Protein Diffusion in the Periplasm of E. coli under Osmotic Stress

Abstract

The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure diffusion of periplasmic green fluorescent protein and probe the fluidity of the periplasm as a function of external osmotic conditions. For cells adapted to growth in complete medium at 0.14-1.02 Osm, the mean diffusion coefficient <D(peri)> increases from 3.4 μm² s⁻¹ to 6.6 μm² s⁻¹ and the distribution of D(peri) broadens as growth osmolality increases. This is consistent with a net gain of water by the periplasm, decreasing its biopolymer volume fraction. This supports a model in which the turgor pressure drops primarily across the thin peptidoglycan layer while the cell actively maintains osmotic balance between periplasm and cytoplasm, thus avoiding a substantial pressure differential across the cytoplasmic membrane. After sudden hyperosmotic shock (plasmolysis), the cytoplasm loses water as the periplasm gains water. Accordingly, <D(peri)> increases threefold. The fluorescence recovery after photobleaching is complete and homogeneous in all cases, but in minimal medium, the periplasm is evidently thicker at the cell tips. For the relevant geometries, Brownian dynamics simulations in model cytoplasmic and periplasmic volumes provide analytical formulae for extraction of accurate diffusion coefficients from readily measurable quantities.

Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37

“…If LL-37 diffused freely within the periplasm after translocation, the thin space would fill in a few seconds (20). Instead, we attribute the slow expansion of phase 2 to continual entry into the periplasmic space at the septal region followed by prompt binding to immobile elements there.…”

“…The green channel monitors emission from two species: GFP, which has been exported to the periplasm (the ∼40-nm-thick space between the OM and CM) by the twin arginine translocase pathway (19,20), and the DNA stain Sytox Green. When the OM becomes permeabilized to GFP, the thin periplasmic GFP image disappears.…”

“…For experiments on periplasmic GFP, TorA-GFP was expressed from the plasmid pJW1 as previously described (20). TorA-GFP consists of a short sequence (43 residues) from trimethylamine Noxide (TMAO) reductase that signals the twin-arginine translocase pathway.…”

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15

“…The background strain is MG1655 in all cases. For experiments on periplasmic GFP, TorA-GFP was expressed from a plasmid pJW1 as previously described (32). APEX2 was expressed using tetracycline in the same manner as TorA-GFP.…”

Resolving Fast, Confined Diffusion in Bacteria with Image Correlation Spectroscopy

“…In live cells, the function of a protein in a biological process can be inferred from its rate of diffusion under different chemical or genetic conditions (5)(6)(7)(8)(9). Traditionally, optical measurements of subcellular diffusion have been done using fluorescence recovery after photobleaching (FRAP) (10)(11)(12), but single-molecule imaging techniques like single-particle tracking (SPT) are being increasingly used to precisely evaluate the motion of a diffusing biomolecule (13)(14)(15).…”