Intermembrane crosstalk drives inner-membrane protein organization in Escherichia coli

Abstract

Gram-negative bacteria depend on energised protein complexes that connect the two membranes of the cell envelope. However, β-barrel outer-membrane proteins (OMPs) and α-helical inner-membrane proteins (IMPs) display quite different organisation. OMPs cluster into islands that restrict their lateral mobility, while IMPs generally diffuse throughout the cell. Here, using live cell imaging of Escherichia coli, we demonstrate that when transient, energy-dependent transmembrane connections are formed, IMPs become subjugated by the inherent organisation of OMPs and that such connections impact IMP function. We show that while establishing a translocon for import, the colicin ColE9 sequesters the IMPs of the proton motive force (PMF)-linked Tol-Pal complex into islands mirroring those of colicin-bound OMPs. Through this imposed organisation, the bacteriocin subverts the outer-membrane stabilising role of Tol-Pal, blocking its recruitment to cell division sites and slowing membrane constriction. The ordering of IMPs by OMPs via an energised inter-membrane bridge represents an emerging functional paradigm in cell envelope biology.

Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism

“…In non-dividing cells ( Figure 5a ), TolQ-TolR-TolA in the inner membrane exhibits unrestricted Brownian motion 32 . We postulate that in this diffusive mode, and via coupling to the PMF, TolA continuously scans the OM for TolB-Pal complexes on which to pull.…”

“…In dividing cells ( Figure 5b ), the TolQ-TolR-TolA assembly is recruited to the divisome 21,32 . An important consequence of this spatial localisation is that the assembly is now no longer available to dissociate TolB-Pal complexes anywhere other than the divisome.…”

“…Plasmid pREN126 encoding E. coli Pal-mCherry was generated by ligating XbaI/SalI digest of pal gene PCR-amplified form MG1655 genomic DNA fused together 33 with the mCherry gene optimised for E. coli expression into the plasmid pDOC-K. The strain RKCK8 ( pal::pal-GGGGS-mCherry ) was engineered using λ-Red recombination based on the gene-doctoring protocol 34 , as described previously 32 . QuickChange mutagenesis was used to introduce point mutations into genes where needed.…”

“…Laser power was set at 10 % to scan the sample over 4 µm thickness (32 slices) without to much photobleaching. Image stacks were reconstructed using Deltavision softWoRx 6.1.1 software with a Wiener filter of 0.002 using wavelength specific experimentally determined OTF functions (see 32 ). Image analysis: Cells were automatically aligned using Image J software so the fluorescent ring appears as a vertical bar from the lateral projection of 3D-SIM imaging.…”

“…Values were then normalized to 0-100 for comparison between cells in Excel. FRAP values were normalized using FRAPnorm plugin as described in Rassam et al 32 . For all experiments, ∼30 cells were analysed from at least three independent repeats per condition.…”