ABSTRACTWe demonstrate the genetic transformation ofChlamydia pneumoniaeusing a plasmid shuttle vector system which generates stable transformants. The equineC. pneumoniaeN16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearingC. pneumoniaetransformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolateC. pneumoniaeLPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolateC. pneumoniaeCV-6 and the human community-acquired pneumonia-associatedC. pneumoniaeIOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown thatChlamydiaspp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-freeC. trachomatis,C. muridarum,C. caviae,C. pecorum, andC. abortus. However, contrary to our expectation, pRSGFPCAT-Cpn did transformC. felis. Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid ofC. felis. Taken together, we provide for the first time an easy-to-handle transformation protocol forC. pneumoniaethat results in stable transformants. In addition, the vector can cross the species barrier toC. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid.IMPORTANCEThe absence of tools for the genetic manipulation ofC. pneumoniaehas hampered research into all aspects of its biology. In this study, we established a novel reproducible method forC. pneumoniaetransformation based on a plasmid shuttle vector system. We constructed aC. pneumoniaeplasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase inC. pneumoniae.C. pneumoniaetransformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation inC. pneumoniaeusing pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions inC. pneumoniaebiology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.