Germination Of Bacterial Spores Research Articles

Mechanism of the inhibition of germination of bacterial spores by gamma-irradiation in the presence of iodoacetamide and iodate.

SUMMARY: Whereas γ-radiation alone either did not affect or stimulated spore germination, spores which had been irradiated in the presence of iodoacetamide (IAM) or potassium iodate were inhibited from germinating in germinants like L-alanine, inosine or yeast glucose broth. Activity of a lytic enzyme which may be involved in germination was mostly lost in spores irradiated in IAM, and a phosphomonoesterase was about 60 % inhibited. Spore alanine racemase was partly inactivated by γ-radiation alone but, with spore protease, survived irradiation in IAM without much further loss of activity. Although spores made non-germinable by γ-radiation plus IAM were non-viable as measured by colony counts, viability could be partly restored by chemical treatments known to by-pass the normal germination reactions. It was concluded that spores were inactivated by γ-radiation in the presence of the potentiators through inactivation of enzyme(s) essential in the degradative reactions of germination. The enzyme inactivation was caused by iodine-containing free radicals rather than directly by the γ-radiation.

Spores of microorganisms. 23. Interdependence of intra- and extra-cellular levels of calcium: its effect on the germination of bacterial spores in different media.

Spores of microorganisms. 23. Interdependence of intra- and extra-cellular levels of calcium: its effect on the germination of bacterial spores in different media.

Initiation of germination of bacterial spores by hydrostatic pressure.

SUMMARY: Hydrostatic pressure initiated germination of bacterial spores in nutrient-free media. Those spores which were most dormant towards chemical germinants at I atmosphere pressure were also the most resistant to germination by pressure treatment. Germination by high pressure treatment was characterized by temperature and pH optima, like germination at atmospheric pressure. Germination initiated by pressure was inhibited by metabolic poisons and was potentiated by low concentrations of various nutrients including some of those which are normally germinative (at higher concentrations) at atmospheric pressure. In particular, L-alanine and closely related α-amino acids, but not their breakdown products, potentiated germination initiated by pressure. Study of potentiation by D-alanine (which strongly inhibits germination initiated by L-alanine at I atmosphere pressure) revealed that high pressures caused an increase in the rate of racemization of alanine by spores. Germination by pressure probably resulted from acceleration of some germination reaction which is normally negligibly slow at a pressure of I atmosphere, and also from an increase in permeability of some barrier within the spore to L-alanine and related α-amino acids.

Inhibition of the amino acid induced initiation of germination of bacterial spores by chlorocresol.

The initiation of germination of Bacillus subtilis spores in complete medium and in phosphate buffer plus the enzyme subtilopeptidase is inhibited by chlorocresol, but the activity of the enzyme alone is not. This suggests that proteolytic activity is not the primary site of inhibition. Germination of spores of this organism can be initiated with one of each of five amino acids, viz. L-alanine, L-cysteine, L-valine, L-leucine, and L-isoleucine. The germination initiated by these amino acids is reversibly inhibited by chlorocresol. These results strongly suggest that the inhibitor prevents the initiation of germination of bacterial spores at the level of amino acid use, both in complete medium and with subtilopeptidase.

The crystal structure of calcium 4H-pyran-2,6-dicarboxylate trihydrate, Ca.C7O5H4.3H2O

