Fig 1.

Effect of pORF deletion on GlgA protein expression and glycogen synthesis. (A) Each of the 8 individual ORFs from the shuttle vector pGFP::SW2 was deleted, with the deletion region indicated. (B) The deletion mutants, designated pGFPΔPgp1 to pGFPΔPgp8, together with the parent plasmid (pGFP::SW2), as listed on top of the gel image, were detected for the presence of each of the 8 pORFs (from top to bottom rows) by PCR. (C) The 9 plasmids were each transformed into plasmid-free L2 (L2R). However, stable L2R transformants were obtained with only 5 out of the 9 plasmids. The 5 stable transformants, designated L2RpGFP::SW2, L2RpGFPΔPgp3, L2RpGFPΔPgp4, L2RpGFPΔPgp5, and L2RpGFPΔPgp7, along with wild-type L2 (L2 Wt) and plasmid-free L2 (L2R) organisms were used to infect HeLa cells. The infected cells were monitored for live-cell bright-field appearance (a to g) and green fluorescence intensity (GFP) (h to n); triple immunofluorescence labeling for GlgA protein (red), chlamydial organisms (green), and DNA (blue) (o to u); and iodine staining of glycogen accumulation (v to bb). 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 both GlgA expression and glycogen accumulation were restored in L2R organisms transformed with plasmid pGFP::SW2 (q and x) and plasmids with a deletion of pgp5 (t and aa) or pgp7 (u and bb) but not in plasmids with a deletion of pgp4 (s and z) or pgp3 (r and y).