Abstract
Bacterial endospores can survive harsh environmental conditions and long-term dormancy in the absence of nutrients, but can rapidly germinate under favorable conditions. In the present study, we employed transposon sequencing (Tn-seq) to identify genes with previously uncharacterized roles in spore germination. Identified genes that encoded spore inner membrane proteins were chosen for study of defined mutants, which exhibited delayed germination in several assays in response to varying germinants. Significantly slowed release of DPA indicated that mutants were affected in Stage I of germination. Several mutants exhibited phenotypic traits consistent with failure of a GerA germinant receptor-mediated response, while others appeared to have a more general loss of response to varied germinants. Use of a gerA-lacZ transcriptional fusion and quantitative western blotting of GerAC allowed mutants to be classified based upon normal or decreased gerA transcription and normal or reduced GerA accumulation. Fourteen genes were identified to have newly described roles within Bacillus spore germination. A more complete understanding of this process can contribute to the development of better spore decontamination procedures.
Highlights
Bacterial endospores are capable of extended periods of dormancy while remaining resistant to a variety of chemical and physical decontamination measures [1]
Seeking to identify additional genes that contribute to spore germination, transposon sequencing (Tn-seq) was used to reveal genes functioning in the early stages of germination
A library of magellan6x transposon insertions [15] was transformed into a B. subtilis wild type strain, PS832, that is highly efficient at spore formation and germination
Summary
Bacterial endospores are capable of extended periods of dormancy while remaining resistant to a variety of chemical and physical decontamination measures [1]. Dormant spores can rapidly germinate when in a suitable environment, returning to a vegetative state [2, 3]. These factors allow endospores produced by certain species of Bacillus and Clostridium to excel as human pathogens, act as potential bioterrorism agents, and contribute to significant food contamination events [4, 5]. Preservation of dehydration of the metabolically inactive spore core is the greatest factor in spore resistance properties and maintenance of spore dormancy [1]. The accumulation of small molecule solutes within the core, such as calcium dipicolinic acid (DPA), contribute to spore dehydration and resistance properties [1]
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