Vladimir Vinter

Spores of microorganisms

The addition of different cysteine or thioproline concentrations (1–5×10−4M) to the culture at the outset of the formation ofBacillus cereus prespores, i.e. before the commencement of dipicolinic acid synthesis, led to the death of some of the cells and injured the thermoprotection mechanism of the surviving spores. In control spores with a high dipicolinic acid content, inactivation by heating at 85°C was preceded by a lag phase, while in cysteine- and thioproline-treated spores this lag phase was completely absent and the death rate of most of the spores (D-value=17) was actually higher than the final death rate of the control spores (D-value=33). A small proportion of the spores in inhibited cultures (less than 10%) displayed almost the same heat resistance as untreated spores. The heat sensitivity of treated spores was greater than might have been anticipated from their dipicolinic acid content. Their resistance to X-rays was not lowered, but was actually slightly raised. The results are discussed with reference to the differentiation of a possible “basal” and “additional” spore thermoprotection mechanism and to differentiation of the nature of heat and radiation resistance in bacterial spores.AbstractПрибавление различных концентраций (1–5.10−4M) цистеина или тиопролина к культуре Bacillus cereus в начале образования преспор, т. е. перед началом синтеза дипиколиновой кислоты, вызывает гибель части клеток, а в дальнейшем—нарушение теплозащитного механизма выживших спор. У контрольных спор с высоким содержанием дипиколиновой кислоты при прогревании до 85° C наблюдалось начальное инактивирование фазы задержки роста; у спор, возникших в присутствии цистеина и тиопролина, эта фаза задержки роста отсутствовала и, с дуугой стороны, у большей части спор наблюдалось ускорение гибели (D85=17) по сравнению с конечной скоростью умирания контрольных спор (D85=33). Небольшая часть (<10%) спор в угнетенных культурах обнаруживала почти такую же устойчивость к теплу, как необработанные цистеином и тиопролином споры. Чувствительность обработанных спор к теплу была больше, чем отвечало бы содержанию в них дипиколиновой кислоты. Их устойчивость к лучам Х не была пониженной, а, наоборот, несколько повышенной. —Результаты обсуждаются с точки зрения дифференциации эвентуальных «базального» и «аддиционального» теплозащитных механизмов спор и определения сущности теплоустойчивости в отличие от устойчивости спор бактерий к облучению.

Sporulation of bacilli

AbstractВ ходе формирования спор о Bacillus мегатерий, цистеина и cystine содержанием белков увеличивается. Способность клеток к включению35S цистеина заметно, поднятые в ходе формирование спор. Белки спор из Bacillus мегатерий содержат в пять раз насколько cystine плюс цистеина в вегетативной клетки до формирования спор. Possibillity что некоторые из цистеина и cystine могут быть синтезированы из метионина в ходе формирования спор, и возможной роли в их cystine белка Структура обсуждается.

External factors involved in the regulation of synthesis of an extracellular proteinase in Bacillus megaterium: effect of temperature

SummaryWe studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35–38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24–28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26–34° C) when the rate of temperature decrease was 0.2–0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32–34° C. The optimum temperature decrease was 0.8° C/h.

SPORES OF MICROORGANISMS. XV. THE ALTERATION OF HEAT SENSITIVITY AND ITS RELATION TO RADIATION RESISTANCE OF BACTERIAL SPORES

The addition of different cysteine or thioproline concentrations (1–5×10−4M) to the culture at the outset of the formation ofBacillus cereus prespores, i.e. before the commencement of dipicolinic acid synthesis, led to the death of some of the cells and injured the thermoprotection mechanism of the surviving spores. In control spores with a high dipicolinic acid content, inactivation by heating at 85°C was preceded by a lag phase, while in cysteine- and thioproline-treated spores this lag phase was completely absent and the death rate of most of the spores (D-value=17) was actually higher than the final death rate of the control spores (D-value=33). A small proportion of the spores in inhibited cultures (less than 10%) displayed almost the same heat resistance as untreated spores. The heat sensitivity of treated spores was greater than might have been anticipated from their dipicolinic acid content. Their resistance to X-rays was not lowered, but was actually slightly raised. The results are discussed with reference to the differentiation of a possible “basal” and “additional” spore thermoprotection mechanism and to differentiation of the nature of heat and radiation resistance in bacterial spores.AbstractПрибавление различных концентраций (1–5.10−4M) цистеина или тиопролина к культуре Bacillus cereus в начале образования преспор, т. е. перед началом синтеза дипиколиновой кислоты, вызывает гибель части клеток, а в дальнейшем—нарушение теплозащитного механизма выживших спор. У контрольных спор с высоким содержанием дипиколиновой кислоты при прогревании до 85° C наблюдалось начальное инактивирование фазы задержки роста; у спор, возникших в присутствии цистеина и тиопролина, эта фаза задержки роста отсутствовала и, с дуугой стороны, у большей части спор наблюдалось ускорение гибели (D85=17) по сравнению с конечной скоростью умирания контрольных спор (D85=33). Небольшая часть (<10%) спор в угнетенных культурах обнаруживала почти такую же устойчивость к теплу, как необработанные цистеином и тиопролином споры. Чувствительность обработанных спор к теплу была больше, чем отвечало бы содержанию в них дипиколиновой кислоты. Их устойчивость к лучам Х не была пониженной, а, наоборот, несколько повышенной. —Результаты обсуждаются с точки зрения дифференциации эвентуальных «базального» и «аддиционального» теплозащитных механизмов спор и определения сущности теплоустойчивости в отличие от устойчивости спор бактерий к облучению.

