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Metabolism and the triggering of germination of Bacillus megaterium. Use of L-[3H]alanine and tritiated water to detect metabolism.

ABSTRACT: L-[2,3-3H]Alanine was used to probe for metabolism of alanine during triggering of germination of spores of Bacillus megaterium KM. No detectable incorporation of label into any compound, including water, was found, indicating that any metabolism involving the alanine germinant must be at a very low rate and also that alanine racemase is absent from spores of this strain. Spores were germinated in 3H2O to find if any of the many metabolic reactions causing irreversible incorporation of 3H into reaction products took place during triggering of germination. No incorporation was detected until 2-3 min after addition of germinants. It is therefore concluded that a wide variety of metabolic routes, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and amino acid metabolism are either not involved in the reactions causing the triggering of germination or operate at an extremely low rate during this process.

SUBMITTER: Scott IR

PROVIDER: S-EPMC1185957 | biostudies-other | 1978 Aug

REPOSITORIES: biostudies-other

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Metabolism and the triggering of germination of Bacillus megaterium. Concentrations of amino acids, organic acids, adenine nucleotides and nicotinamide nucleotides during germination.

Project description:A considerable amount of evidence suggests that metabolism of germinants or metabolism stimulated by them is involved in triggering bacterial-spore germination. On the assumption that such a metabolic trigger might lead to relatively small biochemical changes in the first few minutes of germination, sensitive analytical techniques were used to detect any changes in spore components during the L-alanine-triggered germination of Bacillus megaterium KM spores. These experiments showed that no changes in spore free amino acids or ATP occurred until 2-3 min after L-alanine addition. Spores contained almost no oxo acids (pyruvate, alpha-oxoglutarate, oxaloacetate), malate or reduced NAD. These compounds were again not detectable until 2-3 min after addition of germinants. It is suggested, therefore, that metabolism associated with these intermediates is not involved in the triggering of germination of this organism.

| S-EPMC1185956 | biostudies-other

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

Project description: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.

| S-EPMC1153977 | biostudies-other

Spore germination mediated by Bacillus megaterium QM B1551 SleL and YpeB.

Project description:Previous work demonstrated that Bacillus megaterium QM B1551 spores that are null for the sleB and cwlJ genes, which encode cortex-lytic enzymes (CLEs), either of which is required for efficient cortex hydrolysis in Bacillus spores, could germinate efficiently when complemented with a plasmid-borne copy of ypeB plus the nonlytic portion of sleB encoding the N-terminal domain of SleB (sleB(N)). The current study demonstrates that the defective germination phenotype of B. megaterium sleB cwlJ spores can partially be restored when they are complemented with plasmid-borne ypeB alone. However, efficient germination in this genetic background requires the presence of sleL, which in this species was suggested previously to encode a nonlytic epimerase. Recombinant B. megaterium SleL showed little, or no, activity against purified spore sacculi, cortical fragments, or decoated spore substrates. However, analysis of muropeptides generated by the combined activities of recombinant SleB and SleL against spore sacculi revealed that B. megaterium SleL is actually an N-acetylglucosaminidase, albeit with apparent reduced activity compared to that of the homologous Bacillus cereus protein. Additionally, decoated spores were induced to release a significant proportion of dipicolinic acid (DPA) from the spore core when incubated with recombinant SleL plus YpeB, although optimal DPA release required the presence of endogenous CLEs. The physiological basis that underpins this newly identified dependency between SleL and YpeB is not clear, since pulldown assays indicated that the proteins do not interact physically in vitro.

| S-EPMC3957701 | biostudies-literature

Crystallization and preliminary X-ray analysis of an alanine dehydrogenase from Bacillus megaterium WSH-002.

Project description:Alanine dehydrogenase (L-AlaDH) from Bacillus megaterium WSH-002 catalyses the NAD?-dependent interconversion of L-alanine and pyruvate. The enzyme was expressed in Escherichia coli BL21 (DE3) cells and purified with a His6 tag by Ni²?-chelating affinity chromatography for X-ray crystallographic analysis. Crystals were grown in a solution consisting of 0.1 M HEPES pH 8.0, 12%(w/v) polyethylene glycol 8000, 8%(v/v) ethylene glycol at a concentration of 15 mg ml?¹ purified protein. The crystal diffracted to 2.35 Å resolution and belonged to the trigonal space group R32, with unit-cell parameters a = b = 125.918, c = 144.698 Å.

| S-EPMC3729178 | biostudies-literature

Role of the gerA operon in L-alanine germination of Bacillus licheniformis spores.

Project description:BACKGROUND: The genome of Bacillus licheniformis DSM 13 harbours three neighbouring open reading frames showing protein sequence similarities to the proteins encoded from the Bacillus subtilis subsp. subtilis 168 gerA operon, GerAA, GerAB and GerAC. In B. subtilis, these proteins are assumed to form a germinant receptor involved in spore germination induced by the amino acid L-alanine. RESULTS: In this study we show that disruption of the gerAA gene in B. licheniformis MW3 hamper L-alanine and casein hydrolysate-triggered spore germination, measured by absorbance at 600 nm and confirmed by phase contrast microscopy. This ability was restored by complementation with a plasmid-borne copy of the gerA locus. Addition of D-alanine in the casein hydrolysate germination assay abolished germination of both B. licheniformis MW3 and the complementation mutant. Germination of both B. licheniformis MW3 and the gerA disruption mutant was induced by the non-nutrient germinant Ca2+-Dipicolinic acid. CONCLUSIONS: These results demonstrate that the B. licheniformis MW3 gerA locus is involved in germination induced by L-alanine and potentially other components present in casein hydrolysate.

| S-EPMC3359204 | biostudies-literature

L-alanine-induced germination in Bacillus licheniformis -the impact of native gerA sequences.

