FIG 4.

(A) Western blotting of spore extracts probed with antiserum against mCherry (left) or BxpB (right). The blot was probed with anti-mCherry and then stripped and reprobed with anti-BxpB. The arrow indicates the position of the larger anti-mCherry-reactive species in the Sterne ΔbclA pMK4 bclA-NTD-mcherry lane. (B) Strategy for bclA-NTD-his₁₂ labeling of the BclA partner protein. The BclA NTD sequence (red bar represents residues 1 to 19, and black bar represents residues 20 to 35) with His₁₂ residues at the C terminus was expressed in B. anthracis. During assembly around the spore (20), a proteolytic cleavage event removes the N-terminal 19 residues, with the remaining NTD sequences being stably attached to a partner protein in the exosporium (represented by the blue circle). (C) Western blotting of spore extracts probed with antiserum against the His tag. The pGS4900 clones 1 and 2 refer to two independent isolates bearing the plasmid. The size of the BclA-NTD-His₁₂ protein is 5,287 daltons (Da) (3,290 Da when cleaved). A 35-kDa species was detected with the anti-His tag antiserum, and higher-MW SDS-resistant complexes are evident as well.