Figure 3.

Loss of ct622 expression due to intron insertion and complementation. (A) PCR verification of plaque purified C. trachomatis L2 ctl0886::GII(aadA) clones. PCR was performed using genomic DNA from wild type L2 (ACE051 strain) and the plaque purified AS9 clones to assess intron insertion, loss of the intron donor plasmid, and maintenance of the chlamydial cryptic plasmid. Primers were designed to amplify regions of the cryptic plasmid, ctl0886 locus, intron, and to verify the orientation of the intron. Products were separated on 1% agarose gels and stained with ethidium bromide. The template used in the PCR reaction is listed at the top of each lane. The specificity of the primers used in each reaction is indicated above. Identical results were obtained for the 6 isolates, PCR products for one isolate are shown. (B) Crude EB preparations of each isolate and of the parental strain were analyzed by western blot using anti-CT622 (top), and anti-MOMP (bottom) antibodies. Note that only the contaminant 78 kDa band is still visible in the AS9 isolates. (C) Cells were infected for 30 h with wild type, AS9 or AS9_complCT622 bacteria before fixation and permeabilization. Cells were stained with mouse antibodies against the inclusion protein CT813 and rabbit anti-CT622 antibodies followed with Cy3-conjugated anti-mouse (shown in white) and Alexa647-conjugated anti-rabbit (shown in red) antibodies. DNA was labeled with Hoechst 33342 (blue). Arrows point to inclusions in the AS9 infected cells. (D) Crude lysates of HeLa cells infected or not for 48 h with the indicated strains were analyzed by western blot using anti-CT622 (top), anti-CT621 (middle), and anti-MOMP (bottom) antibodies. (E) Cells infected for 48 h with wild-type (left) or AS9 (right) bacteria were stained with anti-CT813 and anti-CT622 antibodies followed with Cy3-conjugated anti-mouse (shown in white) and Alexa647-conjugated anti-rabbit (red) antibodies. DNA was labeled with Hoechst 33342. Arrows point to CT622 detected in the cytosol. (F) Cells infected for 48 h with AS9_complCT622 bacteria were stained with mouse anti-flag antibodies and rabbit antibodies again the inclusion protein Cap1 followed with Alexa647-conjugated anti-mouse antibodies (red) and Cy3-conjugated anti-rabbit antibodies (white). Bacteria express GFP (green), DNA was labeled with Hoechst 33342 (blue). In panels (E,F), arrows point to CT622 detected in the cytosol. Scale bar = 10 μm.