Characterization and Reassembly of a Regular Array in the Cell Wall of Clostridium difficile GAI 4131

Abstract

The cell wall of Clostridium difficile GAI 4131 was revealed by electron microscopy to have an outer layer composed of a nearly square array and contained the two major proteins with molecular weights of 38kDa and 42kDa. The properties and reassembly of the two major proteins into the regular array were investigated. When the isolated cell walls were treated with hydrophobic bond-disrupting agents or a chelating agent specific for Ca²⁺, the two major proteins were effectively removed and the regularly arranged outer layer disappeared. The amino acid composition of the two major proteins differed from each other. The two major proteins also gave different peptide maps from each other upon proteolysis with Staphylococcus aureus V8 protease. The major proteins solubilized from the isolated cell walls with 8M urea or 4M guanidine hydrochloride could be reassembled into open-ended cylinders possessing the native regular pattern by dialysis against neutral buffer containing 5mM CaCl₂. The reassembled cylinders purified by centrifugation on a Percoll density gradient were composed of almost equal amounts of the 38kDa and 42kDa proteins and freed from the other proteins. These results suggest that the regular array in the outer cell wall layer is constructed from the two major cell wall proteins and requires Ca²⁺ for its assembly.