Phase separation in the outer membrane of
            <i>Escherichia coli</i>

Benn, Georgina; Mikheyeva, Irina V.; Inns, Patrick George; Forster, Joel C.; Ojkic, Nikola; Bortolini, Christian; Ryadnov, Maxim G.; Kleanthous, Colin; Silhavy, Thomas J.; Hoogenboom, Bart W.

doi:10.1073/pnas.2112237118

Cited by 68 publications

(74 citation statements)

References 62 publications

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“…This clearly contrasts with our work, where separate SusC or SusD components have never been observed during purification, regardless of whether the purification tag was located on SusC or SusD. Moreover, while it is not clear how much the E. coli OM resembles that of B. theta, recent work has shown that E. coli OMP and LPS mobility is limited 30,31 , raising questions regarding the efficiency of dynamically (dis)assembling transport complexes in a crowded OM.…”

Section: Discussioncontrasting

confidence: 96%

Outer membrane utilisomes mediate oligosaccharide uptake in gut Bacteroidetes

Berg

White

Silale

et al. 2022

Preprint

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Bacteroidetes are abundant members of the human microbiota, with species occupying the distal gut capable of utilising a myriad of diet- and host-derived glycans. Transport of glycans across the outer membrane (OM) of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, that are thought to operate via a pedal-bin mechanism in which the lids open and close to facilitate substrate binding. However, additional cell surface-exposed lipoproteins, namely surface glycan binding proteins and glycoside hydrolases, play critical roles in the capture and processing of large glycan chains into transport-competent substrates. Despite constituting a crucial mechanism of nutrient acquisition by our colonic microbiota, the interactions between these components in the OM are poorly understood. Here we show that for the levan and dextran utilisation systems of Bacteroides thetaiotaomicron, the additional OM components assemble on the core SusCD transporter, forming stable glycan utilising machines which we term utilisomes. Single particle electron cryogenic electron microscopy (cryo-EM) structures in the absence and presence of substrate reveal concerted conformational changes that rationalise the role of each component for efficient nutrient capture, as well as providing a direct demonstration of the pedal bin mechanism of substrate capture in the intact utilisome.

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Section: Discussioncontrasting

confidence: 96%

Outer membrane utilisomes mediate oligosaccharide uptake in gut Bacteroidetes

Berg

White

Silale

et al. 2022

Preprint

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“…The mechanism of action thus goes beyond a simple detergent-like effect as suggested previously 49 . Recent MD simulations of polymyxin-induced clusters 50 , 51 have proposed an increase in the local order of LPS bilayers by crystal domains in hexagonal coordination, which was also found to be correlated with the bacterial sensitivity to antimicrobials 52 , 53 . The crystalline structures identified in this work likely give a rationale for these observations.…”

Section: Discussionmentioning

confidence: 95%

Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane

et al. 2022

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Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays. We find that polymyxins arrange LPS into hexagonal assemblies to form crystalline structures. Formation of the crystalline structures is correlated with the antibiotic activity, and absent in polymyxin-resistant strains. Crystal lattice parameters alter with variations of the LPS and polymyxin molecules. Quantitative measurements show that the crystalline structures decrease membrane thickness and increase membrane area as well as stiffness. Together, these findings suggest the formation of rigid LPS–polymyxin crystals and subsequent membrane disruption as the mechanism of polymyxin action and provide a benchmark for optimization and de novo design of LPS-targeting antimicrobials.

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“…OMPs are the major components of bacterial OMs, accounting for more than 50% of the OM volume 58,59 . The quantitative analysis of single-cell data suggest ~500 of molecules of OmpF need to be assembled into the outer membrane per minute per cell 60,61 , meaning that factors that promote the efficiency of OmpF assembly are critical to ensure that E. coli can handle this extraordinary rate of substrate protein flux. In order to fight against external insult, all of the Gram-negative bacteria contain BamD, the OMPs assembly accelerator.…”

Section: Discussionmentioning

confidence: 99%

Dual Recognition of Multiple Signals in Bacterial Outer Membrane Proteins Enhance Assembly and Maintain Membrane Integrity

Ding

Imai

Bamert

et al. 2021

Preprint

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SUMMARYGram-negative bacteria, mitochondria and chloroplasts contain outer membrane proteins (OMPs) characterized by a transmembrane domain with a β barrel structure. Most OMPs have a diagnostic “β-signal” imprinted in the amino acid sequence of the final β-strand (−1 strand) of the β barrel. Molecular chaperones of the Omp85 superfamily, such as BamA in the bacterial BAM complex, recognizes the β-signal and then catalyze the folding of the OMP into its membrane-embedded β barrel structure. Here, we reconstituted OMP assembly in vitro to study this process with five model OMPs, revealing that a critical assembly signal exists in the fifth β-strand from the C-terminus (−5 strand). This signal was shown to drive a critical insertion step of the OMP assembly process but is not required for the initial recognition step between the OMP and BamA. Furthermore, we identified the receptor for this “-5 signal” as BamD, a second essential subunit of the BAM complex. Distinct binding sites for the β-signal and the -5 signal, were identified on BamD. Comparative sequence analysis showed that the -5 signal is a conserved feature in bacterial OMPs, and that mitochondrial OMPs also contains -5 signal motif positioned in the fifth from last strand of their β barrel structure. We propose the “-5 rule” as a conserved feature of the process of OMP assembly in bacteria and the organelles of eukaryotes.HIGHLIGHTSGram-negative bacterial OMPs contain a conserved motif at the fifth β-strand from the C-terminus (−5 strand).The information encrypted in the conserved -5 signal is responsible for a rate-limiting step in protein insertion into the outer membraneDistinct binding sites on the outer membrane protein BamD recognize the -5 signal and a canonical β-signal.We propose -5 rule, to explain the function of the -5 signal in OMP assembly.

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Phase separation in the outer membrane of Escherichia coli

Cited by 68 publications

References 62 publications

Outer membrane utilisomes mediate oligosaccharide uptake in gut Bacteroidetes

Outer membrane utilisomes mediate oligosaccharide uptake in gut Bacteroidetes

Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane

Dual Recognition of Multiple Signals in Bacterial Outer Membrane Proteins Enhance Assembly and Maintain Membrane Integrity

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