Axiovision axiovs40 v 4

Overnight cultures of PAO1161 harboring pAKW1125 (tacp–YFP–ParB) were grown overnight in LB or M9 medium with chloramphenicol added at a concentration of 75 µg mL⁻¹. After dilution to the fresh media without antibiotics, samples were collected in time intervals, and cells were fixed with formaldehyde/glutaraldehyde mixture essentially as described before [34 (link)]. Images were captured using a Zeiss Imager M2 equipped with a 100 × 1.30 NA Plan-Neofluar objective, a Zeiss AxioCam MRc5 camera, using AxioVision (AxioVs40 V 4.8.2.0, Carl Zeiss MicroImaging GmbH, Munich, Germany) software. Images were processed using ImageJ, and foci detection was performed using MicrobeJ ver 5.13m [71 (link)], as described [34 (link)]. Since the images obtained at different time points varied significantly in fluorescence background within the cells (possibly due to autofluorescence), the threshold for foci intensity detection in MicrobeJ was adjusted in individual sets of images, and proper selection was validated by manually counting foci in at least 100 cells.

PA2540 protein was localised in P. aeruginosa cells by cloning the PA2504 and sfGFP (super folder GFP) fragments in to the pKGB to give pKGBgfp2504. PA2504 was amplified on the PAO1161 strain genomic DNA template and sfGFP fragment on the pBAD24-sfGFPx1 plasmid [39 (link)]; pKGBgfp2504 was introduced into the P. aeruginosa ΔPA2504 by conjugation [40 (link)]. An overnight culture was diluted 1:150 and incubated for 4 h at 37 °C, then 1.5 mL of the culture was centrifuged and resuspended in 20 µL of fresh LB medium and 1 µL of the suspension was placed on a microscope slide covered with polylysine (Thermo Scientific). Cells were studied using a Zeiss Imager. M2 fluorescence microscope with a 100× 1.30 NA Plan-Neofluar lens and Zeiss AxioCam MRc5 camera with ta 470/40 nm excitation filter, 495 nm dichroic beam-splitter, and 525/50 nm emission filter. AxioVision (AxioVs40 V 4.8.2.0, Carl Zeiss MicroImaging) software was used.

Cells were processed for immunofluorescence as described [31] (link), [51] (link). Briefly, cells cultured on glass coverslips were fixed in 3%-paraformaldehyde/2%-sucrose solution for 15 min at room temperature, permeabilized for 2 min with 0.5% (vol/vol) Triton X-100 in PBS, blocked (10% goat serum in PBS) and incubated with primary antibodies overnight at 4C. The next day, following incubation with secondary antibodies and DAPI (Sigma) or Hoechst (Invitrogen, H3570) for 2 h, coverslips were mounted using Vectashield mounting medium (Vector Lab). Primary and secondary antibodies were used in immunofluorescence as follows: RAD51 (Santa Cruz, sc-8349, 1/50) (Abcam, ab63801, 1/1000), RPA70 (Millipore, NA13, 1/100), γH2AX (Cell Signalling, 9718, 1/50), cyclin-A (Santa Cruz, sc-271,682, 1/100) and Mitosin/CENPF (Abcam, Ab5, 1/200), anti-rabbit FITC (Stratech – Jackson, 711-095-152, 1/100), anti-mouse FITC (Stratech – Jackson, 715-095-151, 1/100), anti-rabbit Texas Red (Stratech – Jackson, 711-075-152, 1/100) and anti-mouse Texas Red (Stratech – Jackson, 715-075-151, 1/100). Images were acquired with an ApoTome fluorescence microscope (Zeiss) with a 40× objective lens and processed with AxioVision AxioVS40 V4.8.1.0 (Zeiss) and Photoshop CS5 (Adobe Systems Inc.).