Spores of microorganisms

The addition of penicillin (300–1,000 units/ml.) to a culture ofBacillus cereus during formation of the refractive prespores leads to lysis of the sporangia and to the release of spore components (calcium and dipicolinic acid) from the cells. Penicillin mildly raises the incorporation of amino acids, including diaminopimelic acid, into hot-TCA precipitate of cells, while chloramphenicol lowers it. In the later phases of penicillin inhibition, DAP-containing structures are also destroyed, including the fraction firmly bound to the envelope structures of the spore (in the control culture this fraction is not released until later, during digestion by enzymes localized in the envelope structures themselves). Penicillin inhibition of sporogenesis can be reversed by adapting the culture to penicillin or by simultaneously adding chloramphenicol. After the presporulation phase, sporogenesis is relatively resistant to chloramphenicol, but the whole process is considerably slowed down. Chloramphenicol also affects the morphology of the spores during their formation and inhibits their release from the sporangia until the late phase of sporulation.     

Spores of microorganisms

Histone (final concentration 200 μg/ml) inhibits the synthesis of both proteins and total ribonucleic acid (RNA) in germinated spores ofBacillus cereus. It also blocks a further cytodifferentiation of germinated spores into vegetative cells. The inhibition takes place both in complex media facilitating the differentiation (media with bactopeptone or casamino acids) and in limited minimal medium which permits only the germination and the synthesis of a limited amount of proteins and RNA. At this concentration, the histone inhibits strongly the synthesis of pulse-labelled RNA and brings about a change in the sedimentation constants of ribosomes. The results are interpreted in terms of the strong affinity of the histone towards electronegative peripheral layers and intracellular structures and macromolecules of the germinated spore.     

Spores of microorganisms

The relationship of the synthesis of new cell wall in the postgerminative development ofBacillus cereus spores to protein and ribonucleic acid synthesis was studied through the incorporation of¹⁴C-diaminopimelic acid. The spores were not capable of synthesizing cell wall immediately after germination. A very short, period of protein synthesis was first needed, the messenger ribonucleic acid for these proteins being formed at the end of the depolymerization phase. On blocking cell wall synthesis with penicillin or cycloserine, swelling and the outset of elongation were normal. In the presence of penicillin, the cells afterwards disintegrated during the elongation phase, while with cycloserine, elongation of the cells was only arrested and later atypical division occurred. The findings are discussed from the aspect of the possibility of the participation of part of the preexisting diaminopimelic acid-containing spore material in the envelope system of the outgrowing cell.     

Spores of microorganisms

Spores ofBacillus cereus (strain NCIB 8122) were germinated in a synthetic germination limited medium (GL-medium), which permitted germination but did not make the termination of post-germinative development possible. Incorporation of¹⁴C-diaminopimelic acid into the newly formed cell wall was followed in this culture. Morphological changes were studied by optical and electron microscopy. Germination was associated with the usual germination changes,i.e. depolymerization of the “bulky” cortex, differentiation of nuclear structure and mesosomes and ribosomes in the cytoplasm. At this stage the spore protoplast is surrounded by several layers: exosporium, laminated coat with four layers, residual spore wall and the protoplast membrane. During incubation in this limited medium the residual wall layer thickens and the nuclear structure, mesosomes and ribosomes were not more detectable. After enrichment of the GL medium (shift up) the thick-walled cells can form additional cell wall material, elongate and an atypical septum formation can occur. The cell wall material forms local thickenings. On long-term cultivation in the GL medium some of the cells in the GL medium lyze. If, in the course of 3–6 h the cells are transferred from the GL-medium to a solid complex medium (Difco Nutrient Agar) the thickwalled cells are transformed into dividing cells. When the cells are transferred later, their colony-forming ability rapidly decreases. The decrease of viability of the thick-walled cells derived directly from spores after their germination in the limited medium indicates that these cellular forms probably do not represent more stable cellular types that would be of considerable importance for survival of the populat ion of bacilli.     

Spores of microorganisms

Preexisting¹⁴C-DAP in vegetative cells ofBacillus cereus is not incorporated into the spores, but is released into the medium after sporogenesis is complete. Exogenous¹⁴C-DAP added to the medium before sporulation is incorporated intensively into the sporangia and practically all of it is taken up by the spores. During sporogenesis, two periods of increased incorporation of¹⁴C into hot TCA-precipitate of cells are found after adding¹⁴C-DAP— one before formation of the spores, when¹⁴C-lysine formed by decarboxylation is incorporated together with¹⁴C-DAP, and one during the “whitening” phase, when any¹⁴C-lysine is no longer incorporated. The incorporation of exogenous¹⁴C-lysine into the sporangial proteins is also markedly elevated during the presporulation phase and at the outset of sporogenesis.     

