Extended Data Fig. 5: PG and OMP biogenesis are also coordinated in other Gram-negative bacterial species.

(A) Timeline of IutA biogenesis in K. pneumoniae. IutA OMP expression was induced by transferring K. pneumoniae cells from overnight LB culture (0 min) into fresh M9. Samples were taken and labelled with CloDF13-AF488 at different time points after IutA induction. (B) Fluoresence intensity of cells shown in panel A. (C) Demograph showing the normalised fluorescence distribution of IutA in multiple cells. Cells were treated as in panel A. (D) Co-labelling of PG and OMP biogenesis 80 min after lutA induction as described in panel A. HADA was added 10 min before sample collection. (E) Pixel by pixel cytofluorogram of a single field of view after co-labelling as in panel D emphasising the correlation of PG and OMP fluorescence. (F) Comparison of the fluorescence intensity profiles of PG and OMP biogenesis in dividing K. pneumoniae cells. Shown are profiles of cells at two different stages of septum formation (Group 1/Group 2). Inset displays the width of individual cells at the designated division plane. n_early = 40 cells, n_late = 20 cells. Statistical significance was calculated using two-tailed Student’s unpaired t-test (P = 0.0001). In the following panels polar displacement of OMPs and PG was used as a measure of their spatially coordinated insertion (see Fig. 4c & Methods). (G) Time lapse images of PG and OMP polar displacement emphasising coordination of these layers during growth of K. pneumoniae. Cells from an overnight culture were labelled and grown on M9 agar pads. Images were taken at the indicated time points. (H) Time course images of PG and OMP polar displacement during P. aeruginosa growth. Cells from an overnight M9 culture were labelled with HADA and resuspended in fresh LB+FeCl₃ to suppress expression of the OMPs FpvAI and FptA. Samples were taken at the indicated time points and each OMP labelled with PyoS2-mCherry and PyoS5-AF488, respectively. (I) Pixel by pixel cytofluorogram of a single field of view after co-labelling as in panel H. Charts demonstrate the co-localisation of both OMPs with one another (top) and with the old PG (bottom). Scale bar, 1 μm in all microscopy images shown.