<p>Heterologous overproduction of Spo0A-6xHis and Spo0A-DBD-6xHis in <i>E. coli</i> Rosetta(DE3) pLysS. M = molecular weight marker, numbers indicate hours after induction with 1 mM IPTG. P = metal affinity purified protein. Lysates were separated on a 12% SDS-PAGE. <b>B </b><i>and </i><b>C.</b> Immunoblot detection of Spo0A in total cell lysates of a <i>C. difficile spo0A</i> mutant (CT::spo0A) and a wild type strain (630Δerm). Times indicated range from early exponential (3 h post inoculation) to late stationary growth phase (48 h post inoculation). Sample volumes were corrected for OD600 to ensure loading of similar amounts of total cell lysate in each lane. For details see Materials and Methods. M = molecular weight marker. <b>B.</b> ECL+ detection. <b>C.</b> Fluorescent detection. Y-axes show peak volumes normalized to values at 48 hours post inoculation (closed diamonds; left axis) and optical density readings at 600 nM (open squares; right axis). Inset shows the blot on which the curve is based. Vertical dashed line indicates the moment Spo0A levels increase sharply (6 hours post inoculation).</p
