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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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Kinetics of auto-inducer release.(A) Wild-type B. cereus spores were germinated with 0.2 mM inosine supplemented with 0 or 1 mM D-cycloserine. Conditioned supernatants were collected at different time points post-inosine addition. Fresh B. cereus spores were resuspended in the conditioned supernatants that contained inosine and either 0 (•) or 1 (○) mM D-cycloserine and germination curves monitored as described above. T1/2 values were plotted against the time point of supernatant collection. (B) Wild-type B. cereus spores were germinated with 0.2 mM inosine supplemented with either 0 (•) or 1 mM D-cycloserine (○). The conditioned supernatants were collected at different intervals post-inosine addition and amino acids were derivatized with isobutyl groups. Total alanine concentration was determined by comparison with known alanine standards.
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pone-0006398-g005: Kinetics of auto-inducer release.(A) Wild-type B. cereus spores were germinated with 0.2 mM inosine supplemented with 0 or 1 mM D-cycloserine. Conditioned supernatants were collected at different time points post-inosine addition. Fresh B. cereus spores were resuspended in the conditioned supernatants that contained inosine and either 0 (•) or 1 (○) mM D-cycloserine and germination curves monitored as described above. T1/2 values were plotted against the time point of supernatant collection. (B) Wild-type B. cereus spores were germinated with 0.2 mM inosine supplemented with either 0 (•) or 1 mM D-cycloserine (○). The conditioned supernatants were collected at different intervals post-inosine addition and amino acids were derivatized with isobutyl groups. Total alanine concentration was determined by comparison with known alanine standards.

Mentions: We also determined the effect of increasing incubation times on the potency of the harvested supernatants on inosine-mediated germination of B. cereus spores. As expected, germination efficiency of harvested supernatants increased with incubation time: germination was most efficient using supernatant collected 30 min post-inosine exposure (Fig. 5A). No increase in germination rate was observed when conditioned media was collected within 5 min of inosine exposure. Taken together, the potency of conditioned media increased with inosine and spore concentrations, as well as with longer incubation times.


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)

Kinetics of auto-inducer release.(A) Wild-type B. cereus spores were germinated with 0.2 mM inosine supplemented with 0 or 1 mM D-cycloserine. Conditioned supernatants were collected at different time points post-inosine addition. Fresh B. cereus spores were resuspended in the conditioned supernatants that contained inosine and either 0 (•) or 1 (○) mM D-cycloserine and germination curves monitored as described above. T1/2 values were plotted against the time point of supernatant collection. (B) Wild-type B. cereus spores were germinated with 0.2 mM inosine supplemented with either 0 (•) or 1 mM D-cycloserine (○). The conditioned supernatants were collected at different intervals post-inosine addition and amino acids were derivatized with isobutyl groups. Total alanine concentration was determined by comparison with known alanine standards.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712684&req=5

pone-0006398-g005: Kinetics of auto-inducer release.(A) Wild-type B. cereus spores were germinated with 0.2 mM inosine supplemented with 0 or 1 mM D-cycloserine. Conditioned supernatants were collected at different time points post-inosine addition. Fresh B. cereus spores were resuspended in the conditioned supernatants that contained inosine and either 0 (•) or 1 (○) mM D-cycloserine and germination curves monitored as described above. T1/2 values were plotted against the time point of supernatant collection. (B) Wild-type B. cereus spores were germinated with 0.2 mM inosine supplemented with either 0 (•) or 1 mM D-cycloserine (○). The conditioned supernatants were collected at different intervals post-inosine addition and amino acids were derivatized with isobutyl groups. Total alanine concentration was determined by comparison with known alanine standards.
Mentions: We also determined the effect of increasing incubation times on the potency of the harvested supernatants on inosine-mediated germination of B. cereus spores. As expected, germination efficiency of harvested supernatants increased with incubation time: germination was most efficient using supernatant collected 30 min post-inosine exposure (Fig. 5A). No increase in germination rate was observed when conditioned media was collected within 5 min of inosine exposure. Taken together, the potency of conditioned media increased with inosine and spore concentrations, as well as with longer incubation times.

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