Spores of microorganisms

A pulse incorporation of radioactive precursors into the cells was used in studying the rate of RNA and protein synthesis during postgerminative development of spores ofBacillus cereus. It was found that the extractable pool of the spores can be enriched by these precursors during swelling and preelongation. During this period of differentiation of a spore to a primary cell,¹⁴C-uracil and to a smaller extent¹⁴C-adenine are more rapidly used for RNA synthesis. Labelled¹⁴C-leucine and³⁶S-methionine are found mostly in the extractable fraction of the cells. However, their rate of incorporation into proteins is not higher. Differences in utilization of precursors for the synthesis of macromolecules are apparently caused by different availability for the metabolic pool of the cell. Of the precursors tested¹⁴C-uracil seems to be selectively incorporated into RNA with a higher rate. This can be explained by a selective permeability into metabolic pool or by an increased need of uracil during preelongation period.     

Spores of microorganisms

1. (1)
   Аланин-2-¹⁴C и³⁵S-метионин требования клеток Bacillus cereus и Bacillus мегатерий не увеличивают в течение sporulation. Существу ет, однако, Увеличение включени я³⁵S-цистеина.     
   
   

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.     

Spores of microorganisms

The spores ofBacillus cereus formed during endotrophic sporulation in the presence of β 2-thienylalanine differ by the curve of heat-inactivation from the spores produced in distilled water containing calcium or in bactopeptone medium: the initial lag phase observed on heat inactivation is missing. The content of dipicolinic acid is comparable with that of control spores. Their UV resistance remains practically unchanged.     

Spores of microorganisms

Bacillus cereus spores labelled with radioactive diaminopimelic acid (DAP) in different phases of spore development germinated and the fate of the individual DAP-containing fractions during germination and postgerminative development was studied. Envelope structures not depolymerized during germination were formed in the prespore stage. During maturation of the spores, structures degraded during germination and released into the medium were mainly formed. Some of the DAP-containing material remained in the cells in a labile form during germination and could probably be re-utilized in synthesis of the cell wall during postgerminative development.     

Spores of microorganisms

6-Azauracil at a concentration of 1 μmole/ml inhibits by 50% the outgrowth of germinated spores of a strain ofBacillus cereus, concentration of 1.5 μmole/ml resulting in 100% inhibition. Two distinct maxima of sensitivity to 6-azauracil are observed during postgerminative development of spores. The first occurs during early stages of development (immediately after depolymerization period) and the second after about 60 min of cultivation (late stage of swelling). Uracil reverses the inhibition of the outgrowth of spores caused by 6-azauracil when added during 0–30 min of the spore development. The addition of uracil after 30 min of the germination does not bring about the reversion of the effect of 6-azauracil. An important role of pyrimidine pathway via orotidine 5′-phosphate in germinating spores was proved, suggesting a possible use of 6-azauracil in synchronization of the postgerminative development of spores.     

Spores of microorganisms

Spores ofBacillus cereus were germinated in a germination limited medium (GL-medium) which facilitates only germination but not the postgerminative development of spores. Under these conditions a limited protein synthesis occurs. However, this protein synthesis is stopped after a short time interval. The rate of synthesis of new proteins, as well as their total amount, is influenced by the length of the activation heat shock. Synthesis of the wall material continues for several hours and thick-walled cells with a changed ultrastructure are formed. Synthesis of the diaminopimelic acid (dap) containing material of the cell wall is sensitive to actinomycin D and relatively resistant to chloramphenicol. Similarly, protein synthesis is relatively chloramphenicol-resistant but is fully inhibited by azauracil or spiramycin. Whereas RNA formed in the control culture is partially decomposed after 30 min of incubation, chloramphenicol accelerates its synthesis and prevents its decay. Exudate components apparently stimulate synthesis of ribonucleic acid, proteins and the wall material. The¹⁴C-dap containing material released by prelabelled spores in the form of the exudate during the germination is not re-utilized by the spores germinated in the GL-medium. The results are discussed with respect to the atypical primary synthetic activities of spores under conditions when the postgerminative development is prevented and from the point of view of participation of the germination exudate during these syntheses.     

Spores of microorganisms XVI

The heat inactivation of spores ofBacillus cereus irradiated and non-irradiated with X-rays was investigated with respect to their dipicolinic acid (DPA) content. Spores with a high DPA content (98 μg./mg. dry weight) displayed a biphasic heat inactivation curve. This biphasic character of the curve was preserved for the heat inactivation of previously irradiated spores even if the length of the initial lag of inactivation rate was reduced. Spores with a lower DPA content (24 μg./mg. dry weight) display a single-phase logarithmic order of dying from the beginning of heating. In both types of spores differing in the DPA content a pronounced sensitization of radiation-surviving spores toward subsequent heating was observed.     

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

Спорообразующие 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.