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Their obligate intracellular niche serves as a barrier for natural genetic exchange via horizontal gene transfer (HGT), and further limits the development and application of genetic tools. To date, the only example for recent inter-phylum HGT among the Chlamydiota is tetracycline resistance in the potentially zoonotic species<jats:italic>Chlamydia suis<\/jats:italic>, a close phylogenetic relative of human<jats:italic>C. trachomatis<\/jats:italic>, which causes bacterial sexually transmitted infections and ocular trachoma. Tetracycline resistance in porcine<jats:italic>C. suis<\/jats:italic>strains has been described worldwide and is always part of a genomic island dividing<jats:italic>invasin<\/jats:italic>(<jats:italic>inv<\/jats:italic>), located within a chromosomal region between the rRNA operon (<jats:italic>rrn<\/jats:italic>) and the<jats:italic>nqrF<\/jats:italic>reductase. Here, we aimed to expand the still modest number of available genetic manipulation systems for<jats:italic>Chlamydia<\/jats:italic>by generating allele-replacement and integration vectors for<jats:italic>C. suis<\/jats:italic>. These vectors comprised homologous<jats:italic>C. suis<\/jats:italic>sequences of the chromosomal region of interest, an<jats:italic>E. coli<\/jats:italic>origin of replication (<jats:italic>ori<\/jats:italic>) and selection markers but lacked the native chlamydial plasmids or its<jats:italic>ori<\/jats:italic>. We first recovered allele-replacement mutants using a vector that targets the tryptophan (<jats:italic>trp<\/jats:italic>) operon of<jats:italic>C. suis<\/jats:italic>. The vector was further successfully maintained as a free plasmid in<jats:italic>C. trachomatis<\/jats:italic>without allele replacement, suggesting complex plasmid dynamics in the absence of a chlamydial<jats:italic>ori<\/jats:italic>. Moreover, we showed that the hypervariable<jats:italic>rrn-nqrF<\/jats:italic>intergenic region of<jats:italic>C. suis<\/jats:italic>is highly susceptible to transformation, resulting in complete vector integration upstream of<jats:italic>nqrF<\/jats:italic>without interruption of the targeted<jats:italic>inv<\/jats:italic>gene.<\/jats:p><jats:sec><jats:title>IMPORTANCE<\/jats:title><jats:p>The obligate intracellular<jats:italic>Chlamydia<\/jats:italic>genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of<jats:italic>Chlamydia<\/jats:italic>with the opportunistic porcine pathogen<jats:italic>Chlamydia suis<\/jats:italic>, the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer.<jats:italic>C. suis<\/jats:italic>is transmissible to humans, posing a potential public health concern. We report that<jats:italic>C. suis<\/jats:italic>can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that<jats:italic>C. trachomatis<\/jats:italic>, the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with<jats:italic>C. suis<\/jats:italic>vectors. Finally, the chromosomal region that harbors the resistance gene of<jats:italic>C. suis<\/jats:italic>is highly susceptible to complete vector integration.<\/jats:p><\/jats:sec>","DOI":"10.1128\/spectrum.02378-23","type":"journal-article","created":{"date-parts":[[2023,10,26]],"date-time":"2023-10-26T13:04:07Z","timestamp":1698325447000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":7,"title":["<i>Chlamydia suis<\/i>displays high transformation capacity with complete cloning vector integration into the chromosomal<i>rrn-nqrF<\/i>plasticity zone"],"prefix":"10.1128","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8398-4708","authenticated-orcid":true,"given":"Hanna","family":"Marti","sequence":"first","affiliation":[{"name":"Institute of Veterinary Pathology, University of Zurich , Zurich, 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