In brief, overnight cultures were diluted 1:100 in 10 ml TB (10 g/l tryptone, 5 g/l NaCl, pH 7.0) containing appropriate antibiotics and inducers (Table 1). After growing at 34°C with 275 rpm to OD600≈0.45-0.5 cells were two times washed

in tethering buffer (10 mM KH2PO4/K2HPO4, 0.1 mM EDTA, 10 mM sodium lactate, 67 mM NaCl, 1 μM methionine, pH 7.0). To minimize growth and protein production, cells were subsequently incubated for at least 1 h at 4°C. FRAP Analyses and NF-��B inhibitor data processing For FRAP experiments cells were immobilized on (poly)L-lysine-coated coverslips for 5 min. Measurements were usually performed at 20°C (RT) or when indicated at 39°C. For that, slides were placed in a metal chamber connected to a water bath. Cells were visualized with the 63× oil objective of a laser-scanning confocal microscope (Leica TCS SP2). Compound C supplier Fluorescent cells were scanned by the 514 nm laser line of a 20 mW argon laser with 1-5% intensity and detected within 525-650 nm at 32-fold magnification. Regions of interest (ROIs) were bleached with two 0.336 s laser scans at 50% laser intensity using the same laser line. The following image series were recorded (Leica Confocal software, Version 2.61) by bidirectional scanning: one prebleach- and 10 postbleach Selleck Small molecule library images every 0.336

s, 10 postbleach images every 3 s and depending on protein 10-40 postbleach images every 30 s. Images were analyzed by using a custom-written plug-in [37] for ImageJ software, Version 1.34l (W. Rasband, National Institutes of Health, Bethesda, MD; http://​rsb.​info.​nih.​gov/​ij). For FRAP evaluation, the polar region was defined as 52 pixles, which is approximately Montelukast Sodium 20% of the average cell length. Fluorescence of the ROI was normalized two times: first to the fluorescence of the entire cell in the same image to compensate for gradual bleaching during scanning, second to the prebleach value of the ROI, to make different experiments comparable. To reduce variability that arises due to varying depth of bleaching, for experiments shown in Figure 1 and 3d

the value of the first post-bleach point was additionally subtracted and the curves were renormalized. Data were processed using KalaidaGraph software, Version 3.6 (Synergy Software). For data fitting in Figure 2, protein exchange at chemotaxis clusters can be treated as a combination of anomalous diffusion and an exponential decay with the characteristic exchange time τ obs and fit with the following equation: where F 0 accounts for the relative fluorescence intensity of free fluorescent protein after bleaching, F ∞ is the corresponding intensity after recovery, t 1/2 is half-time of recovery, α is the factor accounting for anomalous diffusion and C is the relative steady-state concentration of cluster-bound fluorescent protein [37].