In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum

Although the isolation of Treponema pallidum subsp. pallidum (T. pallidum) from a syphilis patient dates to 1912, for the duration of the 20^th century, this pathogen has remained an exceedingly difficult organism to study due to the lack of a system to support its viability in vitro. This limitation, in turn, has precluded the application of genetic engineering techniques via transformation and subsequent selection of T. pallidum transformants. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible for us to experiment with transformation and selection methods. Here we describe the approach that we adopted to successfully transform T. pallidum with foreign DNA and select the resulting recombinant strain using kanamycin. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Transformation of T. pallidum Support Protocol 1: Quantification of T. pallidum in suspensions using dark-field microscopy Support Protocol 2: Counting cells using a hemacytometer Basic Protocol 2: Selection, initial passaging, and expansion of transformed cultures Basic Protocol 3: Isolation of a clonal strain through limiting dilution.