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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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B. cereus spore germination in the presence of Ca-DPA.(A) Wild-type B. cereus spores were germinated in the presence of 0.2 mM inosine (□). B. cereus spores were also germinated in the presence of conditioned supernatants containing 0.2 mM inosine (+). Spores were also germinated with 0.2 mM inosine supplemented with 0.18 mM Ca-DPA (▪).
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pone-0006398-g004: B. cereus spore germination in the presence of Ca-DPA.(A) Wild-type B. cereus spores were germinated in the presence of 0.2 mM inosine (□). B. cereus spores were also germinated in the presence of conditioned supernatants containing 0.2 mM inosine (+). Spores were also germinated with 0.2 mM inosine supplemented with 0.18 mM Ca-DPA (▪).

Mentions: A release of DPA has been linked to increased germination efficiencies, presumably through the activation of cortex-lytic enzymes [22]–[25]. While B. cereus spores germinate in the presence of 60 mM extracellular calcium-DPA [23], [24], [25], the final DPA concentration in the conditioned medium of germinated B. cereus spores was only 0.18 mM [26]. To test whether released DPA and/or calcium could account for the enhanced germination kinetics observed in the presence of conditioned supernatants, we exposed spores to 0.2 mM inosine supplemented with Ca-DPA (Fig. 4). As a control, we germinated spores in the presence of inosine alone. The presence of 0.18 mM Ca-DPA did not accelerate inosine-mediated germination (Fig. 4), suggesting that DPA is not a co-germinant in this process.


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)

B. cereus spore germination in the presence of Ca-DPA.(A) Wild-type B. cereus spores were germinated in the presence of 0.2 mM inosine (□). B. cereus spores were also germinated in the presence of conditioned supernatants containing 0.2 mM inosine (+). Spores were also germinated with 0.2 mM inosine supplemented with 0.18 mM Ca-DPA (▪).
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Related In: Results  -  Collection

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pone-0006398-g004: B. cereus spore germination in the presence of Ca-DPA.(A) Wild-type B. cereus spores were germinated in the presence of 0.2 mM inosine (□). B. cereus spores were also germinated in the presence of conditioned supernatants containing 0.2 mM inosine (+). Spores were also germinated with 0.2 mM inosine supplemented with 0.18 mM Ca-DPA (▪).
Mentions: A release of DPA has been linked to increased germination efficiencies, presumably through the activation of cortex-lytic enzymes [22]–[25]. While B. cereus spores germinate in the presence of 60 mM extracellular calcium-DPA [23], [24], [25], the final DPA concentration in the conditioned medium of germinated B. cereus spores was only 0.18 mM [26]. To test whether released DPA and/or calcium could account for the enhanced germination kinetics observed in the presence of conditioned supernatants, we exposed spores to 0.2 mM inosine supplemented with Ca-DPA (Fig. 4). As a control, we germinated spores in the presence of inosine alone. The presence of 0.18 mM Ca-DPA did not accelerate inosine-mediated germination (Fig. 4), suggesting that DPA is not a co-germinant in this process.

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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