Swarm agar assays TB swarm agar plates (1% bacto-tryptone, 0.8% NaCl; 0.35%
bacto-agar) containing 0.2% arabinose or 0.2% fructose, respectively, were inoculated with a single colony of E. coli MM500 or MM500 harbouring one of the plasmids pBAD-Ppr, pBAD-Pph, pBAD-PphH670A, pBADKdpE and pBAD, respectively. The plates were incubated for 6 hours at 37°C. Chemotaxis assay using a chemotactic chamber 2 ml minimal medium A (MMA) [56] containing an amino acid mixture (threonine, leucine, histidine, methionine), vitamin B1 (final concentration 10 μg/ml each), 200 μg/ml ampicillin and 0.2% fructose were inoculated with an overnight culture of E. coli MM500 or cells harbouring pBAD-Pph, pBAD-PphH670A, SC79 pBAD-KdpE or pBAD18, respectively. When the cultures reached an OD600 = 0.6 the cells were washed twice with MMA SBI-0206965 datasheet without sugar and finally either 0.2% arabinose to induce protein expression or 0.2% fructose (as a control) were added. The cultures were incubated for 60 min at
37°C. For the kinetic analysis the Bcl-2 inhibitor incubation times are indicated in Figure 3B. Again, the cells were washed twice with MMA without carbon source and were back diluted to an OD600 = 0.6. The chemotactic assays were performed as follows. 300 μl of the cell suspension were filled in each drilling of the chamber and a capillary containing either 2 μl 1 mM aspartate or 2 μl H2O as a control was placed into the channel between the two cylindrical compartments. The chamber was incubated at 37°C for 30 minutes. The outside of the capillary was washed Palbociclib extensively with sterile water and the content of the capillary was blown out and a dilution series was streaked on agar plates. After overnight incubation at 37°C the colonies were counted and the chemotactic inhibition
(CI) was calculated as the ratio of colonies of the water containing capillary to the colonies from the aspartate containing capillary. Therefore, a low CI indicates an undisturbed chemotactic response whereas a high CI reflects an inhibition of the E. coli chemotactic system. Expression and purification of Pph protein from inclusion bodies E. coli strain C41 [52] harbouring the plasmid pET16b-Pph were grown at 37°C in 1 l LB medium containing 200 μg/ml ampicillin. When cells reached the midlogarithmic phase, IPTG was added at a final concentration of 1 mM and the cells were grown for an additional 4 hours at 37°C. Then the cells were harvested by centrifugation. The resulting pellets were resuspended in 100 mM Tris-HCl pH 8.0, 150 mM NaCl (buffer W) and lysed by a French Press. Inclusion bodies were precipitated by centrifugation and resuspended in buffer W containing 0.5% N-lauroylsarcosine. The inclusion bodies were solubilized overnight at 4°C with gentle shaking.