{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T16:48:35Z","timestamp":1790095715163,"version":"4.0.1"},"reference-count":66,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2025,9,2]],"date-time":"2025-09-02T00:00:00Z","timestamp":1756771200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,9,2]],"date-time":"2025-09-02T00:00:00Z","timestamp":1756771200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,9,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      In veterinary medicine, the obligate intracellular bacteria\n                      <jats:italic toggle=\"yes\">Chlamydia<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">C.<\/jats:italic>\n                      )\n                      <jats:italic toggle=\"yes\">abortus<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">Chlamydia caviae<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">Chlamydia pecorum<\/jats:italic>\n                      are known to cause ovine enzootic abortion, conjunctivitis in guinea pigs, and ocular\/urogenital disease in koalas, respectively. Studying the biology of these bacteria has been challenging due to a dearth of genetic tools. This study aimed to establish stable transformation systems for\n                      <jats:italic toggle=\"yes\">C. abortus<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">C. pecorum<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">C. caviae<\/jats:italic>\n                      by introducing shuttle vectors carrying green fluorescent proteins. With the aim to select the most suitable green fluorescent protein for the tracking of chlamydiae\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      , we further compared the fluorescence intensity of GFP to that of mNeonGreen. Transformed shuttle vectors comprised the native plasmid of the chlamydial species of interest, an\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      origin of replication (\n                      <jats:italic toggle=\"yes\">ori<\/jats:italic>\n                      ), a beta-lactamase (\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      ) or spectinomycin (\n                      <jats:italic toggle=\"yes\">aadA<\/jats:italic>\n                      ) resistance gene, and GFP or mNeonGreen for heterologous fluorescence expression. We compared the success of a\n                      <jats:italic toggle=\"yes\">C. suis<\/jats:italic>\n                      -tailored transformation protocol (Protocol A) to that of an alternative protocol for\n                      <jats:italic toggle=\"yes\">C. psittaci<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">C. trachomatis<\/jats:italic>\n                      (Protocol B), both of which employ calcium chloride for competence induction. Stable transformants were obtained for\n                      <jats:italic toggle=\"yes\">C. pecorum<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">C. caviae<\/jats:italic>\n                      using protocols A and B, respectively, and we found that the fluorescence intensity of heterologously expressed GFP is higher than that of mNeonGreen. In contrast, pre-incubation with trypsin-EDTA prior to the application of calcium chloride was needed to obtain transformants of\n                      <jats:italic toggle=\"yes\">C. abortus<\/jats:italic>\n                      . In summary, we established protocols for stable calcium chloride-mediated transformation for\n                      <jats:italic toggle=\"yes\">C. pecorum<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">C. abortus<\/jats:italic>\n                      and expanded upon the genetic toolbox of\n                      <jats:italic toggle=\"yes\">C. caviae<\/jats:italic>\n                      .\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Chlamydiae are a diverse group of bacteria impacting human and animal health. Many of the veterinary species, such as\n                        <jats:italic toggle=\"yes\">Chlamydia abortus<\/jats:italic>\n                        ,\n                        <jats:italic toggle=\"yes\">Chlamydia caviae<\/jats:italic>\n                        , and\n                        <jats:italic toggle=\"yes\">Chlamydia pecorum<\/jats:italic>\n                        , which cause reproductive disorders and\/or conjunctivitis, are zoonotic pathogens leading to a potentially life-threatening disease in humans. Our understanding of these species has been hampered due to a lack of genetic tools. In this study, we developed calcium chloride-mediated transformation protocols for each of these species: chlamydiae are mixed with shuttle vectors containing the complete species-specific plasmid sequence, an\n                        <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                        origin of replication, and an antibiotic resistance gene for selection. We could further show that certain chlamydial species become more susceptible to genetic modification if they are pre-treated with trypsin-EDTA prior to the addition of calcium chloride and the vector of interest. Overall, we demonstrate that species-specific protocol refinement is indispensable to render chlamydiae competent for genetic transformation.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01641-25","type":"journal-article","created":{"date-parts":[[2025,7,23]],"date-time":"2025-07-23T12:00:29Z","timestamp":1753272029000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":3,"title":["Development of shuttle vector-based transformation systems for veterinary and zoonotic chlamydiae"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-7246-6902","authenticated-orcid":false,"given":"Nadja","family":"F\u00e4ssler","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02crff812","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Veterinary Pathology, Vetsuisse-Faculty, University of 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