Spores of microorganisms. X. Interference of tetracycline antibiotics with sporogenesis of bacilli.

SummarySporulating cells of bacilli characterized by enormous accumulation of calcium were used as a model for determining the basis of the effect of tetracycline anti biotics on whole cells. Increased amounts of calcium are bound on the cells during the prespore phase. At the same time the ability of cells ofBacillus cereus andBacillus megaterium to bind chiortetra cycline is also markedly increased. In this phase both calcium and tetracycline are probably bound to the cell surface and can be removed by different washing procedures. Cultivation of sporulating cells with citric acid highly increased the binding of chlortetracycline on or in the cells. Chlortetracycline, oxytetracycline and tetracycline, in a concentration of 5 µg./ml., markedly inhibited the incorporation of calcium into the cells and dipicolinic acid synthesis in the sporangium probably by blocking the cell surface. They also influenced the morphology of the spores and autoly of the sporangia. Their effect depends largely on the stage of development of the culture at the moment when the antibiotic is added. Excess calcium in the medium somewhat stimulated dipicolinic acid synthesis in the presence of chiortetracycline and in the controls. Excess magnesium, manganese and glucose did not reverse inhibition. The effect of tetracycline antibiotics on sporulation and autolysis is compared with the effect of ethylenediaminetetraacetic acid. The results are discussed from the aspect of the theory of a metalmediated block of macromolecules or structures by tetracycline antibiotics.AbstractСпорообразующие клетки бацилл, характеризующиеся чрезвычайно сильным накоплением кальция, служили моделью для изучения сущности действия тетрациклиновых антибиотиков на цельные клетки. В период образования преспор наблюдается усиление связывания клетками кальция. В тот же период резко повышается способность клеток Bacillus cereus и Bacillus megaterium связывать хлортетрациклин. Повидимому, как кальций, так и тетрациклин адсорбируются в этот период на поверхность клеток и могут быть отмыты различными методами промывания. Культивирование спорообразующих клеток с лимонной кислотой в значительной степени усиливает связывание хлортетрациклина с клетками. Хлортетрациклин, окситетрациклин и тетрациклин (в концентрации 3 µг/мл), блокируя поверхность клетки, по-видимому, резко тормозят внедрение кальция в клетки и синтез дипиколиновой кислоты в спорангиях, влияя так на морфологию оюразуюшихся спор и на автолиз спорангиев. Это их действие в значительной степени зависит от стадии развития культуры в момент прибавления к ней антибиотика. Избыток кальция в среде несколько повыwает образование дипиколиновой кислоты как в присутствии хлортетрациклина, так и в контроле. Избыток магния, марганци илп глюкозы не меняет торможения внедрения кальция. — Действие тетрациклиновых антибиотиков на спорообразование и на автолиз спорангиев сравнивается с действием этилендиаминтетрау ксусной кислоты. Полученные результаты обсуждаются с точки зрения теории блокады макромолекул и структур тетрациклиновыми антибиотиками через посредство металлов.

Factors influencing the activity of cellular alkaline phosphatase during growth and sporulation of Bacillus cereus.

Alkaline phosphatase (EC 3.1.3.1) is synthesized in media with a low phosphate concentration (0.37 mmm of total and 19 μm of inorganic phosphate, respectively) already during the exponential phase of growth ofBacillus cereus. The enzyme is repressed by higher phosphate concentrations (3.7 mm) during the whole growth period; during sporogenesis the enzyme activity in cells slightly increases even under these conditions. During growth the enzyme is not secreted into the medium, a minor amount being released after cessation of growth. The enzyme activity can be increased by adding Zn2+ ions (10 μm). When during growth without phosphate the pH of the medium decreases below 5.0, the enzyme activity temporarily decreases and growth is slowed down, followed by a subsequent increase of the enzyme activity. In this case the onset of sporulation is also delayed.

Dual effect of amino acids on the development of intracellular proteolytic activity in the irreversible sporulation phase ofBacillus megaterium

Amino acids added to a population ofBacillus megaterium immediately after its transfer to a sporulation medium stimulated growth, delayed sporulation by 1 h, and delayed the development of intracellular cytoplasmic serine proteinase (ISP) activity. However, the ISP activity in late sporulation stages exceeded twice that of the control population. Amino acids supplemented at T3, i.e., at the time when engulfed forespores were developing, caused a decrease of specific ISP activity. The course of the phenylmethane sulfonyl fluoride (PMSF)-resistant activity in the cytoplasm was not affected by amino acids. Intracellular degradation of proteins prelabeled at the end of the growth phase was decreased by amino acids during the reversible sporulation phase but was only slightly affected later.

