Title

Characterization of protein cross-links in the coats of Bacillus subtilis spores and analysis of spore coat rinds resistant to degradative enzymes*

Date of Completion

January 2008

Keywords

Biology, Microbiology|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The Bacillus subtilis spore coat is a multilayered, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. GerQ, the spore coat protein examined in this study is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an &egr;-(γ-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In this work, we have identified the residues in GerQ that are essential for the transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization. ^ In addition, we have also examined the proteins of the spore coat on a more global level. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a "rind" that looks like coat material remains intact. These rinds are transparent, spherical or hemispherical structures that appear devoid of internal contents. Atomic force microscopy and chemical analyses have now shown that: (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers; and (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion. ^ *Portions of this dissertation were previously published. See appendix for copyright permission.^