs and RNAs as substrates. The hyperchromic increment is different from that of pancreatic endo-nuclease I and phosphodiesterase.

SOME FACTORS STIMULATING AND INHIBITING PANCREATIC DEOXYRIBONUCLEASE.

AbstractДля выяснения роли ДН-азы в реакции трансформации исследовали действие различных сред, применяемых при трансформации пневмококков, на активность панкреатической ДН-азы. Эти виды среды резко повышали активность фермента. Далее, было установлено, что это повышение вызывается yeast-экстрактом, входящим в состав этих сред. С помщяю спектрального анализа и пламенного спектрофотометра в yeast-экстракте были обнаружены Mg2+, Ca2+, K+ и Na+. Изучалось действие этих ионов на активность ДН-азы и было установлено, что в присутствии Mg2+ (5×10−3m)иCa2+(вконцентрации 2×10−4m) резко повышается активность фермента, тогда как прибавление к этой системе KCl в концентрации 2×10−1, 2×10−2, 2×10−3 и 2×10−4m оказывает угнетающее действие.—Обсуждается возможное влияние этих ионов на peaкцию трансформации.

Temporary, reversible binding of the competence factor to cellular receptors ofPneumococcus during induction of competence

The binding of the competence factor to cellular receptors of physiologically non-competent cells ofPneumococcus was followed as a function of time. A transformation medium without bovine serum albumin was used to study the binding of the competence factor. Control cells without the added factor remained completely non-competent under these conditions. The maximal binding of the factor to the cellular receptors took place already after 3 min of contact of the cells with the factor at 37°C. After 10 min, when the maximum induction of competence occurs in the system used, the competence factor is fully released from the receptors to the medium. It follows that within the period between the 3rd and 10th min, when the cells are being modified for the irreversible binding of DNA, the presence of the competence factor on the cells is no longer necessary.

Prevention of cell agglutination and competence in a genetically transformablc strain ofPneumococcus byd-glucosamine andd-galactosamine

In the presence of amino sugarsd-glucosamine andd-galactosamine no spontaneous competence could be observed in the highly transformable R6bd strain ofPneumococcus or it was decreased by several orders of magnitude. The highest inhibition of competence was detected when the amino sugar at a concentration 5 mg/ml of the medium was added not only to the transformation but also to the pretransformation medium. After a 150 min growth in the transformation medium in the presence of the amino sugar a 3–4-fold greater number of cells (as a viable count) could be detected as compared with the control without the amino sugar. It was found microscopically that the amino sugar prevents natural agglutination, which normally occurs in the competent culture. The role of specific amino sugar determinants for binding of the competence factor on the cell surface and the resulting inhibitory effect of these sugars on the development of competence are discussed.