Calcium dipicolinate is believed to play an important role in the heat resistance and metabolic inertness of bacterial spores (Murrell & Warth, 1965). The discovery by Strahs & Dickerson (1968) of a strong dimeric linkage in the crystal structure of calcium dipicolinate (Ca. DPA) trihydrate has encouraged speculation as to the role of Ca. DPA in bacterial spores. Investigation of analogues of dipicolinic acid has shown that 4H-pyran-2,6-dicarboxylic acid (PDC) substitutes very effectively for D P A in the germination of bacterial spores induced by 0-04 M C a . D P A (Lewis, 1967; Riemann & Ordal, 1961) and in the formation of spores by a DPA-requiring mutant of Bacillus megaterium (Fukuda & Gilvarg, 1968; Fukuda et al., 1969). Because of the interest in determining the structural features responsible for the observed spore properties (Murrell, 1969), we have undertaken the determination of the crystal structure of several salts of D P A and of a few compounds which have a close chemical relationship to DPA. One of these latter is Ca. PDC, synthesized by Richard M. Seifert of this Laboratory. The molecular structures of Ca. D P A and Ca. PDC are shown in Fig. 1. C a . P D C , like C a . D P A , crystallizes from water as the trihydrate. The unit-cell dimensions, density, and space group for C a . P D C indicate that it is isostructural with Ca. DPA. Preliminary crystallographic data for Ca. PDC. 3H20 were obtained from Weissenberg and precession photographs. The unit-cell dimensions were refined from high angle 20 scans with an automatic diffractometer at a take-off angle of 1 °. The final values are listed in Table 1, along with the published values for Ca. DPA. 3H20 (Strahs & Dickerson, 1968).

The chemical inhibition of an enzyme induced germination of bacterial spores.

Spores of Bacillus subtilis germinate rapidly in phosphate buffer upon the addition of the enzyme subtilopeptidase-A. This enzyme induced germination is inhibited by p-choloro-m-cresol. The effects have been found of preincubating intact spores with the inhibitor, of preincubating enzyme with the inhibitor, and of preincubating spores, enzyme, and inhibitor together. In every case when the inhibitor is suitably neutralized the spores will germinate normally.

Germination of single bacterial spores.

Changes in refractility and optical density occurring in individual spores of Bacillus cereus T and B. megaterium QM B1551 during germination were investigated by use of a Zeiss microscope photometer. The curves revealed that the germination process in single spores had two distinct phases; an initial rapid phase was followed by a second slower phase. Under the experimental condition employed, the first phase of germination of B. cereus spores lasted for approximately 75 +/- 15 sec, whereas the second phase lasted for 3 to 4.5 min. In B. megaterium spores, the first phase was observed to last for approximately 2 min and the second phase for more than 7 min. The duration of the second phase was dependent on conditions employed for germination. The kinetics of the first phase were strikingly similar under all conditions of physiological germination. Time-lapse phase-contrast microscopy of germinating spores also revealed the biphasic nature of germination. It was postulated that the first phase represents changes induced by an initial partial hydration of the spore and release into the medium of dipicolinic acid, whereas the second phase reflects degradation of the cortex and hydration of the core.

Mechanism and factors influencing the veronal inhibition of bacterial spore germination.

The inhibitory effect of sodium 5,5-diethyl barbiturate (Veronal) on the L-alanine-induced initiation of germination of Bacillus subtilis spores was examined. Veronal reversibly inhibited the initiation of germination by a noncompetitive mechanism. The inhibition was time-independent and it took place whether L-alanine was or was not allowed to permeate the spore before the addition of the inhibitor. The concentration of the inhibitor and the pH of the initiation system were important factors determining the effectiveness of Veronal as an inhibitor. The magnitude of the inhibition increased linearly with decreasing pH at constant concentration and with increasing concentration at constant pH. These results suggest that the inhibition involves a permeability phenomenon related to the access of drug to the active sites in the spore and that the entry of Veronal into the spores is regulated by the concentration of undissociated molecule. At the physiologically important pH of 7.4, initiation with alanine in phosphate buffer at high spore densities (about 10(9) spores per ml) was 50% inhibited by 4 mM Veronal, and 8mM Veronal inhibited initiation completely. L-Alanine initiation in tris(hydroxymethyl)amino-methane-hydrochloride buffer was completely inhibited by 5 mM Veronal. The inhibition could be partially reversed by the combined addition of D-fructose, D-glucose, and K(+). Possible reasons for the failure of otherwise inhibitory concentrations of Veronal to inhibit completely the L-alanine-induced initiation when a combination of fructose, glucose, and K(+) was present and a suggested relationship to two functional roles of L-alanine in the initiation of germination are discussed.

