GST-FliI migrated at approximately 73 kDa, its predicated molecular mass. Numbers refer to the eluted fraction. B: i) Time course of purified GST-FliI ATP hydrolysis (diamonds) and GST-CopN ATP hydrolysis as a negative control (squares). ii) Inorganic phosphate released at different concentrations of GST-FliI (diamonds) and GST-CopN as a negative control (squares) iii) GST-FliI ATPase activity at either 4°C, 16°C, 23°C, 37°C or 42°C. iv) GST-FliI ATPase activity at varying pH.

FlhA interacts with FliF FlhA is known to interact with the MS ring protein, FliF, in other flagellar systems [33, 34]. We explored the Doramapimod cell line interactions of these two proteins in C. pneumoniae. Two fragments of FliF were cloned and expressed as His-tagged proteins. His-FliF1-271 lacked the distal C-terminal 70 amino acids while His-FliF35-341 lacked

the N-terminal 35 amino acids. Each fragment contained only one selleck chemical of the two predicted TM regions. FliF1-271 migrated with an apparent molecular weight of 30 kDa, while His-FliF35-341 migrated at 34 kDa. FlhA was cloned and expressed as a soluble fragment with either a GST or His tag. FlhA308-583 encoded the C-terminal half of the protein, lacking the stretch of seven TM domains. Expression and detection of His-FlhA308-583 used as the bait check details protein in GST pull-down assays migrated at the expected molecular weight of 30 kDa. We used the bacterial-2-hybrid assay to test for interactions between FliF and FlhA. Full length FlhA interacted significantly with full length FliF, with a β-galactosidase activity of 847.2 ± 21.2 units of activity, as compared with a negative control value of 412.0 ± 82.4 units of activity (Table Resminostat 1). We next used GST pull-downs to confirm the interactions found by the bacterial-2-hybrid system and to determine the exact regions of the proteins mediating these interactions (Figure 3A). All protein complexes were washed with either low or high salt buffers containing 0.1% Triton X-100 to dissociate spurious protein-protein interactions. GST-FlhA308-583

co-purified with His-FliF35-341 but not His-FliF1-271, suggesting that the C-terminus of FliF (amino acids 271-341) is required for interactions with the cytoplasmic portion of FlhA. Table 1 Interaction between the flagellar proteins of C. pneumoniae using the Bacterial-2-hybrid System Plasmids β-Galactosidase Activity in units/mg bacteria Protein Functions Negative Control     pT18 + pT25 412.0 ± 82.4 pT18: Empty vector Positive Control:   pT25: Empty vector pT18-PknD + pT25-CdsD-FHA-2 996.3 ± 50.0 FliI: Putative flagellar ATPase Negative Interactions:   FliF: Putative flagellar MS ring protein pT18-FliI + pT25-FliF 396.4 ± 32.1 FlhA: Putative flagellar integral membrane pT18-FliF + pT25-Cpn0859 421.1 ± 25.9 protein pT18-FliI + pT25-Cpn0706 404.4 ± 19.5 Cpn0859: Hypothetical C. pneumoniae pT18-Cpn0706 + pT25-FlhA 443.0 ± 32.