Site-Directed Spin Labeling Reveals Multiple Modes for Regulating Protein-Protein Interactions in Bacterial Outer Membrane Transport

ABSTRACT Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.

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Three-dimensional structure of a regular bacterial surface layer: The HPI-layer of Deinococcus radiodurans

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075920 ◽

1983 ◽

Vol 41 ◽

pp. 438-439

Author(s):

W. Baumeister ◽

M. Hahn ◽

W.O. Saxton

Keyword(s):

Outer Membrane ◽

Protein Interactions ◽

Deinococcus Radiodurans ◽

Bacterial Species ◽

Hexagonal Lattice ◽

Three Dimensional ◽

Dimensional Structure ◽

Protein Protein Interactions ◽

Bacterial Surface ◽

Freeze Dried

Regularly organized surface (RS) layers are a feature common to many bacterial species; they are clearly more abundant than was anticipated even a few years ago. The RS-layers are believed to fulfil a variety of functions in the interaction between the cell and its environment (see e.g. [1]). The so-called HPI-layer of the radiotolerant bacterium Deinococcus radiodurans is a typical example of such a layer: It is composed of a single polypeptide species (Mr 105 kDa) arranged on a hexagonal lattice to form a network that covers the entire surface of the bacterium; it is associated with the outer membrane via hydrophobic protein-protein interactions.Isolated HPI-layer sheets, released from the outer membrane by detergent treatment, have been studied in the electron microscope making extensive use of the present arsenal of preparation techniques: negative staining, (auro- thio)glucose embedding, freeze-dried/unstained, freeze-dried/metal shadowed etc.Because of the notorious problem of lattice imperfections image processing usually followed the strategy of correlation averaging as outlined in some detail elsewhere.

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Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport inEscherichia coli

Science Advances ◽

10.1126/sciadv.aau2634 ◽

2018 ◽

Vol 4 (11) ◽

pp. eaau2634 ◽

Cited By ~ 33

Author(s):

Stefan U. Vetterli ◽

Katja Zerbe ◽

Maik Müller ◽

Matthias Urfer ◽

Milon Mondal ◽

...

Keyword(s):

Protein Interactions ◽

Macromolecular Complex ◽

Gram Negative Bacteria ◽

Periplasmic Protein ◽

Protein Protein Interactions ◽

Inner Membrane ◽

Gram Negative ◽

Research Priority ◽

Naturally Occurring ◽

Bacterial Outer Membrane

With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5′-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB2FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis inEscherichia coli.

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IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

Infection and Immunity ◽

10.1128/iai.00595-13 ◽

2013 ◽

Vol 81 (11) ◽

pp. 4026-4040 ◽

Cited By ~ 19

Author(s):

Gregory T. Robertson ◽

Robert Child ◽

Christine Ingle ◽

Jean Celli ◽

Michael V. Norgard

Keyword(s):

Outer Membrane ◽

Francisella Tularensis ◽

Protein Interactions ◽

Single Copy ◽

Hypothetical Protein ◽

Sucrose Density Gradient ◽

Intracellular Replication ◽

Protein Protein Interactions ◽

Content Type ◽

Schu S4

ABSTRACTIglE is a small, hypothetical protein encoded by the duplicatedFrancisellapathogenicity island (FPI). Inactivation of both copies ofiglErenderedFrancisella tularensissubsp.tularensisSchu S4 avirulent and incapable of intracellular replication, owing to an inability to escape the phagosome. This defect was fully reversed following single-copy expression ofiglEintransfromattTn7under the control of theFrancisella rpsLpromoter, thereby establishing that the loss ofiglE, and not polar effects on downstreamvgrGgene expression, was responsible for the defect. IglE is exported to theFrancisellaouter membrane as an ∼13.9-kDa lipoprotein, determined on the basis of a combination of selective Triton X-114 solubilization, radiolabeling with [3H]palmitic acid, and sucrose density gradient membrane partitioning studies. Lastly, a genetic screen using theiglE-null live vaccine strain resulted in the identification of key regions in the carboxyl terminus of IglE that are required for intracellular replication ofFrancisella tularensisin J774A.1 macrophages. Thus, IglE is essential forFrancisella tularensisvirulence. Our data support a model that likely includes protein-protein interactions at or near the bacterial cell surface that are unknown at present.

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How nanoscale protein interactions determine the mesoscale dynamic organisation of bacterial outer membrane proteins

Nature Communications ◽

10.1038/s41467-018-05255-9 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 20

Author(s):

Matthieu Chavent ◽

Anna L. Duncan ◽

Patrice Rassam ◽

Oliver Birkholz ◽

Jean Hélie ◽

...

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Protein Interactions ◽

Outer Membrane Proteins ◽

Bacterial Outer Membrane

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Accessing the distance range of interest in biomolecules: Site-directed spin labeling and DEER spectroscopy

Spectroscopy An International Journal ◽

10.1155/2010/729060 ◽

2010 ◽

Vol 24 (3-4) ◽

pp. 283-288 ◽

Cited By ~ 8

Author(s):

Sabine Böhme ◽

Heinz-Jürgen Steinhoff ◽

Johann P. Klare

Keyword(s):

Conformational Changes ◽

Protein Interactions ◽

Spin Labeling ◽

Paramagnetic Resonance ◽

Topological Information ◽

Pulsed Epr ◽

Site Directed Spin Labeling ◽

Intermolecular Distances ◽

And Function ◽

Electron Paramagnetic

Investigations on the structure and function of biomolecules often depend on the availability of topological information to build up structural models or to characterize conformational changes during function. Electron paramagnetic resonance (EPR) spectroscopy in combination with site–directed spin labeling (SDSL) allow to determine intra- and intermolecular distances in the range from 4–70 Å, covering the range of interest for biomolecules. The approach does not require crystalline samples and is well suited also for molecules exhibiting intrinsic flexibility. This article is intended to give an overview on pulsed EPR in conjunction with SDSL to study protein interactions as well as conformational changes, exemplified on the tRNA modifying enzyme MnmE.

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