In chlamydiae,

the identity of other proteins (if they exist) that play important roles in the flagellar apparatus is currently pending, but it is possible that the flagellar apparatus, if it exists, is a hybrid structure of C. pneumoniae T3S and flagellar proteins. Another possibility is that flagellar proteins are involved in T3S, aiding in secretion of effector proteins or structural components. In Pseudomonas, there is evidence to support that flagellar assembly actually antagonizes the T3SS, suggesting a negative cross-regulation of the two systems [30]. No interaction between chlamydial T3S and flagellar components, however, has been reported to our knowledge. The protein interactions

that occur within the bacterial flagellar system have been characterized previously [29, 31, 32]. Genetic evidence, followed by direct biochemical assays, suggests an interaction of FlhA and FliF [33, Angiogenesis inhibitor 34]. The C-terminal end of FlhA, which is CHIR98014 manufacturer predicted to be cytoplasmic, is known to interact with the soluble components of the flagellar system such as FliI, FliH and FliJ [34, 35]. FliH acts as a negative regulator of the flagellar ATPase, FliI, and binds FliI as a homodimer, forming a trimeric (FliI)(FliH)2 complex [36–38]. FliJ, a second soluble component which interacts with FlhA, acts as a general chaperone for the flagellar system to prevent premature aggregation of export substrates in the cytoplasm, and also interacts with the FliH/FliI complex [39]. This complex of FliI/FliH/FliJ is believed to

be crucial for selection of export substrates and construction of the flagellar apparatus, although the proton motive force Osimertinib concentration could play a role in the actual secretion of flagellar proteins [28, 40]. In C. pneumoniae, FliH and FliJ have not been annotated in the genome. FliI, the putative C. pneumoniae flagellar ATPase ortholog, has significant amino acid similarity with both CdsN, the C. pneumoniae T3S ATPase, and FliI, the Salmonella flagellar ATPase, suggesting that it possesses enzymatic activity. Here we report an initial characterization of FliI, the flagellar ATPase, and show that it hydrolyzes ATP at a rate similar to that of its T3S ATPase paralog CdsN as well as orthologs in other bacteria [16, 41, 42]. We have also characterized the protein-interactions occurring between FliI, FliF and FlhA, demonstrating a direct interaction of FliI and FlhA, and FlhA and FliF. As well as interactions between the flagellar proteins, we have also characterized four novel interactions between the flagellar and T3S components. The role of these interactions in the chlamydial replication cycle is discussed. Results Sequence analysis of FliI, FlhA and FliF FliI (Cpn0858) is 434 amino acids in length with a predicted molecular mass of 47.5 kDa and a pI of 8.00.