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Bacillus subtilis

Spore germination and outgrowth

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germinationstate

8. Spore germination and outgrowth

Despite their dormancy and metabolic inactivity, spores can monitor changes in their surroundings and respond to these, resulting in spore germination. Similar to sporulation, the germination process consists of various stages marked by key morphological changes. Initiation of germination starts with the sensing of trigger molecules in the form of nutrients or peptidoglycan fragments and ends with shedding of the protective coat layers and outgrowth to a vegetative cell. Germination (Ger) receptor complexes in the inner membrane of the spore are known to play a crucial role in the initiation of germination through binding of nutrient germinants175. Alternatively, signals derived from surrounding growing cells, such as shed peptidoglycan fragments, also indicate improved conditions that support growth and are sensed by a Ser/Thr kinase called PrkC 176. Via an as of yet unknown mechanism, a signal to exit dormancy is subsequently transferred into the spore causing commitment to germination. Only minutes later the Ca-DPA depot is released from the spore core and replaced by water for rehydration. Then, germination proceeds through degradation of the cortex and further swelling of the spore core. Metabolic activity is restored to allow for outgrowth into a vegetative state. For a more detailed description of the process of germination we refer to several reviews and reports available in literature 161, 160, 177.

 

Although dormant spores were long thought to be devoid of mRNA and mRNA has been shown to be degraded upon dormancy of the spore 174, still transcripts of at least 23 genes were identified in dormant spores of B. subtilis 180. All of these genes are expressed under the control of σG during late-stage sporulation and code for small acid-soluble proteins or proteins of unknown function. The reason for the presence of these transcripts is unknown, although it is possible that their degradation provides building blocks for de novo RNA synthesis upon germination and outgrowth180.

Exit from dormancy starts with the synthesis of RNA and proteins while synthesis of DNA and chromosome replication do not occur until at least 30 minutes into the germination process 181. Similar to stage-directed gene expression during sporulation, gene transcription and protein production during outgrowth of the germinated spore also take place in an orderly fashion 180. So far, the exact cascade of events, regulatory mechanisms involved and the correlation between observed gene transcription and phenotypic development remain poorly understood. Nevertheless, a detailed study published in 2007 on gene transcription during several stages of spore outgrowth, offered a first overview on the activation of functional classes of genes during this phase in the B. subtilis life cycle 180. Upon germination, some of the first genes to be activated are those belonging to functional classes of transport, regulation of transcription, DNA repair and replication 180. Dependent on the nutrient availability in the environment, the germinated spore can undergo the first round of DNA replication approximately 30 minutes after initiation of germination. This stages marks the second phase in outgrowth. This phase is marked by gene transcription directed towards cell wall and cell membrane expansion, cell division and general stress responses 180 As the coat layers are subsequently shed from the outgrowing spore, cell morphology changes and the first round of chromosomal replication is completed. Gene clusters involved in metabolism as well as in cell motility are activated, allowing the cell to gradually re-enter a vegetative state 180 concluding the sporulation cycle of B. subtilis.

Sporulation cycle of Bacillus subtilis

 

 

 

Spore germination and outgrowth