Functional consequences of amino acid substitutions to GerVB, a component of the Bacillus megaterium spore germinant receptor

Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, United Kingdom

^* To whom correspondence should be addressed. Email: gc301{at}cam.ac.uk.

	Abstract

The extreme metabolic dormancy and resistance properties of spores formed by members of the Bacillus and Clostridium genera are lost upon exposure to a variety of small molecule germinants. Germinants are known to interact in an as yet undefined manner with cognate receptor complexes that reside in the inner-membrane that surrounds the spore protoplast. The receptor itself is a complex of at least three proteins, and in this study we identify amino acid residues, predicted to lie in loop regions of GerVB on the exterior aspect of the membrane, that influence the Bacillus megaterium spore germination response. Three consecutive residues adjacent to putative transmembrane (TM) domain 10 were demonstrated to mediate to varying degrees the proline germinative response, while also influencing germination in response to leucine, glucose and inorganic salts, suggesting that this region may be part of a ligand binding pocket. Alternatively, substitutions in this region may affect the conformation of associated functionally important TM regions. Leucine and KBr mediated germination was also influenced by substitutions in other outer loop regions. These observations, when considered with accompanying kinetic analyses that demonstrate cooperativity between germinants, suggest that binding sites for the respective germinants are in close spatial proximity in the receptor, but do not overlap. Additionally, proline recognition was conferred to a chimeric receptor when TM regions associated with the putative binding loop were present, indicating that residues in TM9 and/or TM10 of GerVB are also of functional importance in the proline induced germinative response.