Cellsens version 11

The mutant X. citri parB-gfp::rbsw was cultivated in 5.0 mL of NYG medium for approximately 16 h at 30 °C and 200 rpm. The cultures were adjusted to the OD600nm of 0.1 with fresh NYG medium for a final volume of 5.0 mL and subsequently cultivated in the same conditions for approximately four hours. At this point, theophylline was added to the medium to a final concentration of 2 mM and the culture was kept at 30 °C and 200 rpm to monitor the ParB-GFP dynamics within the cells. Negative controls without the addition of theophylline were also prepared in the same growth conditions as described above. At the points of 4 h (T0 of theophylline exposure), 6 h (2 h of theophylline exposure), 8 h (4 h of theophylline exposure), and 16 h (12 h of theophylline exposure) drops of 5 µL of cell culture were placed on microscope slides prepared for microscope imaging as described by [33 (link)]. Bacteria were visualized using an Olympus BX61 microscope equipped with a monochromatic camera OrcaFlash2.8 (Hamamatsu, Japan) and GFP filter. Data collection and analysis were performed with the software CellSens Version 11 (Olympus, Hamburg, Germany).

X. citri, Δ4296 mutant, and Δ4296c strains were cultivated in NB media until O.D. 600-nm reached around 0.3 ABS at 29 °C. We performed analysis in different conditions: with ampicillin (20 µg/mL), sucrose 2% (w/v) and glutamic acid 2% (w/v). For morphological analysis, strains were collected by centrifugation, and cells were resuspended in 0.85% NaCl. We used 4′,6-diamidino-2-phenylindole DAPI staining at a final concentration of 0.01% to visualize chromosome organization. Cultures were treated with propidium iodide (IP) for cell viability investigations at a 0.001 mg/mL final concentration. Cells were immobilized in agarose-covered slides for microscope observation [39 (link)]. We performed the assays three times and quantified cells individually (n = 800). Following treatments, cells were immediately visualized using an Olympus BX61 microscope equipped with a monochromatic camera OrcaFlash 2.8 (Hamamatsu, Japan). The software CellSens Version 11 (Olympus, Tokyo, Japan) was used for data collection and analysis.

Bacteria were cultivated in 5.0 mL of NB medium for approximately 16 hours at 30°C and 200 rpm. The cultures were adjusted to the OD 600 nm of 0.1 with NB medium for a final volume of 5.0 mL and subsequently cultivated in the same conditions until the OD 600 nm of 0.3. At this point, arabinose was added to the medium to a final concentration of 0.05% and the culture was kept at 30°C and 200 rpm. After a minimum of two hours of induction, drops of 5 μL of cell culture were placed on microscope slides covered with a slide cover slip as described by Martins et al. [23 (link)]. Bacteria were immediately visualized using an Olympus BX61 microscope equipped with a monochromatic OrcaFlash2.8 camera (Hamamatsu, Japan) and TxRed filter. Data collection and analysis were performed with the software CellSens Version 11 (Olympus).