Germination Of Bacterial Spores Research Articles

Reversal of the inhibition of bacterial spore germination and outgrowth by antibacterial agents

The effects of some antibiotics and other antibacterial agents on the germination and outgrowth of Bacillus subtilis are described. The antibiotics tested (benzylpenicillin, cephalothin, chloramphenicol, minocycline, actinomycin D and mitomycin C) had no effect on germination but inhibited outgrowth to varying degrees. Of the preservative-type compounds, phenol and cresol acted predominantly at the germination stage, whereas the quaternary ammonium compound, benzalkonium chloride (BZK), and the organomercurial, phenylmercuric nitrate, inhibited out-growth but not germination. The inhibitory effect of phenol was reversed by membrane filtration, but not by 2% polysorbate 80, whereas BZK appeared to be tightly bound to the cells and could not be removed by filtration.

A stochastic model of bacterial spore germination.

A stochastic approach is utilized to develop a model equation capable of describing the time course of germination in a sample of bacterial spores. The time required by a spore to complete the change characteristic of germination consists of an initial interval of no change followed immediately by the duration of the change itself. The experimental basis of the proposed model is the observation that each of these time intervals is distributed over a range of values in a spore sample. Mixed continuous and discrete probabilities are employed in arriving at an average single-spore germination curve which, to a different scale, describes the sample in time.

A stochastic model of bacterial spore germination

A stochastic approach is utilized to develop a model equation capable of describing the time course of germination in a sample of bacterial spores. The time required by a spore to complete the change characteristic of germination consists of an initial interval of no change followed immediately by the duration of the change itself. The experimental basis of the proposed model is the observation that each of these time intervals is distributed over a range of values in a spore sample. Mixed continuous and discrete probabilities are employed in arriving at an average single-spore germination curve which, to a different scale, describes the sample in time.

Zinc release and the sequence of biochemical events during triggering of Bacillus megaterium KM spore germination.

Zinc release is the first quantitatively significant event detected during the triggering of Bacillus megaterium KM spore germination. Of the total spore Zn2+ pool 25% is released from non-heat-activated spores within 4 min of triggering germination. During this period only 10% of the spore population becomes irreversibly committed to germinate. The investigation of a putative role for Zn2+ in the germination trigger mechanism has established a relationship between the rate and extent of Zn2+ release and the stimulation of spore germination by heat activation. Furthermore, a correlation can be demonstrated between the extent of zinc release from spore populations and the time required to obtain 50% commitment of these populations to germinate over a wide temperature range. These findings have been used to expand a recently published model for the triggering of bacterial spore germination.

Bacillus megaterium spore protease: purification, radioimmunoassay, and analysis of antigen level and localization during growth, sporulation, and spore germination.

The protease which initiates the massive protein degradation early in bacterial spore germination has been purified from Bacillus megaterium spores. The enzyme has a molecular weight of 160,000 and contains four apparently identical subunits, but only the tetramer is enzymatically active. A radioimmunoassay has been developed for this enzyme and has been used to show that the protease is absent from growing cells, but appears early in sporulation within the developing forespore. In contrast, the protease antigen disappears rapidly during spore germination, in parallel with the loss in enzyme activity.

Inhibition and Control of Bacterial Spore Germination

Factors affecting germination of bacterial spores as well as chemical inhibition of germination are reviewed. Current theories on the nature of initiation of the germination process are also presented. Transformation of a dormant bacterial spore into a metabolically active vegetative cell involves a myriad of complex biochemical events of which the “trigger reaction” is thought to be the prime event. The ability to control or prevent this complex sequence of events from occurring by manipulation of environmental factors or use of chemical inhibitors has been the objective of numerous research endeavors. A thorough understanding of these factors is important to both maintenance and future development of an adequate, safe and wholesome food supply.

Covalent structure of protein A. A low molecular weight protein degraded during germination of Bacillus megaterium spores.

