Fig 5.

Expression of mCherry red fluorescent protein using chlamydial shuttle vector pGFP::SW2. The mCherry gene was used to replace the coding sequences of pgp3, pgp4, and pgp5. The replacement constructs were transformed into L2R, and stable transformants, designated L2RpGFPmCherry(pgp3), L2RpGFPmCherry(pgp4), and L2RpGFPmCherry(pgp5), respectively, were obtained. These transformants together with wild-type L2 [L2 (Wt)], plasmid-free L2 (L2R), and L2R transformed with a plasmid (L2RpGFP::SW2), as listed on top of the images, were used to infect HeLa cells. (A) The infected cultures were processed for monitoring red fluorescence intensity (mCherry; red) (a to f) and green fluorescence intensity (GFP; green) (g to l), labeling DNA (blue) (m to r), and, finally, staining for glycogen accumulation with iodine (s to x). Inclusions positive for glycogen staining are marked with white arrows, while those with a lack of glycogen staining are marked with white arrowheads. Note that red fluorescence was detected when the mCherry gene was used to replace pgp3 (d), pgp4 (e), or pgp5 (f). (B) The expression levels of pgp3, pgp4, and pgp5 as well as of the glgA and ompA genes were quantitated by quantitative RT-PCR. Note that the mCherry replacement mutants largely phenocopied the corresponding ORF deletion mutants, including L2RpGFPΔPgp3(72), L2RpGFPΔPgp4, and L2RpGFPΔPgp5.