Fig 1. A new set of versatile spectinomycin resistant expression vectors for C. trachomatis.

Vectors maps of p2TK2_Spec-SW2 mCh(Gro_L2) (A), p2TK2_Spec-SW2 mCh(Gro_L2)-MCS-3xFLAG (B), p2TK2_Spec-SW2 mCh(Gro_L2) Tet-IncV-3xFLAG (C), and p2TK2_Spec-SW2 mCh(Gro_L2) Tet-KpnI-IncV-3xFLAG (D). The pSW2 plasmid (SW2) is shown in black; the incDEFG operon promoter in dark blue; the aadA ORF that confers spectinomycin resistance (Spec) in blue; aadA terminator light blue; the E.coli origin of replication (Ori) in grey; the groESL operon promoter in dark red; the mCherry ORF in red; the groESL operon terminator in light red; unique restriction sites in violet; the tet repressor in orange; the tetA promoter in light orange; the incV ORF in yellow; the 3xFLAG tag in teal; the incDEFG operon terminator in light green. (E, G) Immuno-blots of cell lysates from HeLa cells infected with strains of C. trachomatis transformed with p2TK2_Spec-SW2 mCh(Gro_L2) Tet-IncV-3xFLAG (E) or p2TK2_Spec-SW2 mCh(Gro_L2) Tet-KpnI-IncV-3xFLAG (G) expressing mCherry constitutively and IncV-3xFLAG under the control of the aTc inducible promoter. The cells were infected for 24h in the absence (-aTc) or in the presence (+aTc) of 10ng/ml aTc added at 20h p.i. for 4h. Immuno-blots of the corresponding lysates were probed using antibodies against FLAG, mCherry, and actin. (F, H) Confocal micrographs of inclusions of the C. trachomatis strains as described in E and G, respectively. The cells were fixed 24h p.i., immunostained with anti-FLAG antibodies and imaged using a confocal microscope. A single plane crossing the middle of the inclusion is shown. The left panels correspond to the bacteria (Ct mCherry, red) and the middle panels to the 3xFLAG signal (IncV-3xF, green). The merge is shown on the right. Scale bar: 5μm.