Figure 2.

Experimental approach for assaying E. coli secretion of HlyA chimeric proteins bearing the C-termini of putative T1SS substrates in an HlyBD-dependent manner. E. coli BL21 (DE3) cells, which express TolC but not HlyB or HlyD, were transformed with pLG575 to constitutively express HlyB and HlyD and thereby functionally reconstitute the T1SS. E. coli with or without pLG575 were transformed with plasmids that express IPTG-inducible chimeric HlyA proteins having their 60 C-terminal residues (amino acids 964–1024) replaced with the 60–70 C-terminal residues of each O. tsutsugamushi Ank or control protein. The E. coli cultures were induced and centrifuged, and the resulting cell pellets and filter-sterilized supernatants were analyzed by Western blot using HlyA antibody to detect HlyA-fusion proteins that the bacteria expressed and secreted into the medium.