The intended mutation sequence was overhung at the 5′ end of the downstream fragment. For the convenience of manipulation, BamHI recognition sequence was engineered at the 5′ end of the Selleck VX 770 upstream fragment, and HindIII at the 3′ end of the downstream fragment. The two fragments were then phosphorylated, treated with BamHI or HindIII, and inserted into pBBR1MCS to generate pZX series plasmids (Table 1). All mutants were confirmed by DNA sequencing. Protein expression analysis of FlbD and the FliX alleles Overnight cultures of C. crescentus were transferred to fresh PYE media Sirtuin activator inhibitor at a ratio of 1 to 10 (v/v) and were allowed to grow at 31°C until mid-log phase. Culture biomass was measured as optical density
at 600 nm (OD600), normalized, and was subject to 14% (w/v) SDS-PAGE. After electrophoresis, protein profiles were transferred to nitrocellulose membranes and were detected using anti-FliX or anti-FlbD antibodies purified with affinity columns (AminoLink® Plus Immobilization Kit, Thermo Fisher Scientific Inc., Rockford, IL, USA). Measurement of the transcription of flagellar genes The pZX serial plasmids bearing various fliX mutants were introduced into the wild-type strain LS107 or the ΔfliX stain JG1172 via conjugation, along with the reporter genes fliF-lacZ or fliK-lacZ. β-Galactosidase learn more activity was measured as described previously . Co-immunoprecipitation
(co-IP) Cells in middle log stage were harvested, normalized, and treated with 5 mg/ml lysozyme. The clear cell extract was incubated with Agarose-Protein A beads (Roche Applied Science, Indianapolis, IN, USA) to eliminate non-specific associated proteins. The pre-cleared cell lysate was then incubated overnight with Agarose-Protein A-anti-FlbD complexes prepared as instructed by the manufacturer. After extensive Casein kinase 1 washing, the bead complexes were spun down, resuspended in SDS-PAGE sample buffer and were subjected to electrophoresis followed by immunoblotting with anti-FliX antibodies. Results FlbD forms stable in vivo complex with FliX Previous experiments have shown that FliX and FlbD interact in a two-hybrid
assay , FliX can be precipitated from cell extracts of Caulobater by anti-FlbD antibodies, and that FliX regulates FlbD-activated transcription in vitro . In order to gain further understanding of the physical recognition between the two and to find out whether there are other proteins associated with FliX-FlbD complex, we performed an affinity pull-down experiment in which cell extracts of Caulobacter were treated with sepharose beads coated with histidine-tagged wild-type FliX. Cellular proteins that physically associated to FliX were then retrieved from the bead complexes and resolved by electrophoresis (Figure 1). Five major bands with corresponding molecular weights of approximately 70, 60, 48, 44, and 19 kilodaltons were observed.