Inhibitory effect of 1-methyldodecyldimethylamine oxide and N,N-bis(dodecyldimethyl)-1,2-ethanediammonium dibromide on the spores of Bacillus cereus.

Abstract1-Methyldodecyldimethylamine oxide (MDDO) and N, N’-bis(dodecyldimethyl)-l, 2-ethanediammonium dibromide (BDED) exhibit a significant affinity for the surface ofBacillus cereus spores and adsorb very rapidly to the cells; they have a pronounced inhibitory effect on spore outgrowth. In order to alter the affinity of the spore surface for these inhibitors, the spores were pretreated with sodium dodecyl sulfate (SDS), and with an electronegative (Tween 80) and electropositive (histone) compound. In SDS-pretreated spores the inhibitory effect of MDDO and BDED was abolished to a considerable extent. Whereas the development of intact spores was inhibited already after germination, in SDS-pretreated spores the postgermination development continued but was not completed. In Tween.80-pretreated spores the addition of BDED led only to a retardation of outgrowth and division; BDED added only during the division stage interrupted further development completely. Histone-pretreated spores stopped their development instantaneously after the addition of BDED at any phase of the postgermination development. The possible mechanisms of the interaction of the compounds used with spore surface or rather with the state of its structures are discussed.

Origin and morphology of atypical forms ofStreptomyces granaticolor

Nonfilamentous forms ofStreptomyces granaticolor are formed in a medium with amino acids and glucose. They form filaments again after transfer to a medium with glucose and peptone. The nonfilamentous forms do not produce granaticin. Formation of nonfilamentous forms depends on the concentration of the inoculum, on the cultivation temperature and on the presence of simple sugars. Ultrathin sections revealed atypical septation in the nonmycelial forms and non-uniform accumulation of the wall material.

Sporulation of Bacilli. IV.

РезюмеДобавить аминокисл оты цистеина или (Конечная концентр ация 1 × 10−4 M) к культуре из мегатерий до фор мирования спор зам етно этом процессе начало затягивается и процесс остановлен для значительного времени на стадии и что из форми рований которые мы называем дефектных споры. Ингибировани е спор о формировани и сопровождается со кращением поглощен ие кальция в клетках. Ячейки неисправных спор не содержат больше кальция чем вегетативные клетк и Эффект от формирование спор из мегатерий завис ит от точки на котор ой она будет добавле на в культуре Ингибирование спор о формировании проя вляется только тогд а когда добавляется к культуре в период степень инг ибирования находящ ихся в прямой пропор ции квозраста культ уры в момент когда добавляется. Культи вирования ме гатерий присутствие повышает сопротивл ение культуре этой аминокислоты добави л когда вновь до в спор формирования может быть частично восстанавливается добавив значительн ое превышение уровн я глюкозы вместе с аминокисло той. Сульфид натрия имеет аналогичные ингибирующее влиян ие на формирование спор от мегатерий что и цистеина и.SummaryThe addition of the amino acids cystine or cysteine (final concentration 1 × 10−4 M) to a culture of Bacillusmegatherium prior to the formation of the spores markedly disorganises this process The commencement of sporulation is delayed and the process is halted for considerable time at the prespore stage and that of formations which we have termed “defective spores”. Inhibition of spore formation is accompanied by a reduction in the uptake of calcium into the sporulating cells. The cells with “defective spores” do not contain more calcium than vegetative cells The effect of cystine on the formation of spores ofBacillus megatherium depends on the point at which it is added to the culture Inhibition of spore formation is manifested only when cystine is added to the culture in the presporulation period, the degree of inhibition being in direct proportion to the age of the culture at the moment when the cystine is added. The cultivation ofBacillus megatherium the presence of cystine increases resistance of the culture to the actionof this amino acid when added again prior to sporulation.Inhabition of spore formation by cystine can be partially reversed by adding a considerable excess of glucose together with the amino acid. Sodium sulphide has a similar inhibitory effect on the formation of spores otBacillus megatherium to that of cysteine and cystine.The addition of the amino acids cystine or cysteine (final concentration 1 × 10−4 M) to a culture of Bacillusmegatherium prior to the formation of the spores markedly disorganises this process The commencement of sporulation is delayed and the process is halted for considerable time at the prespore stage and that of formations which we have termed “defective spores”. Inhibition of spore formation is accompanied by a reduction in the uptake of calcium into the sporulating cells. The cells with “defective spores” do not contain more calcium than vegetative cells The effect of cystine on the formation of spores ofBacillus megatherium depends on the point at which it is added to the culture Inhibition of spore formation is manifested only when cystine is added to the culture in the presporulation period, the degree of inhibition being in direct proportion to the age of the culture at the moment when the cystine is added. The cultivation ofBacillus megatherium the presence of cystine increases resistance of the culture to the actionof this amino acid when added again prior to sporulation.Inhabition of spore formation by cystine can be partially reversed by adding a considerable excess of glucose together with the amino acid. Sodium sulphide has a similar inhibitory effect on the formation of spores otBacillus megatherium to that of cysteine and cystine.