Figure 3.

YplA is an extracellular protein whose production requires expression of the flagellar regulon. (A) Concentrated whole-cell lysates and culture supernatants of strain JB580v grown in LB or T medium were examined for phospholipase activity by a radial diffusion assay. A positive reaction resulted in the formation of a precipitate emanating from the sample well in gels containing the artificial substrate Tween 80. Each well contained an equivalent of cells or supernatants from 1.5 ml culture at an OD₆₀₀ of 3.0. Row 1, culture supernatant after growth in T medium (Left), culture supernatant after growth in T medium and preheated to 90°C for 10 min (Center), and cell lysate after growth in T medium (Right). Row 2, culture supernatant after growth in LB (Left), culture supernatant after growth in LB and preheated to 90°C for 10 min (Center), and cell lysate after growth in T medium and preheated to 90°C for 10 min (Right). Row 3, cell lysates after growth in LB (Left), cell lysate after growth in LB and preheated to 90°C for 10 min (Center), and buffer control (Right). (B) Phospholipase activity of secreted YplA was measured for strain GY460 containing plasmid pVLT33 (vector control), pGY20 (ptac-flhDC from Y. enterocolitica), and pMG600 (ptac-flhDC from S. liquefaciens). Conditions of the assay were as described in A. Each well contained concentrated culture supernatant for each strain grown in T medium, with the inducer IPTG at a concentration of 0, 5, or 50 μM (indicated at the top). From top to bottom each well was loaded with an increased dilution of each sample.