, 2007) using primers HGFPF and HGFPR (Table 1). The DNA sequences coding for eGFP and PilACt were fused using overlapping PCR (Sambrook & Russell, 2001), and primers HGA-1, 2, 3 and 4 (listed in Table 1). All three constructs have a 6× Panobinostat His tag at the N-terminus of the proteins of interest to facilitate purification. Each protein was expressed overnight at 16 °C as previously described (Li et al., 2005), and the cells were harvested and lysed. The lysate was

loaded onto a 5-mL HisTrap-HP column (GE Healthcare) and eluted with a linear gradient of 0–0.5 M imidazole in elution buffer (20 mM Tris, 0.5 mM NaCl, pH 8.0); the total volume of buffer equaled 20-column volumes. To remove the His-tag, 1/1000 volume of Turbo3C protease

(2 mg mL−1 stock; Accelagen) was added to the pooled fractions and incubated at 4 °C overnight. The digested sample was passed over a 5-mL HisTrap-HP column and the flow-through was collected, containing the proteins without the His tag. Pooled proteins following His-tag removal were concentrated and loaded onto a Hiload 16/60 Superdex 200 pg column (GE Healthcare) equilibrated in SEC buffer (20 mM Tris/pH 8.0, 100 mM NaCl). Peak fractions were pooled and concentrated to 4 mg mL−1. SDS-PAGE and Western blots were performed following standard procedures (Harlow, 1988). Primary polyclonal anti-PilA antibody (Li et al., 2005) was used at a 1 : 10 000 dilution. Primary polyclonal anti-eGFP antibody (Fisher) was used at a 1 : 2000 dilution. Anti-rabbit horseradish peroxidase-conjugated Oligomycin A ic50 secondary antibody (Pierce) was used at a 1 : 10 000 dilution. Cyclin-dependent kinase 3 Blots were developed using the Supersignal West Pico chemiluminescence reagent (Pierce). Images were obtained with the ChemiDoc XRS system (Bio-Rad). A pilus precipitation assay was performed as previously described (Li et al., 2003). Cell-surface pili/pilin were sheared off from 1010 SW504 cells by vigorous vortexing for 20 min and centrifuged for 5 min to remove the cell pellet. Protein-free EPS was isolated from DK1622 and quantified as previously described (Chang & Dworkin, 1994; Li et al., 2003). The isolated pili/pilin

and purified proteins (final concentration 0.2 mg mL−1 for both) were incubated with either MOPS buffer or purified EPS (final concentration 0.5 mg mL−1) at 32 °C for 1 h. The mixtures were pelleted by centrifugation at 10 000 g for 10 min. The supernatants were discarded, and the pellets were resuspended in 80 μL of 1% SDS followed by boiling with protein-loading dye (final concentration 1×) for SDS-PAGE and Western blotting. A PASCAL 5 CLSM (Zeiss, Germany) equipped with a 40× oil-immersion objective (Plan-Neofluar/NA 1.3) was employed to analyze M. xanthus submerged biofilms and fruiting bodies. Excitation at 488 nm with an argon laser in combination with a 505–530-nm bandpass emission filter was used for imaging eGFP.