The resulting cloned elementary bodies (EBs) were grown to high titers and were partially purified by centrifugation of lysates of infected cells through a 30% MD-Gastroview® pad (Mallinckrodt Inc. St Louis). Generation of recombinant

clones for complete genome sequence analysis Recombinants isolated for genome analysis were generated from two sets of crosses (Table 1). The first of these involved two parental strains; L2-434ofl and F(s)/70rif and the second was a three-way cross with the parental strains F(s)/70tet-rif, J/6276rif and L2-434ofl. Recombination experiments were conducted as previously described [5]. Briefly, crosses were performed in McCoy cells seeded in sets of individual shell vials. The monolayers SN-38 price were then infected with

different combinations of drug-resistant strains each at an MOI = 2, ensuring infections of cells with both strains. Cultures were incubated for 48 h post-infection in the absence of antibiotics and were then detached and lysed using a -80C/37C selleck inhibitor freeze-thaw cycle [5]. Potential recombinants were selected by inoculating 50 μl of the freeze-thaw lysates from each shell vial onto a new shell vial monolayer and overlaying with a medium containing antibiotics at 1/4 the MIC for each resistant parental strain. In the case of the three-way cross [F(s), J, L2], three different combinations of drug were applied to the infected monolayers (MOI = 2). These combinations included ofloxacin/rifampicin, Etomidate ofloxacin/tetracycline, and ofloxacin/rifampicin/tetracycline. Generation of recombinant chlamydial strains for analysis of recombination hot spots Multiple independent shell vials containing confluent McCoy cells were inoculated sequentially

with ofloxacin-resistant D/UW3Cx and rifampin-resistant L1/440/LN or L3/404/LN strains, and incubated 48 h in medium lacking antibiotics. Monolayers were lysed and used as inocula onto fresh McCoy cells at MOI = 1, and incubated in the presence of 4X the MIC of the drugs used for selection, rifampin and ofloxacin. These concentrations were previously determined to be sufficient to select for individual recombinant strains resistant to both drugs. Incubation of either parent in this combination and concentration of antibiotics at MOI = 1 never yielded a doubly resistant mutant parent. Chlamydial recombinants growing in this mixture of antibiotics were propagated and cloned by limiting dilution. Only a single recombinant progeny was collected from each lineage from a single original inoculated shell vial. DNA was harvested from these clones, and PCR primers were created that flanked regions of suspected recombination Nec-1s nmr hotspots identified by Srinivasan and colleagues [24]. The Phusion high fidelity DNA polymerase (New England Biolabs, Ipswich, MA) was used to generate PCR products from these regions, and these were sequenced at the Oregon State University Center for Genomics Research and Biocomputing.