TITLE: Calcium chloride-mediated transformation of different Chlamydia species
AUTHORS: Nadja Faessler, Kensuke Shima, Hanna Marti

[DESCRIPTION]
Summary
The genetic toolbox for the obligate intracellular bacterial species Chlamydia trachomatis and C. muridarum has rapidly expanded in recent years (Sixt and Valdivia, 2016; O’Neill et al., 2020; Fields et al., 2022), and has now extended to C. caviae, C. felis, C. pecorum, C. pneumoniae, C. psittaci and C. suis (Shima et al., 2018, 2020; Filcek et al., 2019; Marti et al., 2023; Faessler et al., In Preparation). This document comprises details for the design, construction, and transformation of various vectors into different Chlamydia species and was adapted from the original publication on calcium chloride-mediated transformation (Wang et al., 2011).


References:
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Faessler, N., Biggel, M., Jelocnik, M., Borel, N., and Marti, H. (In Preparation). Development of shuttle vector-based transformation systems for Chlamydia pecorum and Chlamydia caviae.

Fields, K. A., Bodero, M. D., Scanlon, K. R., Jewett, T. J., and Wolf, K. (2022). A Minimal Replicon Enables Efficacious, Species-Specific Gene Deletion in Chlamydia and Extension of Gene Knockout Studies to the Animal Model of Infection Using Chlamydia muridarum. Infection and Immunity 90, e00453-22. doi: 10.1128/iai.00453-22

Filcek, K., Vielfort, K., Muraleedharan, S., Henriksson, J., Valdivia, R. H., Bavoil, P. M., et al. (2019). Insertional mutagenesis in the zoonotic pathogen Chlamydia caviae. PLoS One 14, e0224324. doi: 10.1371/journal.pone.0224324

Marti, H., Biggel, M., Shima, K., Onorini, D., Rupp, J., Charette, S. J., et al. (2023). Chlamydia suis displays high transformation capacity with complete cloning vector integration into the chromosomal rrn-nqrF plasticity zone. Microbiol Spectr 11, e02378-23. doi: 10.1128/spectrum.02378-23

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Shima, K., Weber, M. M., Schnee, C., Sachse, K., Käding, N., Klinger, M., et al. (2020). Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci. mSphere 5, e00787-20. doi: 10.1128/mSphere.00787-20

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Wang, Y., Kahane, S., Cutcliffe, L. T., Skilton, R.J., Lambden, P. R., and Clarke, I. N. (2011). Development of a Transformation System for Chlamydia trachomatis: Restoration of Glycogen Biosynthesis by Acquisition of a Plasmid Shuttle Vector. PLoS Pathogens 7, e1002258. doi: 10.1371/journal.ppat.1002258

[BEFORE_START]
Before implementing this protocol in your laboratory, check the biosafety regulations of your
laboratory/country.

[STEPS]