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Bacillus cereus spores release alanine that synergizes with inosine to promote germination.

Dodatko T, Akoachere M, Muehlbauer SM, Helfrich F, Howerton A, Ross C, Wysocki V, Brojatsch J, Abel-Santos E - PLoS ONE (2009)

Bottom Line: We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores.Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine.We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT

Background: The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and findings: We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions: We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.

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Germination rates as a function of inosine and spore concentrations.(A) Wild-type B. cereus spores were germinated with increasing inosine concentrations (•). B. cereus spores were also germinated in conditioned supernatants collected 30 min after exposure to increasing inosine concentrations (○). T1/2 values were calculated for each one of the spore sample germinated in inosine and conditioned buffer and plotted against the initial inosine concentration. T1/2 values correspond to the time it takes to reach half-maximal values. (B) B. cereus spores were resuspended at increasing optical densities (OD580) and germinated in the presence of 0.2 mM inosine. After 30 min, conditioned supernatants were isolated from the germinated spores. Fresh spore aliquots were resuspended in the conditioned supernatants to an optical density of 1. Germination curves were monitored as described above. T1/2 values were calculated from each spore sample germinated in conditioned buffer and plotted against the initial spore optical densities.
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pone-0006398-g002: Germination rates as a function of inosine and spore concentrations.(A) Wild-type B. cereus spores were germinated with increasing inosine concentrations (•). B. cereus spores were also germinated in conditioned supernatants collected 30 min after exposure to increasing inosine concentrations (○). T1/2 values were calculated for each one of the spore sample germinated in inosine and conditioned buffer and plotted against the initial inosine concentration. T1/2 values correspond to the time it takes to reach half-maximal values. (B) B. cereus spores were resuspended at increasing optical densities (OD580) and germinated in the presence of 0.2 mM inosine. After 30 min, conditioned supernatants were isolated from the germinated spores. Fresh spore aliquots were resuspended in the conditioned supernatants to an optical density of 1. Germination curves were monitored as described above. T1/2 values were calculated from each spore sample germinated in conditioned buffer and plotted against the initial spore optical densities.

Mentions: To determine conditions that promote germination, B. cereus spores were germinated at different spore densities, or in the presence of increasing inosine concentrations. Conditioned supernatants collected 30 min post-germination were added to fresh spores and T1/2 values were determined. T1/2 values represent the time point when the optical density has reached 50% of its final value. As expected, germination T1/2 times decreased with increasing inosine concentrations (Fig. 2A). Similarly, the potency of conditioned supernatants increased when they were harvested from spores germinated at increasing spore concentrations as indicated by decreased T1/2 values (Fig. 2B).


Bacillus cereus spores release alanine that synergizes with inosine to promote germination.

Dodatko T, Akoachere M, Muehlbauer SM, Helfrich F, Howerton A, Ross C, Wysocki V, Brojatsch J, Abel-Santos E - PLoS ONE (2009)

Germination rates as a function of inosine and spore concentrations.(A) Wild-type B. cereus spores were germinated with increasing inosine concentrations (•). B. cereus spores were also germinated in conditioned supernatants collected 30 min after exposure to increasing inosine concentrations (○). T1/2 values were calculated for each one of the spore sample germinated in inosine and conditioned buffer and plotted against the initial inosine concentration. T1/2 values correspond to the time it takes to reach half-maximal values. (B) B. cereus spores were resuspended at increasing optical densities (OD580) and germinated in the presence of 0.2 mM inosine. After 30 min, conditioned supernatants were isolated from the germinated spores. Fresh spore aliquots were resuspended in the conditioned supernatants to an optical density of 1. Germination curves were monitored as described above. T1/2 values were calculated from each spore sample germinated in conditioned buffer and plotted against the initial spore optical densities.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712684&req=5

pone-0006398-g002: Germination rates as a function of inosine and spore concentrations.(A) Wild-type B. cereus spores were germinated with increasing inosine concentrations (•). B. cereus spores were also germinated in conditioned supernatants collected 30 min after exposure to increasing inosine concentrations (○). T1/2 values were calculated for each one of the spore sample germinated in inosine and conditioned buffer and plotted against the initial inosine concentration. T1/2 values correspond to the time it takes to reach half-maximal values. (B) B. cereus spores were resuspended at increasing optical densities (OD580) and germinated in the presence of 0.2 mM inosine. After 30 min, conditioned supernatants were isolated from the germinated spores. Fresh spore aliquots were resuspended in the conditioned supernatants to an optical density of 1. Germination curves were monitored as described above. T1/2 values were calculated from each spore sample germinated in conditioned buffer and plotted against the initial spore optical densities.
Mentions: To determine conditions that promote germination, B. cereus spores were germinated at different spore densities, or in the presence of increasing inosine concentrations. Conditioned supernatants collected 30 min post-germination were added to fresh spores and T1/2 values were determined. T1/2 values represent the time point when the optical density has reached 50% of its final value. As expected, germination T1/2 times decreased with increasing inosine concentrations (Fig. 2A). Similarly, the potency of conditioned supernatants increased when they were harvested from spores germinated at increasing spore concentrations as indicated by decreased T1/2 values (Fig. 2B).

Bottom Line: We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores.Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine.We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT

Background: The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and findings: We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions: We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.

Show MeSH
Related in: MedlinePlus