Another possibility is that there are other, as yet

unannotated proteins that play a role in a putative flagellar system in C. pneumoniae. For example, along with the FliH/FliI complex that is formed in other bacteria, another protein, FliJ, which is a general chaperone, is believed to be involved in this complex [39, 44]. FliJ has not been identified in C. pneumoniae. In the absence of a genetic manipulation system for the chlamydiae, direct evidence for the role of these flagellar proteins remains elusive. The fact that FliI is enzymatically active and forms complexes in vitro with other flagellar proteins, all of which are present in all other chlamydiae sps. studied to date, suggests that these proteins play an important role in chlamydial replication or survival. Further PD173074 supplier studies using heterologous systems and genetic complementation could help to decipher the exact role of these flagellar proteins in chlamydiae. Methods Talazoparib in vitro Expression Plasmids C. pneumoniae CWL029 (VR1310:ATCC) (GenBank accession # AE001363) was the strain used to isolate genomic DNA for cloning and protein expression. Full length fliI, Cpn0859, cdsL, copN, Cpn0322, and fragments of flhA, fliF, and fliI were amplified from CWL029 using AttB-containing primers (Gateway; Invitrogen).

The amplified products were cloned into pDONR201 (Gateway; Invitrogen) to generate pENT vectors. The pENT vectors were then {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| used in LR reactions (Gateway; Invitrogen) to produce pEX vectors containing the genes of interest. We used either pEX17 (N terminal His tag) or pEX15 (N terminal GST tag) vectors for our protein expression. All constructs were confirmed by sequencing at the Molecular Biology Facility at McMaster University. To identify protein interactions we utilized the bacterial-2-hybrid Methane monooxygenase system [39]. Genes of interest were

cloned into either pT18 or pT25 plasmids, each of which expresses a different fragment of adenylate cyclase. When these two plasmids are co-transformed, expressing the protein of interest fused to the adenylate cyclase fragment, any interaction between the two proteins results in production of cAMP. Increases in cAMP results in an increase in the β-galactosidase gene that can be monitored using β-galactosidase activity assays. pT18 and pT25 were digested with KpnI (New England Biolabs) as well as genes amplified from CWL029 (fliI, flhA, fliF, cdsL, Cpn0322, copN) that had a KpnI site designed into the primers. Ligation was performed overnight at 16°C using T4 Ligase (Invitrogen) and the resulting mixture was used to transform E. coli XL-1 cells and transformants were selected on 100 μg/μL ampicillin and 34 μg/μL chloramphenicol (Luria Bertani) LB plates. Plasmids were prepared using the GenElute Plasmid Miniprep Kit (Sigma). Protein Expression All constructs were expressed in E. coli Rosetta pLysS. Expression plasmids were used to transform E.