Plan apo 100x

Imaging of L. pneumophila expressing Dot/Icm fluorescent fusions was carried out as previously described (Chetrit et al., 2018 ). Briefly, 2 day heavy patches were suspended in water, after which they were spotted on a thin pad of 1% agarose, covered with a cover slip and immediately imaged at room temperature. Fluorescence micrographs were captured using a Nikon Eclipse TE2000‐S inverted microscope equipped with a Spectra X light engine from Lumencor, CoolSNAP EZ 20 MHz digital monochrome camera from Photometrics and a Nikon Plan Apo100x objective lens (1.4 numerical aperture) under the control of SlideBook 6.0 (Intelligent Imaging Innovations). Samples were imaged using a 196 mW 485 nm LED light, with typical exposure times of 500–1,000 ms and 2 × 2 binning. sfGFP‐DotZ and its derivative strains were exposed for 5,000 ms. Polarity scores were calculated by measuring the ratio between the variance and the mean of the fluorescence signal at region of interest located between the cell poles.

Time-lapse fluorescence microscopy assays were carried out as previously described (Oka et al, 2022 (link)). Briefly, after the cultivation of the X. citri inocula in 2xTY supplemented with the appropriate antibiotics, cells were harvested by centrifugation (5000 rpm, 5 min), washed in water twice and the OD_600nm was normalized to 0.5. Then, 1 µL of each X. citri strain were pipetted onto a thin LB-agarose support supplemented with propidium iodide (1 µg/mL), appropriate antibiotic and observed with a Nikon Eclipse Ti microscope equipped with filters for GFP (GFP-3035B-000-ZERO, Semrock) and propidium iodide (TxRed-4040B, Semrock) and a Nikon Plan APO 100x objective. Images were collected every 10 min. Image processing and quantitative analysis of the number of cells having a damaged cell envelope and cell counting related to Movies EV1–5 were performed manually using Fiji software (Schindelin et al, 2012 (link)) multipoint tool. Time-lapse microscopy was also performed for bacterial competition experiments between X. citri wild-type transformed with pBBR-RFP and X. citri Δ8Δ2609-GFP transformed with pBBR-GFP (Movie EV6). Cells were grown and washed as above and the cultures were mixed at a 1:1 ratio and microscopy was performed as described above, in the absence of propidium iodide.

Bacteria were isolated from soil in Kagawa University, Japan. Since the area of the university is public, no specific permission was required to collect samples that did not include any endangered nor protected species. The isolated strain JHA19 (material number, QM2015–0042) has been deposited in the Material Management Center (MMC; http://mmc-u.jp/en/). YMG medium (0.4% yeast extract, 1% malt extract, 0.4% glucose and 2% agar, pH 7.3) was used for bacterial growth on plates and in liquid cultures, which were performed at 30°C with shaking at 200 rpm. Cells of the isolated strain JHA19 were cultured in YMG liquid medium for 3 days and observed under an Eclipse 80i microscope (Nikon) with a Plan Apo 100x/1.40 NA oil objective lens (Nikon). Images were acquired with a CoolSNAP EZ CCD camera (Photometrics) and the software MetaVue (Molecular Devices).