Transformation of Chlamydia trachomatis should greatly advance the chlamydial research. However, significant progress has been hindered by the failure of C. trachomatis to induce clinically relevant pathology in animal models. Chlamydia muridarum, which naturally infects mice, can induce hydrosalpinx in mice, a tubal pathology also seen in women infected with C. trachomatis. We have developed a C. muridarum transformation system and confirmed Pgp1, -2, -6, and -8 as plasmid maintenance factors, Pgp3, -5, and -7 as dispensable for in vitro growth, and Pgp4 as a positive regulator of genes that are dependent on plasmid for expression. More importantly, we have discovered that Pgp5 can negatively regulate the same plasmid-dependent genes. Deletion of Pgp5 led to a significant increase in expression of the plasmid-dependent genes, suggesting that Pgp5 can suppress the expression of these genes. Replacement of pgp5 with a mCherry gene, or premature termination of pgp5 translation, also increased expression of the plasmid-dependent genes, indicating that Pgp5 protein but not its DNA sequence is required for the inhibitory effect. Replacing C. muridarum pgp5 with a C. trachomatis pgp5 still inhibited the plasmid-dependent gene expression, indicating that the negative regulation of plasmid-dependent genes is a common feature of all Pgp5 regardless of its origin. Nevertheless, C. muridarum Pgp5 is more potent than C. trachomatis Pgp5 in suppressing gene expression. Thus, we have uncovered a novel function of Pgp5 and developed a C. muridarum transformation system for further mapping chlamydial pathogenic and protective determinants in animal models.