Calcium chloride-mediated transformation of different Chlamydia species v1
Authors: Faessler Nadja , Shima Kensuke , Marti Hanna 
Published: July 2024

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: Bauler, L. D., and Hackstadt, T. (2014). Expression and Targeting of secreted Proteins from Chlamydia trachomatis. Journal of Bacteriology, 196, 1325–1334. doi:10.1128/JB.01290-13 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 O’Neill, C. E., Clarke, I. N., and Fisher, D. J. (2020). “Chlamydia Genetics,” in Chlamydia Biology: From Genome to Disease, eds. M. Tan, J. H. Hegeman, and C. Sütterlin (Norfolk: Caister Academic Press), 241–262. doi: 10.21775/9781912530281.11 Shima, K., Wanker, M., Skilton, R. J., Cutcliffe, L.T., Schnee, C., Kohl, T. A., et al. (2018). The Genetic Transformation of Chlamydia pneumoniae. mSphere 3, e00412-18. doi: 10.1128/mSphere.00412-18 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 Sixt, B. S., and Valdivia, R. H. (2016). Molecular Genetic Analysis of Chlamydia Species. Annual Review of Microbiology 70, 179–198. doi: 10.1146/annurev-micro-102215-095539 Tam, J. E., Davis, C. H., Thresher, R. J., and Wyrick, P. B. (1992). Location of the origin of replication for the 7.5-kb Chlamydia trachomatis plasmid. Plasmid 27, 231–236. doi: 10.1016/0147-619x(92)90025-6 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