Generation of Tetracycline and Rifamycin Resistant Recombinants.

The are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture , allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only (.) naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of to exchange DNA and especially antibiotic resistance, is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination . With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant , which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced . Here, we describe the protocol with which RifR strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in , a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed genome plasticity by revealing variable, apparently non-specific areas of recombination.