Analysis of the Pla enzymatic activity using a fluorometric assay

The Pla activity was evaluated using a fluorometric assay as previously described [46, 47]. Y. pestis strains were grown on HIB agar at 26°C for 36 h, replated to fresh HIB, and incubated at either 26°C or 37°C for 24 h. Bacteria were harvested from the plates, suspended in PBS and adjusted to optical densities (OD₆₀₀) of 0.1 (5 × 10⁷ cfu/ml) and 0.05 (2.5 × 10⁷ cfu/ml) for the cultures grown at 26°C and 37°C, respectively. The bacterial titers were verified by plating the suspensions on HIB agar in 10-fold dilutions. The assay was conducted with 50 μl of bacterial suspension containing 2.5 μg of the fluorescently labeled hexapeptide substrate DABCYL-Arg-Arg-Ile-Asn-Arg-Glu (EDANS)-NH₂ [48] in the black 96-well microplate (Costar Corning Inc., Corning, NY) in quadruplicates. The kinetics of substrate cleavage by the Pla expressed on the surface of Y. pestis cells was measured every 10 min for 6 h at 37°C by using a BioTek Synergy HT reader (BioTek Instruments Inc., Winooski, VT) at excitation/emission wavelength of 360 nm/460 nm.