The complete covalent structure of Protein A, a protein degraded during bacterial spore germination, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 21 and 22 to 61. The larger peptide was further cleaved into two fragments with either cyanogen bromide or by trypsin cleavage following arginine modification with cyclohexanedione. The peptides derived from cyanogen bromide fragmentation encompassed residues 22 to 53 and 54 to 61 while trypsin hydrolysis yielded overlapping fragments comprising residues 22 to 48 and 49 to 61. Automated sequenator analysis together with carboxypeptidase Y digestion of the intact protein and the peptide fragments provided data from which the following unique amino acid sequence was deduced. NH2-Ala-Asn-Thr-Asn-Lys-Leu-Val-Ala-Pro-Gly10-Ser-Ala-Ala-Ala-Ile-Asp-Gln-Met-Lys-Tyr20-Glu-Ile-Ala-Ser-Glu-Phe-Gly-Val-Asn-Leu30-Gly-Pro-Glu-Ala-Thr-Ala-Arg-Ala-Asn-Gly40-Ser-Val-Gly-Gly-Glu-Ile-Thr-Lys-Arg-Leu50-Val-Gln-Met-Ala-Glu-Gln-Gln-Leu-Gly-Gly60-Lys-COOH.

An autocatalytic model for bacterial spore germination

The sigmoidal response observed in kinetic studies of germinating bacterial spores, using nephelometric techniques and phase contrast microscope photometry, is analysed by postulating an autocatalytic process. A scheme A to B to C, where B catalyses the degradation of A, is applied to describe the response accurately by three rate constants. The autocatalytic model is compared with previous proposals for quantifying germination studies and for interpreting the effect of different experimental conditions on initiation of germination. Its effectiveness is demonstrated by quantifying published results for a germinating single spore determined by phase contrast microscopy, and nephelometric results for spore suspensions, including the effect of temperature on the rate of initiation of germination. Although developed for quantitative analysis of spore germination, the model is applicable to other autocatalytic phenomena. To assist the experimentalist, a simple accurate method for deriving the three constants specifying the sigmoidal characteristic is described.

The time dependence of bacterial spore germination

The absorbance of a suspension of germinating bacterial spores describes a sigmoidal time dependence. This behavior is accurately described by a model which considers that the spore must pass through an intermediate state before reaching its final, germinated state. By attributing to each of these three states a characteristic turbidity coefficient, it is possible to write an expression for the absorbance of the suspension at any time after initiation of germination. The transition between neighboring states is characterized by a probability coefficient which is a function of the experimental variables, but which is independent of time. It is thus possible to separate the underlying time dependence of germination itself, characterized by the changes of state, from the observed manifestation of this phenomenon.

The influence of high concentrations of carbon dioxide on the germination of bacterial spores.

The influence of carbon dioxide at 1–55 atm on the germination of Clostridium sporogenes, Clostridium perfringens and Bacillus cereus spores in a complex medium was studied. The germination studies at atmospheric pressure were done in the pH range 5.2–6.7. Controls at the same pH were done in 100% nitrogen. Carbon dioxide at atmospheric pressure (1 atm) inhibited the spore germination of B. cereus spores but strongly enhanced the germination rate of those of the clostridia. Spore germination of Cl. sporogenes and Cl. perfringens was inhibited completely at 10 atm and at 25 atm, respectively. The germination rate in carbon dioxide or nitrogen was generally higher at pH 6.7 than at 5.2–6.0.

Study of calcium dipicolinate release during bacterial spore germination by using a new, sensitive assay for dipicolinate.

The release of calcium and dipicolinic acid from spores of Bacillus megaterium KM during L-alanine-induced triggering of germination has been studied using a new, simple, and rapid assay for dipicolinic acid capable of detecting a concentration of 0.5 micron. The release of both calcium and dipicolinate started within seconds of exposure of the spores to L-alanine, thus preceding other measurable changes associated with germination. From the earliest times, the two substances were released in equimolar quantities, although later in germination calcium predominated.

Metabolism of Membrane Lipids during Bacterial Spore Germination and Outgrowth

Metabolism of Membrane Lipids during Bacterial Spore Germination and Outgrowth

Temperature–pH effect upon germination of bacterial spores

Using several kinds of criteria for the germination of bacterial spores, germination-pH curves were drawn for Bacillus subtilis spores observed at different temperatures. The experiments revealed that optimum pH for spore germination was markedly changed by changing the incubation temperature; the optimum pH for germination was 7.4 at 37 degrees C and 5.4 at 10 degrees C. A possible mechanism involved in this phenomenon is discussed.

Calcium Release and Germination of Bacterial Spores

Calcium Release and Germination of Bacterial Spores

Biochemical studies on germination of bacterial spores. II. The inhibitory mechanism of D-alanine on L-alanine-induced germination of Bacillus thiaminolyticus.

