Characterization of Chlamydia trachomatis plasmid-encoded open reading frames.

The recent success in transformation of Chlamydia trachomatis represents a major advancement in Chlamydia research. Plasmid-free C. trachomatis serovar L2 organisms can be transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT. Deletion of plasmid genes coding for Pgp1 to Pgp8 in pBRCT led to the identification of Pgp1, -2, -6, and -8 as plasmid maintenance factors; Pgp4 as a transcriptional regulator of chlamydial virulence-associated gene expression; and Pgp3, -5, and -7 as being dispensable for chlamydial growth in vitro. Using the pGFP::SW2 vector system, we confirmed these findings in the current report. To further dissect the roles of pgp coding sequences and Pgp proteins in plasmid maintenance, we introduced premature stop codons into the pgp genes. Stable transformants were obtained with pGFP::SW2 derivatives carrying premature stop codons in pgp8 but not in pgp1, pgp2, and pgp6, suggesting that the pgp8 coding sequence but not the Pgp8 protein is required for maintaining the plasmid, while Pgp1, -2, and -6 proteins are necessary for plasmid maintenance. We also found that a minimum of 30 nucleotides in the pgp3 coding region was required for pgp4 expression. Finally, mCherry red fluorescent protein was successfully expressed when the mCherry gene was used to replace the pgp3, pgp4, or pgp5 coding region, indicating that these regions can be used to express nonchlamydial genes in chlamydial organisms. These novel observations have provided information for further use of chlamydial plasmid shuttle vectors as genetic tools to understand chlamydial biology and pathogenicity as well as to develop attenuated chlamydial vaccines.