THE EXPRESSION OF THE PROTECTIVE ANTIGEN OF BACILLUS-ANTHRACIS IN BACILLUS-SUBTILIS

The expression of Bacillus anthracis protective antigen (PA) in B. sub tilis from the pag gene in pPA101-1 was explored in different genetic backgrounds in an attempt to identify opportunities to maximize expres sion. Introduction of AtxA, which positively regulates PA expression i n B. anthracis did not improve expression levels in the protease-defic ient strain WB600. Plasmid pPA101-1 was found to carry a deletion whic h created a new fusion point between vector and insert sequence, and w hich removed part of the AtxA binding site. The deletion map have occu rred as a consequence of recombination between TCTAT sequences within both the vector and insert. Host mutations could influence expression; PA levels from pPA101-1 are threefold higher in a ccpA mutant than in an otherwise isogenic parent, and eightfold higher in an abrB mutant. These data demonstrate that the introduction of mutations affecting c atabolite repression and growth phase regulation results in an increas e in the yield of PA in this host-vector system. Combining these mutat ions with a multiply protease-negative background could potentially al low further improvements in PA yield.