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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 conditioned supernatants from ΔgerQ or wild type spores.(A) Wild type or ΔgerQ B. cereus spores were treated with 0.2 mM inosine. Conditioned supernatants were collected 30 min post-inosine addition. Fresh ΔgerQ B. cereus spores were germinated in the supernatants obtained from wild type (▪) or ΔgerQ (•) spores. ΔgerQ B. cereus spores were also treated with 0.2 mM inosine (○) and 0.2 mM inosine supplemented with 20 µM alanine (□). (B) Wild type and ΔgerQ B. cereus supernatants were individually prepared as described above. Fresh wild type B. cereus spores were resuspended in conditioned supernatants from wild type (▪) or ΔgerQ (•) B. cereus spores. Wild type B. cereus spores were also treated with 0.2 mM inosine (○).
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pone-0006398-g006: B. cereus spore germination in the presence of conditioned supernatants from ΔgerQ or wild type spores.(A) Wild type or ΔgerQ B. cereus spores were treated with 0.2 mM inosine. Conditioned supernatants were collected 30 min post-inosine addition. Fresh ΔgerQ B. cereus spores were germinated in the supernatants obtained from wild type (▪) or ΔgerQ (•) spores. ΔgerQ B. cereus spores were also treated with 0.2 mM inosine (○) and 0.2 mM inosine supplemented with 20 µM alanine (□). (B) Wild type and ΔgerQ B. cereus supernatants were individually prepared as described above. Fresh wild type B. cereus spores were resuspended in conditioned supernatants from wild type (▪) or ΔgerQ (•) B. cereus spores. Wild type B. cereus spores were also treated with 0.2 mM inosine (○).

Mentions: The GerI and GerQ receptors of B. cereus are required for efficient germination in the presence of inosine [13], [14]. We found that B. cereus spores containing a deletion in the GerQ receptor gene (ΔgerQ spores) did not germinate in the presence of inosine as the sole germinant (Fig. 6A). However, ΔgerQ spores germinated efficiently when inosine was supplemented with alanine or with conditioned media from germinated wild-type B. cereus spores. In fact, the germination kinetics of ΔgerQ spores obtained with conditioned media were similar to those acquired with inosine and alanine (Fig. 6A). Our results are consistent with findings showing that ΔgerQ spores germinate normally when inosine is supplemented with alanine [13], [14]. These results indicate that the responsiveness to primary (inosine) and secondary (alanine) germinants is not compromised in ΔgerQ spores, and that these spores germinate normally in the presence of both germinants.


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 conditioned supernatants from ΔgerQ or wild type spores.(A) Wild type or ΔgerQ B. cereus spores were treated with 0.2 mM inosine. Conditioned supernatants were collected 30 min post-inosine addition. Fresh ΔgerQ B. cereus spores were germinated in the supernatants obtained from wild type (▪) or ΔgerQ (•) spores. ΔgerQ B. cereus spores were also treated with 0.2 mM inosine (○) and 0.2 mM inosine supplemented with 20 µM alanine (□). (B) Wild type and ΔgerQ B. cereus supernatants were individually prepared as described above. Fresh wild type B. cereus spores were resuspended in conditioned supernatants from wild type (▪) or ΔgerQ (•) B. cereus spores. Wild type B. cereus spores were also treated with 0.2 mM inosine (○).
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pone-0006398-g006: B. cereus spore germination in the presence of conditioned supernatants from ΔgerQ or wild type spores.(A) Wild type or ΔgerQ B. cereus spores were treated with 0.2 mM inosine. Conditioned supernatants were collected 30 min post-inosine addition. Fresh ΔgerQ B. cereus spores were germinated in the supernatants obtained from wild type (▪) or ΔgerQ (•) spores. ΔgerQ B. cereus spores were also treated with 0.2 mM inosine (○) and 0.2 mM inosine supplemented with 20 µM alanine (□). (B) Wild type and ΔgerQ B. cereus supernatants were individually prepared as described above. Fresh wild type B. cereus spores were resuspended in conditioned supernatants from wild type (▪) or ΔgerQ (•) B. cereus spores. Wild type B. cereus spores were also treated with 0.2 mM inosine (○).
Mentions: The GerI and GerQ receptors of B. cereus are required for efficient germination in the presence of inosine [13], [14]. We found that B. cereus spores containing a deletion in the GerQ receptor gene (ΔgerQ spores) did not germinate in the presence of inosine as the sole germinant (Fig. 6A). However, ΔgerQ spores germinated efficiently when inosine was supplemented with alanine or with conditioned media from germinated wild-type B. cereus spores. In fact, the germination kinetics of ΔgerQ spores obtained with conditioned media were similar to those acquired with inosine and alanine (Fig. 6A). Our results are consistent with findings showing that ΔgerQ spores germinate normally when inosine is supplemented with alanine [13], [14]. These results indicate that the responsiveness to primary (inosine) and secondary (alanine) germinants is not compromised in ΔgerQ spores, and that these spores germinate normally in the presence of both germinants.

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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