Limits...
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.

Show MeSH
Enhanced germination rate of B. cereus spores germinated in conditioned supernatants.Wild-type B. cereus spores were germinated with a 0.2 mM inosine solution (•) or in conditioned supernatants containing 0.2 mM inosine (▪). B. cereus spores were also germinated with 0.2 mM inosine and 20 µM alanine (X).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2712684&req=5

pone-0006398-g001: Enhanced germination rate of B. cereus spores germinated in conditioned supernatants.Wild-type B. cereus spores were germinated with a 0.2 mM inosine solution (•) or in conditioned supernatants containing 0.2 mM inosine (▪). B. cereus spores were also germinated with 0.2 mM inosine and 20 µM alanine (X).

Mentions: We recently described that B. cereus 569 spores germinate with a time lag when inosine is used as the sole germinant [17]. This lag phase is significantly reduced and germination rates increase considerably when inosine is supplemented with alanine (1). Following the lag phase, B. cereus spores treated with inosine germinate with non-linear kinetics. We hypothesized that cofactors released from germinating spores during the lag phase enhance germination kinetics. To test this, we treated B. cereus 569 spores with 0.2 mM inosine, and collected supernatants 30 min post-inosine exposure. The conditioned supernatants derived from germinated spores were then added to fresh B. cereus spores. As shown in Fig. 1, conditioned supernatants collected from germinated spores significantly accelerated germination of fresh B. cereus spores. The lag phase was greatly shortened in the presence of conditioned supernatants, and the resulting germination kinetics resembled those obtained when 0.2 mM inosine was supplemented with 20 µM alanine (Fig. 1). Heat-treated (90°C for 15 min) or micro-filtrated (5 kDa MWCO) conditioned supernatants showed similar acceleration of the germination rate as untreated conditioned supernatants (data not shown). Together, these findings indicate that B. cereus spores release low molecular weight and heat-stable germination cofactors that promote inosine-mediated germination.


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)

Enhanced germination rate of B. cereus spores germinated in conditioned supernatants.Wild-type B. cereus spores were germinated with a 0.2 mM inosine solution (•) or in conditioned supernatants containing 0.2 mM inosine (▪). B. cereus spores were also germinated with 0.2 mM inosine and 20 µM alanine (X).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712684&req=5

pone-0006398-g001: Enhanced germination rate of B. cereus spores germinated in conditioned supernatants.Wild-type B. cereus spores were germinated with a 0.2 mM inosine solution (•) or in conditioned supernatants containing 0.2 mM inosine (▪). B. cereus spores were also germinated with 0.2 mM inosine and 20 µM alanine (X).
Mentions: We recently described that B. cereus 569 spores germinate with a time lag when inosine is used as the sole germinant [17]. This lag phase is significantly reduced and germination rates increase considerably when inosine is supplemented with alanine (1). Following the lag phase, B. cereus spores treated with inosine germinate with non-linear kinetics. We hypothesized that cofactors released from germinating spores during the lag phase enhance germination kinetics. To test this, we treated B. cereus 569 spores with 0.2 mM inosine, and collected supernatants 30 min post-inosine exposure. The conditioned supernatants derived from germinated spores were then added to fresh B. cereus spores. As shown in Fig. 1, conditioned supernatants collected from germinated spores significantly accelerated germination of fresh B. cereus spores. The lag phase was greatly shortened in the presence of conditioned supernatants, and the resulting germination kinetics resembled those obtained when 0.2 mM inosine was supplemented with 20 µM alanine (Fig. 1). Heat-treated (90°C for 15 min) or micro-filtrated (5 kDa MWCO) conditioned supernatants showed similar acceleration of the germination rate as untreated conditioned supernatants (data not shown). Together, these findings indicate that B. cereus spores release low molecular weight and heat-stable germination cofactors that promote inosine-mediated germination.

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