Anaerobic Culture of Treponema denticola

T. denticola ATCC 35405 was anaerobically grown in Oral Bacterial Growth Medium (OBGM) containing 10% heat-inactivated rabbit serum at 37°C^{23 (link),30 ,31 (link)}. For semisolid medium, 0.7% low-melting-point SeaPlaque agarose was incorporated into the OBGM medium, and the plates were poured after inoculating a bacterial suspension into the medium at 37°C^{24 (link)}. Escherichia coli TOP10 strain (Invitrogen, Carlsbad, CA) was used for routine plasmid constructions and preparations; an E. coli dam⁻/dcm⁻ strain (New England BioLabs, Ipswich, MA) was used to prepare unmethylated plasmids, and the E. coli BL21 Star™ (DE3) strain (Invitrogen) was used for preparing recombinant proteins. Unless noted, E. coli strains were cultured in lysogeny broth (LB) supplemented with appropriate concentrations of antibiotics. The fliK mutation, which results in PH production^{14 (link)}, was originally constructed in ATCC strain 33520, and was transferred by electroporation and erythromycin selection into ATCC 35405. This strain is designated as TdFliK. Description, growth, and construction of the B. burgdorferi ΔfliK mutant, here named B. burgdorferi FliK, have been previously described^{12 (link)}.