Studies on Determination of Germinant Specificity of Bacillus Spore Germinant Receptors
Abstract
Bacterial endospores are major environmental contaminants and some are vectors leading to food spoilage, foodpoisoning and infectious diseases. Discovery and development of novel agents that promote highly efficient germination of spores in populations could facilitate spore decontamination efforts. Specific nutrient germinants that trigger spore germination are recognized by their cognate germinant receptors (GRs); for example, major Bacillus subtilis GRs recognize L-alanine/L-valine, or a combination of L-asparagine/D-glucose/D-fructose/K+. GRs typically consist of three subunits referred to as A, B and C, however, the precise function of each GR subunit and how they recognize and bind to germinants have remained elusive. Our recent work on the conserved N-terminal domain (NTD) of the A subunit of the Bacillus megaterium GerK3 GR (GerK3ANTD) has shed new lights on this mystery. The crystal structure of GerK3ANTD revealed that GerK3ANTD possesses two distinct globular subdomains separated by a deep cleft, a fold with strong homology to substrate-binding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, structure-guided mutagenesis and germination analyses support a model in which the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in germination. Motivated by our recent discovery, the objectives of this project are to apply a multi-disciplinary approach including microbiology, structural biology and protein engineering to: 1) learn molecular details of germinant recognition by GRs that trigger germination of Bacillus spores; and 2) use this information to generate B. subtilis spores with GRs recognizing novel germinants. To achieve our goals, we have developed the following specific aims: 1. To explore GR and germinant exchangeability: constructing B. subtilis spores possessing GRs from Bacillus cereus (GerI) and B. megaterium (GerU) and assessing their capabilities to trigger germination by their respective germinants. 2. To explore the structural mechanism of germinant recognition: identifying and characterizing the germinant binding site in GRs by determining the structures of the NTD of B. cereus GerI A subunit (GerIANTD) alone and in complex with its cognate germinant inosine. 3. To explore novel germinant binding activity: generating mutated and engineered B. subtilis spores with modified germinant specificity and affinity by comparing the structure of GerIANTD bound to inosine with those of the bacterial purine nucleoside receptor and the D-xylose binding protein complexed with their respective ligands. Ultimately, this proposed work is expected to advance our understanding of the germinant recognition mechanism underlying the GR-mediated spore germination pathways and unravel a general principal to design and generate spores that can serve as stable and easily deployed biosensors by responding to any desired small molecules.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 30, 2020
- Source ID
- W911NF2010034
Entities
People
- Peter Setlow
Organizations
- Army Contracting Command
- United States Army
- University of Connecticut Health Center