Haemolytic activity of G. anatis culture supernatants and expression of GtxA. (A) Growth and haemolytic activity of cell-free culture supernatant of G. anatis 12656-12. An overnight culture was diluted 1:100 and growth (cell density measured by absorbance at 600 nm) and haemolytic activity (HA) in cell-free culture supernatant were recorded. The haemolytic activity of supernatant diluted 100-fold in BHI is shown. Extracellular proteins for Western blotting (Fig. 1B) were harvested in parallel. The experiment was repeated three times, the level of haemolytic activity varied but the relative pattern was consistent. (B) Levels of GtxA determined by Western blotting with ApxI-antiserum. Supernatants were harvested at the indicated time points, concentrated 100-fold as described in Materials and Methods and separated by SDS-PAGE in a 3–8% gel prior to blotting. Extracellular proteins from ΔgtxA were harvested at OD600 = 2 (lane marked ΔgtxA). WC = whole cell lysate from wild-type, the cells were harvested 5 h after inoculation. Size markers are indicated on the left. The experiment was repeated with the same result.