MOLECULAR CHARACTERIZATION OF THE BACILLUS-ANTHRACIS MAIN S-LAYER COMPONENT - EVIDENCE THAT IT IS THE MAJOR CELL-ASSOCIATED ANTIGEN

Bacillus anthracis, the aetiological agent of anthrax, is a Gram-posit ive spore-forming bacterium, The cell wall of vegetative cells of B. a nthracis is surrounded by an S-layer. An array remained when sap, a ge ne described as encoding an S-layer component, was deleted. The remain ing S-layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escher ichia coil and shown to be contiguous to the sap gene. The EA1 amino a cid sequence, deduced from the eag nucleotide sequence, shows classica l S-layer protein features (no cysteine, only 0.1% methionine, 10% lys ine, and a weakly acidic pI). Similar to Sap and other Gram-positive s urface proteins, EA1 has three 'S-layer-homology' motifs immediately d ownstream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of the wild-type bacilli. However, EA1 was more tightly bound than Sap t o the bacteria. Electron microscopy studies and in vivo experiments wi th the constructed mutants showed that EA1 constitutes the main lattic e of the B. anthracis S-layer, and is the major cell-associated antige n.