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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Mol Microbiol. 2011 Mar 14;80(3):641–654. doi: 10.1111/j.1365-2958.2011.07608.x

Figure 4.

Figure 4

Cac0903 and Cac3319 autophosphorylation and phosphotransfer to Spo0A. (A) Cac0903 and Cac3319 were incubated with [γ-32P]-ATP in the absence (autophosphorylation) or presence (phosphate transfer) of C. acetobutylicum Spo0A (CaSpo0A) for the times indicated. The minor band (Cac3319*) at 29 kDa in lanes containing Cac3319 is a truncated form of the protein that retains both autophosphorylation and phosphotransfer activity. The first two lanes in each panel are controls showing autophosphorylation activity of B. subtilis KinA (BsKinA) and phosphotransfer to B. subtilis Spo0F (BsSpo0F), which is known to dimerise (BsSpo0FD). (B) Verification of interaction specificity. The 30-min control reactions shown establish the specificity of the interaction between the clostridial kinases and Spo0A. (Note: Spo0A from B. subtilis (BsSpo0A) or C. acetobutylicum was equally effective as phosphoacceptor.) (C) Kinetics of phosphotransfer to Spo0A. Phosphotransfer to CaSpo0A was monitored following labelling of Cac0903 and Cac3319 with [γ-32P]-ATP for 30 min. CaSpo0A was added at zero time together with unlabelled ATP and samples were removed after the indicated periods of time. Quantified band intensities are given in the lower two panels: CaSpo0A (squares), Cac0903 (circles), and Cac3319 (triangles). The sizes of the proteins are as follows: BsKinA (69 kDa), BsSpo0F (14 kDa), BsSpo0FD (28 kDa), BsSpo0A (29.5 kDa), Cac0903 (53 kDa), Cac3319 (53 kDa), CaSpo0A (34 kDa).