The cellular protein level of Pph was verified in parallel by SDS-PAGE and Westernblot analysis (data not shown). Taken together, the results strongly indicate that the Pph interferes with the chemotactic pathway in E. coli. Figure 3 E. coli cells expressing the Pph protein are unable to respond to aspartate. (A) The chemotactic response to aspartate of

E. coli MM500 cells expressing the various Pph-derived proteins was investigated with a chemotactic chamber. The chemotactic inhibition (CI) was calculated as described in Materials and Methods. The CI-value of cells grown in the presence of fructose (hatched columns) was about 0.35, whereas cells grown in the presence of arabinose and expressing the Pph or the Pph-H670A protein (white columns)

were calculated to 0.73 or 0.58, respectively. The error bars indicate AR-13324 molecular weight the standard deviations of three independent experiments. (B) E. coli cells with pBAD-Pph were incubated for the indicated times with 0.2% arabinose or 0.2% fructose, respectively, and their chemotactic response to aspartate was investigated in a chemotactic chamber. The chemotactic inhibition rate was calculated after induction either with fructose (hatched columns) or learn more arabinose (white columns) for the indicated time points. The error bars indicate the standard deviations of three independent experiments. The protein expression profiles (inlet) were analysed at 10 min (lanes 1, 2), 40 min (lanes 3, 4) and 60 min (lanes 5, 6) after induction. The odd numbered lanes are the non-induced controls. The Pph protein interacts with Rc-CheW in an ATP-dependent manner To investigate in detail with which components of the Rc chemotactic pathway Ppr and its C-terminal histidine kinase domain Pph interact, the binding to Rc-CheW or Rc-CheA was analyzed. First, purified R. centenaria CheW (Rc-CheW) containing an N-terminal his-tag and in vitro translated and radiolabelled Pph protein were tested for interaction by matrix-assisted coelution. The Rc-CheW protein as

a bait was heterologously expressed in E. coli C41 and purified by immobilized metal affinity chromatography (Cu-IMAC). The prey protein Pph was translated in vitro and labelled with [35S]-L-methionine (Figure 4A, lanes 1 and 4). To avoid unspecific binding of Pph to the PIK3C2G Cu Sepharose, a buffer containing 50 mM imidazole was used. In the assay, both the bait and prey protein were mixed, incubated overnight at 37°C and then bound to the Cu DMXAA Sepharose column. After intensive washing the bound protein was eluted, separated by SDS-PAGE and analysed by autoradiography. As shown in Figure 4A, the Pph protein co-elutes in the elution fractions containing Rc-CheW (lane 6) whereas no Pph protein was detected in the elution fraction of the control without Rc-CheW (lane 3). The co-elution rate was calculated to 13% of the input Pph protein (lane 4).