Bioluminescence dynamics in single germinating bacterial spores reveal metabolic heterogeneity.
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ABSTRACT:
SUBMITTER: Frentz Z
PROVIDER: S-EPMC7536063 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bacterial spores are metabolically dormant, resistant to microbicides, and vectors of food spoilage and diseases, while germinated spores are easy to kill. Consequently, understanding germination mechanisms may facilitate the development of "germinate-to-eradicate" strategies. Spores germinate in response to many compounds (called germinants). They can also retain the memory of a germinant exposure, such that a second exposure triggers more efficient germination, but how is not clear. A recent high-profile paper [Science (2022) 378:43] suggested that increasing spore electrochemical potential is how memory is "stored" based on measurements of Bacillus subtilis spores' accumulation of the dye thioflavin-T after germinant exposure. Indeed, we found that wild-type spores of three Bacillus and one Clostridioides species all exhibited this early thioflavin-T accumulation during nutrient pulses. However, our data indicate that inferring spores' electrochemical potential from thioflavin-T accumulation is problematic. We found that B. subtilis spores lacking their proteinaceous coat exhibited memory but did not accumulate thioflavin-T prior to germinant addition or during nutrient pulses. Furthermore, wild-type Bacillus spores germinating with dodecylamine, which also elicits memory, showed no thioflavin-T accumulation. Finally, we found that thioflavin-T accumulation by a germinating spore is outside the spore core at early stages but inside the spore core as germination proceeds. These findings suggest that thioflavin-T accumulation during the early stages of germination is due to its binding to one or more protein in the spore coat rather than to changes in spores' electrochemical potential; thus, thioflavin-T is not a potentiometric dye for the study of spore memory of germinant pulses. IMPORTANCE Bacillus and Clostridium spores cause food spoilage and disease because of spores' dormancy and resistance to microbicides. However, when spores "come back to life" in germination, their resistance properties are lost. Thus, understanding the mechanisms of spore germination could facilitate the development of "germinate to eradicate" strategies. One germination feature is the memory of a pulsed germinant stimulus leading to greater germination following a second pulse. Recent observations of increases in spore binding of the potentiometric dye thioflavin-T early in their germination of spores led to the suggestion that increasing electrochemical potential is how spores "remember" germinant pulses. However, new work finds no increased thioflavin-T binding in the physiological germination of Coatless spores or of intact spores germinating with dodecylamine, even though spore memory is seen in both cases. Thus, using thioflavin-T uptake by germinating spores to assess the involvement of electrochemical potential in memory of germinant exposure, as suggested recently, is questionable.
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Project description:Bacterial spores can remain dormant for decades, yet harbor the exceptional capacity to rapidly resume metabolic activity and recommence life. Although germinants and their corresponding receptors have been known for more than 30 years, the molecular events underlying this remarkable cellular transition from dormancy to full metabolic activity are only partially defined.Here, we examined whether protein phospho-modifications occur during germination, the first step of exiting dormancy, thereby facilitating spore revival. Utilizing Bacillus subtilis as a model organism, we performed phosphoproteomic analysis to define the Ser/Thr/Tyr phosphoproteome of a reviving spore. The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism, and spore-specific determinants. Quantitative comparison of dormant and germinating spore phosphoproteomes revealed phosphorylation dynamics, indicating that phospho-modifications could modulate protein activity during this cellular transition. Furthermore, by mutating select phosphorylation sites located within proteins representative of key biological processes, we established a functional connection between phosphorylation and the progression of spore revival.Herein, we provide, for the first time, a phosphoproteomic view of a germinating bacterial spore. We further show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.
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Project description:Background: Bacterial spores can remain dormant for decades, yet harbor the exceptional capacity to rapidly resume metabolic activity and recommence life. Although germinants and their corresponding receptors have been known for more than 30 years, the molecular events underlying this remarkable cellular transition from dormancy to full metabolic activity are only partially defined. Results: Here, we examined whether protein phospho-modifications occur during germination, the first step of exiting dormancy, thereby facilitating spore revival. Utilizing Bacillus subtilis as a model organism, we performed phosphoproteomic analysis to define the Ser/Thr/Tyr phosphoproteome of a reviving spore. The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism and spore-specific determinants. Quantitative comparison of dormant and germinating spore phosphoproteomes revealed phosphorylation dynamics, indicating that phospho-modifications could modulate protein activity during this cellular transition. Furthermore, by mutating select phosphorylation sites located within proteins representative of key biological processes, we established a functional connection between phosphorylation and the progression of spore revival. Conclusions: In this study we provide for the first time a phosphoproteomic view of a germinating bacterial spore. We show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.
2015-08-27 | PXD002559 | Pride