Ribosomal ribonucleic acid maturation during bacterial spore germination.

All the ribosomal ribonucleic acid made during the early stages of germination of spores of Bacillus subtilis is of the "precursor" type, i.e., that type appearing in the incomplete forms of the ribosome. Shortly before the onset of deoxyribonucleic acid synthesis in germination, this precursor ribonucleic acid changed to the mature ribosomal ribonucleic acid characteristic of the 30S and 50S ribosomal subunits.

Mechanism and Factors Influencing the Veronal Inhibition of Bacterial Spore Germination

The inhibitory effect of sodium 5,5-diethyl barbiturate (Veronal) on the L-alanine-induced initiation of germination of Bacillus subtilis spores was examined. Veronal reversibly inhibited the initiation of germination by a noncompetitive mechanism. The inhibition was time-independent and it took place whether L-alanine was or was not allowed to permeate the spore before the addition of the inhibitor. The concentration of the inhibitor and the pH of the initiation system were important factors determining the effectiveness of Veronal as an inhibitor. The magnitude of the inhibition increased linearly with decreasing pH at constant concentration and with increasing concentration at constant pH. These results suggest that the inhibition involves a permeability phenomenon related to the access of drug to the active sites in the spore and that the entry of Veronal into the spores is regulated by the concentration of undissociated molecule. At the physiologically important pH of 7.4, initiation with alanine in phosphate buffer at high spore densities (about 10(9) spores per ml) was 50% inhibited by 4 mM Veronal, and 8mM Veronal inhibited initiation completely. L-Alanine initiation in tris(hydroxymethyl)amino-methane-hydrochloride buffer was completely inhibited by 5 mM Veronal. The inhibition could be partially reversed by the combined addition of D-fructose, D-glucose, and K(+). Possible reasons for the failure of otherwise inhibitory concentrations of Veronal to inhibit completely the L-alanine-induced initiation when a combination of fructose, glucose, and K(+) was present and a suggested relationship to two functional roles of L-alanine in the initiation of germination are discussed.

Stimulation of Germination of Bacterial Spores by Analogues of D-Alanine

SUMMARY: L-Alanine-initiated germination of spores of Bacillus cereus was potentiated by the following structural analogues of alanine: O-carbamyl-D-serine (OCDS), D-cycloserine (DCS), β-alanylhydroxamic acid (βAHA) and glycyl hydroxamic acid (GHA); but not by D-α-alanyl-hydroxamic acid (DAHA). Potentiation of germination resulted from inhibition of alanine racemase in the spores with consequent suppression of the formation of D-alanine, an inhibitor of L-alanine-initiated germination. OCDS was the most effective potentiator of germination and inhibitor of racemase. βAHA and GHA were more effective potentiators of germination than could be explained solely by their weak inhibition of the racemase. The extra effectiveness was associated with slow binding of the analogues to the spores, and possibly also with formation of hydroxylamine which also inhibited alanine racemase and potentiated L-alanine-initiated germination. Germination initiated by ribosides and amino acids other than L-alanine was not strongly potentiated by OCDS, arguing against a role for L-alanine as an intermediate. However, germination initiated by adenosine+D-alanine was strongly inhibited by OCDS, which argues for the role of D-alanine being to supply the L-isomer by racemization. Antibacterial activities of the analogues did not mirror their activities as potentiators of germination.

Limitations of the initiation of germination of bacterial spores as a spore control procedure.

Limitations of the initiation of germination of bacterial spores as a spore control procedure.

A reversible inhibition of the germination of bacterial spores.

The germination of Bacillus subtilis spores in stationary aerobic culture is inhibited by p-chloro-m-cresol. Spores inhibited in this way can be recovered and induced to germinate by the addition to stabilized cultures of the non-ionic surfactant, polysorbate (Tween) 80. In this way the stabilization of the germination process by the inhibitor may be reversed after periods of stabilization of up to 21 hours. The inhibitor may interfere with an enzymatic lytic activity responsible for germination initiation. A denaturation process is not indicated.

