FIGURE 3.

Subcellular location of TrwB_R388GFP and TMD_TraJCD_TrwBGFP fusion-proteins by confocal fluorescence microscopy. Proteins were expressed in E. coli BL21C41 (DE3) strain by induction with 1 mM IPTG for 4 (left panels) and 20 h (right panels) at 25°C. Subcellular location of TrwB_R388GFP and TMD_TraJCD_TrwBGFP was determined in E. coli strains containing plasmid pSU1456 that expresses all R388 conjugative proteins except TrwB_R388, or without plasmid pSU1456. The images were acquired in a Leica TCS SP5 confocal fluorescence microscope, with a 60× oil immersion objective. Sample excitation was performed with 488 nm wavelength, while fluorescence emission was measured between 505 and 525 nm. The images were analyzed using Huygens and ImageJ softwares. Arrrowheads indicate the eGFP fluorescence through the periphery (1st column) and at a single cell pole (2nd column). Scale bar: 5 μm.