Project description:BACKGROUND: L-alanine, acting through the GerA receptor, was recently found to be an efficient germinant in Bacillus licheniformis ATCC14580/DSM13. RESULTS: In this study, we show that several of 46 examined B. licheniformis strains germinate remarkably slower than the type strain when exposed to L-alanine. These strains are not necessarily closely related, as determined by MLST (multi-locus sequence typing). Three of the slow-germinating strains were further examined in order to see whether nucleotide substitutions in the gerA sequences were responsible for the slow L-alanine germination. This was performed by complementing the transformable type strain derivate MW3?gerAA with gerA variants from the three slow-germinating strains; NVH1032, NVH1112 and NVH800. CONCLUSIONS: A wide selection of B. licheniformis strains was evaluated for L-alanine-induced germination efficiency. Our results show that gerA substitutions could only partially explain why spores of some B. licheniformis strains responded slower than others in the presence of L-alanine.

| S-EPMC4021175 | biostudies-literature

Role of uricase in the triggering of germination of Bacillus fastidiosus spores.

Project description:The likelihood that uric acid was the only compound capable of triggering germination of Bacillus fastidiosus spores was reinforced by the finding that ureidoglycollic acid, urea, NH4Cl, 2,8-dihydroxypurine and a combination of L-alanine and O-carbamoyl-D-serine were ineffective as germinants. Uric acid-triggered germination of B. fastidiosus was prevented by a range of inhibitors that also inhibited uricase activity in dormant spore extracts. O2 uptake during germination started immediately after addition of uric acid, possibly as a consequence of the oxidation of uric acid by the enzyme uricase. Germination showed a dependence on uric acid concentration, with a relatively high Km (4-5 mM). During the first 10 min of germination of heat-activated spores there was no detectable change in the number of spore-cortex reducing groups, indicating that selective cortex hydrolysis is not involved in the trigger mechanism of germination of B. fastidiosus. On the basis of the results, a model is proposed in which re-initiation of uricase activity is the mechanism by which B. fastidiosus spores are triggered to emerge from the dormant state.

| S-EPMC1145173 | biostudies-other

The use of inhibitors to identify early events during Bacillus megaterium KM spore germination.

Project description:The germination response of spores of Bacillus megaterium KM, as measured by loss of A600, is more than 95% inhibited by 1 mM-HgCl2. Two Hg2+-sensitive sites (referred to as 'sites I and II') have been identified during germination. Site I represents a pre-commitment event and can be protected from HgCl2 by 50 mM-D-alanine, whereas site II represents a post-commitment event and is not D-alanine-protectable. At 1 mM-HgCl2, 25% of the spore population becomes committed to germinate, but an A600 loss of less than 5% occurs. In this system, loss of heat resistance was associated with commitment, whereas selective cortex hydrolysis, release of pyridine-2,6-dicarboxylic acid, Zn2+ and soluble peptidoglycan, as well as loss of refractility, were identified as post-commitment events. The commitment event was reversibly inhibited by several proteinase inhibitors and a membrane bulking agent. A model of spore germination based on these results is presented.

| S-EPMC1147068 | biostudies-other

U(VI) adsorption in water by sodium alginate modified <i>Bacillus megaterium</i>.

Project description:The surface of Bacillus megaterium was modified by coating sodium alginate. The modified B. megaterium before and after adsorption were characterized by SEM, FTIR and XPS. The effects of pH, reaction time, initial U(VI) concentration and adsorbent dosage on the adsorption of U(VI) by the modified B. megaterium were studied by batch adsorption experiments. The adsorption process was studied by pseudo-first-order kinetics and pseudo-second-order kinetic models, Langmuir and Freundlich isotherms. The results showed that the maximum adsorption capacity of U(VI) was 74.61 mg g-1 under the conditions of pH 5.0, adsorbent 0.2 g l-1, 30°C and initial U(VI) concentration of 15 mg l-1. The adsorption process accords with pseudo-first-order kinetics and Langmuir isotherm. The adsorption capacity of U(VI) by the modified B. megaterium was still higher than 80% after five times of desorption and reuse experiments. In conclusion, the sodium alginate modified B. megaterium was an ideal material for U(VI) biosorption.

| S-EPMC8074630 | biostudies-literature

Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM.

Project description:Two peptidoglycan-lytic enzyme activities were isolated from spores of Bacillus megaterium KM. Surface-bound lytic enzyme was extracted from dormant spores and hydrolysed a variety of peptidoglycan substrates including isolated spore cortex, but did not cause refractility changes in permeabilized spores. Germination-specific lytic enzyme activity appeared early in germination and had minimal activity on isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus species. The germination-specific lytic enzyme was shown to be a heat-sensitive 29 kDa protein with maximal activity at pH 6.5. It catalysed post-commitment muramic acid delta-lactam synthesis and displayed an inhibitor profile similar to that for post-commitment A600 loss. The relationship of the germination-specific enzyme to a recently proposed model of spore germination is discussed.

| S-EPMC1147743 | biostudies-other

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