Initiation of bacterial spore germination.

To investigate the problem of initiation in bacterial spore germination, we isolated, from extracts of dormant spores of Bacillus cereus strain T and B. licheniformis, a protein that initiated spore germination when added to a suspension of heat-activated spores. The optimal conditions for initiatory activity of this protein (the initiator) were 30 C in 0.01 to 0.04 m NaCl and 0.01 m tris(hydroxymethyl)aminomethane (pH 8.5). The initiator was inhibited by phosphate but required two co-factors, l-alanine (1/7 of K(m) for l-alanine-inhibited germination) and nicotinamide adenine dinucleotide (1.25 x 10(-4)m). In the crude extract, the initiator activity was increased 3.5-fold by heating the extract at 65 C for 10 min, but the partially purified initiator preparation was completely heat-sensitive (65 C for 5 min). Heat stability could be conferred on the purified initiator by adding 10(-3)m dipicolinic acid. A fractionation of this protein that excluded l-alanine dehydrogenase and adenosine deaminase from the initiator activity was developed. The molecular weight of the initiator was estimated as 7 x 10(4). The kinetics of germination in the presence of initiator were examined at various concentrations of l-alanine and nicotinamide adenine dinucleotide.

Photochemical evidence for conformation changes in DNA during germination of bacterial spores.

Photochemical evidence for conformation changes in DNA during germination of bacterial spores.

A kinetic model for bacterial spore germination.

A kinetic model for bacterial spore germination.

Splitting line of bacterial spore.

THE mechanisms involved in the germination of bacterial spores have been studied extensively from the biochemical and morphological points of view11,2. Details of morphological changes occurring during germination, however, have not yet been demonstrated clearly, It is not known whether or not, when a new vegetative cell emerges from the spore, the spore coat splits by some specific mechanism.

細菌胞子の発芽に及ぼす加熱の効果

It has generally been believed that bacterial endospores were activated by beat treatment at considerably high temperature (about 80°C). Such treatment of the spores is, therefore, often called “heat activation”. In some particular cases, however, the germination of bacterial spores was sometimes found to be delayed by such a heat treatment. In order to ascertain whether the heat treatment does stimulate the germination of bac-terial spores or not, we observed in this work the germination of both heated and unheated spores of Bacillus subtilis under various conditions. The results obtained may be summarized as follows. (1) In the case of the freshly harvested spores, the germination was delayed by heat treatment under all the conditions employed; the heat treatment had always an inhibitory effect on the germination regardless of the compositions of sporulating and germinating media or the temperature used for the heat treatment. (2) The spores which had been stored at 0°C for more than 66 days were remarkably activated by the heat treatment at 80°C for 20 minutes; germination of the heated spores was more rapid than that of the unheated spores in the germination media which contained L-alanine in the concentration of higher than 1mM. These results suggest that the effect of heat treatment on the germination of spores of Bacillus subtilis essentially varies with the storage condition of the spores.

Role of amino-acids in the germination of bacterial spores by subtilisn.

BACTERIAL spores are characteristically resistant to desiccation, heat, irradiation, chemicals and to attack by enzymes. Resistance to enzymes is an attribute that must have important survival value for spores, because spores may come into contact with a variety of hydrolytic enzymes secreted by micro-organisms and other flora and fauna in their natural habitat, the soil. Previous reports that two lytic enzymes, lysozyme and an enzyme which can be extracted from B. cereus spores, will attack spores, were based on experiments in which the spores were first treated with reagents which rupture disulphide bonds1,2. This sensitization treatment was probably necessary to expose the substrates to the action of the enzymes3,4.

Bacterial spore germination with special reference to Bacillus megaterium.

Bacterial spore germination with special reference to Bacillus megaterium.