Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals where it can be both a commensal and a pathogen1–3. Intricate regulatory mechanisms ensure bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat4,5. Yet no mechanism is known for replacing OMPs in the outer membrane (OM), a biological enigma further confounded by the lack of an energy source and the high stability6 and abundance of OMPs5. Here, we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature wherein old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics (MD) simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form islands of ~0.5 μm diameter where their diffusion was restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the OM7,8. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence the OM of a Gram-negative bacterium is a spatially and temporally organised structure and this organisation lies at the heart of how OMPs are turned over in the membrane.

SignificanceThe outer membrane (OM) excludes antibiotics such as vancomycin that kill gram-positive bacteria, and so is a major contributor to multidrug resistance in gram-negative bacteria. Yet, the OM is readily bypassed by protein bacteriocins, which are toxins released by bacteria to kill their neighbors during competition for resources. Discovered over 60 y ago, it has been a mystery how these proteins cross the OM to deliver their toxic payload. We have discovered how the bacteriocin pyocin S2 (pyoS2), which degrades DNA, enters Pseudomonas aeruginosa cells. PyoS2 tricks the iron transporter FpvAI into transporting it across the OM by a process that is remarkably similar to that used by its endogenous ligand, the siderophore ferripyoverdine.

Porins are β-barrel outer membrane proteins through which small solutes and metabolites diffuse that are also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) assembles a cytotoxic translocon at the surface of Escherichia coli that incorporates the trimeric porin OmpF. Formation of the translocon involved ColE9’s unstructured N-terminal domain threading in opposite directions through two OmpF subunits, capturing its target TolB on the other side of the membrane in a fixed orientation that triggers colicin import. Thus an intrinsically disordered protein can tunnel through the narrow pores of an oligomeric porin to deliver an epitope signal to the cell to initiate cell death.

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

Tryptase and chymase are the major serine proteinases of skin mast cells but their biologic significance depends on their activity. In this study, we demonstrate the release of soluble activity of tryptase, but not markedly that of chymase, into skin blister fluids induced by freezing with liquid nitrogen as well as into supernatant during incubation of 8 whole skin specimens with compound 48/80 for up to 2 days followed by sonication. Incubation of 3 other skin specimens in compound 48/80 for up to 2 days revealed that the number of mast cells displaying tryptase activity decreased significantly on day 2, and the number of mast cells showing chymase activity (but not those showing chymase immunoreactivity) decreased significantly on day 1 but not thereafter on day 2. The results of 3 skin organ cultures for up to 14 days showed steady decrease in the number of tryptase-positive cells but persistence of mast cells containing chymase activity. Chymase in solution was sensitively inhibited by 0.01 mg/ml alpha1-antichymotrypsin but higher concentrations (0.3-3.0 mg/ml) were needed for inhibiting chymase on skin sections. In conclusion, after mast cell degranulation tryptase activity is substantially solubilized and it may potentially affect both local and distant skin structures. Instead, chymase is partially inactivated and the remaining chymase activity persists at the site of degranulation having only local effects.

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria. IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

Mast cell proteases are believed to participate in the basement membrane destruction in blistering diseases. Thus, normal human skin specimens were incubated with purified human skin tryptase or compound 48/80 (a mast cell degranulator) for up to 24 h. Thereafter, the specimens were studied immunohistochemically. Tryptase caused, in the presence and absence of 1,10-phenanthroline, focal dermal-epidermal separation above laminin and almost complete disappearance of the staining of the extra domain A region of cellular fibronectin in and beneath the basement membrane. The immunopositivity of the cell-binding region of fibronectin, laminin, and collagens IV and VII, however, was unaltered. Compound 48/80 induced almost complete dermal-epidermal separation above intact laminin and only focal reduction in the extra domain A region of cellular fibronectin staining. These alterations by compound 48/80 were prevented partially by Nalpha-p-tosyl-L-lysine chloromethyl ketone or 1,10-phenanthroline alone but completely when both inhibitors were present suggesting the involvement of tryptic serine proteinases, probably also tryptase, and metalloproteinases. Preventive effect of N-tosyl-L-phenylalanine chloromethyl ketone was weak suggesting minor function of chymotryptic serine proteinases. When tryptase was incubated with heparin and pure plasma fibronectin, an abrupt decrease in the adherence of cultured keratinocytes on to plastic surface coated with these substances and a gradual plasma fibronectin cleavage to 173, 161, and 28 kDa fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis were found. In conclusion, tryptase can cause focal dermal-epidermal separation above laminin in skin specimens but it is not known to what extent the decreased keratinocyte adherence in vitro and fibronectin cleavage are related to this dermal-epidermal separation.

How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups.