FliI hydrolyzes ATP in a linear, time- and dose-dependant manner at a rate of 0.15 ± .02 μmol min-1 mg-1. This rate is typical of other secretion ATPases such as CdsN, EscN, or FliI from other bacterial species [16, 41, 42]. The optimal Selleck 4SC-202 pH for FliI ATPase activity is 8.0, which is the same as that for other flagellar ATPases . Extreme low or high pH greatly reduced the activity, possibly due to protein denaturation. Also, the enzyme activity peaked at a temperature of 37°C and declined substantially beyond that. Although the formation of higher-order complexes was not explored
here, other flagellar ATPases are thought to form a hexameric complex . The presence of three flagellar genes in chlamydiae is intriguing since chlamydiae are thought to be non-motile and not to possess flagella. FliF, FlhA and FliI alone do not contain all the necessary APR-246 components for a functional flagella or secretion apparatus, however, a rudimentary basal body or pore complex could be formed by these three components. It is known that the most rudimentary flagellar structure that can be assembled is the MS ring, which consists of only the FliF protein . We have shown that
these proteins interact with one another (FliI, FlhA and FliF), most likely at the inner membrane of C. pneumoniae. The interaction between FliI and FlhA is mediated by the N-terminal 150 amino acids of FliI and appears to be specific since it is not disrupted by high salt (500 mM). Only the cytoplasmic domain of FlhA (amino acids 308-583) was utilized in the GST pull-down, suggesting that any protein selleck chemicals interactions that occur are within this region. Protein interaction studies with the full length FlhA protein
are Parvulin difficult due to the presence of seven transmembrane domains rendering full length FlhA insoluble and making this portion of the protein unable to bind to soluble flagellar components. Since FlhA is known to interact with soluble components of the flagellar apparatus in other bacteria, it is expected that the cytoplasmic domain mediates an interaction with FliI . FliF is known to form the MS ring in flagellated bacteria, and is one of the first components of the flagellar basal body to be incorporated into the membrane [26, 29]. We detected an interaction of the C-terminal 70 amino acids of FliF with the cytoplasmic domain of FlhA. These interactions were also stable in 500 mM NaCl, suggesting that the interaction is specific. We did not, however, detect any interaction between FliI and FliF, suggesting that any interaction between those two components may be mediated through the action of another protein, possibly FlhA In C. pneumoniae, Cpn0859 is encoded directly downstream of the ATPase, which led us to explore any interactions Cpn0859 may have with other flagellar proteins.