The initiating mechanism in the germination of Bacillus thiaminolyticus spores was studied with 14C-L-alanine. A characteristic pattern of incorporation of L-alanine into the spores was observed during the early stages of germination with two incorporation peaks, one occurred just after contact with L-alanine (first incorporation) and the other 5 min later (second incorporation). L-Glutamine, L-valine, or L-serine substituted for the incorporation of L-alanine during the first stage of germination. Although, L-alanine taken up during the first incorporation phase was extractable with trichloroacetic acid (TCA), that taken up during the second incorporation phase was not extractable. The distribution of radioactivity showed that incorporated L-alanine was located in the spore coat, mainly in the paracrystal fraction. The radioactive material which remained in the germination medium or was extractable from the spore coat fraction with TCA treatment or pronase digestion was identified as alanine. Significance of incorporation of L-alanine and its location in the spore in reference to the initiation of germination is discussed.

Biochemical Studies on Germination of Bacterial Spores

Biochemical Studies on Germination of Bacterial Spores

Deoxyribonucleic acid synthesis and deoxynucleotide metabolism during bacterial spore germination.

Deoxyribonucleic acid (DNA) synthesis during germination of Bacillus megaterium spores takes place in two stages. In stage I (0-55 min) DNA synthesis is slow and there is no detectable net synthesis, whereas in stage II (from 55 min on) the rate of synthesis is much faster and net DNA synthesis occurs. Deoxyribonucleotide pool sizes match the rates of DNA synthesis in stages I and II. The level of deoxyribonucleotide triphosphates is not correlated with the level of deoxyribonucleotide kinases, but rather with that of ribonucleotide reductase activity.

Germination of Bacterial Spores by Calcium Chelates of Dipicolinic Acid Analogues

Abstract Calcium salts of analogues of dipicolinic acid were substituted for calcium dipicolinate in tests of germination of spores of Bacillus megaterium ATCC 10778. Only 4H-pyran-2,6-dicarboxylic acid was as active as dipicolinic acid. 3-Methyldipicolinic acid and 4-methyl-4H-pyran-2,6-dicarboxylic acid were active but the germination proceeded less rapidly. In the presence of a threshold concentration (0.020 m) of calcium dipicolinate, calcium salts of pyrimidine-2,4-dicarboxylate, pyrazine-2,6-dicarboxylate, 4-hydroxydipicolinate, and furan-2,5-dicarboxylate also showed activity. A hypothesis is proposed for mobilization of native calcium dipicolinate of dormant spores during germination, by way of a dimerization like that exhibited in crystals of calcium dipicolinate trihydrate and the isostructural calcium pyrandicarboxylate trihydrate. The compounds 4-bis(2-hydroxyethyl)iminopyridine-2,6-dicarboxylic acid and ethyl ester, 3-methyl-2,6-distyryl-pyridine, 3-methylpyridine-2,6-dicarboxylic acid, and pyrimidine-2,4-dicarboxylic acid are reported for the first time. Calcium and proton association constants are given for various ligands.

Germination and heat resistance of bacterial spores

Germination and heat resistance of bacterial spores

Increase of thymidine, thymidylate and deoxycytidine kinase activities during germination of bacterial spores

1. 1. Very low activities of thymidine kinase (ATP: thymidine 5′-phosphotransferase, EC 2.7.1.21), thymidylate kinase (ATP: 5′-thymidylic acid phosphotransferase) and deoxycytidine kinase (ATP: deoxycytidine 5′-phosphotransferase) were found in crude extracts from bacterial spores as compared with those from vegetative cells. This was repeatedly confirmed by applying various extraction methods. 2. 2. Activities of thymidine kinase and deoxycytidine kinase increased during germination of spores simultaneously with the initiation of DNA synthesis, but the increase of thymidylate kinase activity was delayed. 3. 3. The correlation between the increase of kinase activities and the rate of DNA synthesis was seen in spores germinated in starved as well as in complete conditions. This correlation, however, was not held in germination of heavily irradiated spores, suggesting that the formation of the kinases is under different control from that for DNA polymerization in cells. 4. 4. The increase in the kinase activities was inhibited by 100 μg/ml of chloramphenicol, indicating de novo formation of the kinases. By delayed addition of chloramphenicol the kinases were distinguished from early proteins synthesized during the first 30–80 min of germination. 5. 5. The possibilities that an inhibitor of thymidine kinase is present in spores and that a thymidine kinase activator is present in vegetative cells were eliminated.