Research Article: Identification of L-Valine-initiated-germination-active genes in Bacillus subtilis using Tn-seq

Date Published: June 14, 2019

Publisher: Public Library of Science

Author(s): Cameron V. Sayer, Bidisha Barat, David L. Popham, Marie-Joelle Virolle.


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.

Partial Text

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]. This dormant state is maintained by the inner spore membrane, which exists in a largely non-fluid state [6], and a thick layer of peptidoglycan termed the cortex [7]. Additionally, the accumulation of small molecule solutes within the core, such as calcium dipicolinic acid (DPA), contribute to spore dehydration and resistance properties [1].

The germination and return to growth of bacterial spores is an essential step in the initiation of several diseases and of some causes of food spoilage. This Tn-seq analysis identified 42 B. subtilis genes that had not previously been associated with germination but are required for a highly efficient germination response to L-Val. As the majority of proteins previously found to play major roles in germination are membrane-associated, fourteen of these genes, whose products had also been identified in studies of the spore membrane proteome [44, 45], were further characterized. Well-defined mutations in these genes caused significantly reduced responses to L-Val, and in some cases a decreased response to other nutrient germinants. Several of these strains also exhibited slowed vegetative growth; such a growth defect could certainly alter gene expression and progression through the sporulation process, potentially affecting the germination apparatus. Future work on the specific mechanism by which these mutations alter germination may reveal such effects. For all these mutants, the germination defect appears to largely be a slow initiation of germination rather than a specific slowing of a subsequent step in the germination process. The reduced percentage of spores within the population that do initiate germination appear to progress through Stages I and II of germination at a near normal pace; rates of OD loss and phase darkening are largely mirrored by rates of DPA and NAM release. This suggests that the genes under study play a role in the earliest steps of germination initiation.