The molecular basis of regulation of bacterial capsule assembly by Wzc

AbstractBacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein “Wzx-Wzy” system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.

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Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

Journal of Bacteriology ◽

10.1128/jb.00135-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 4001-4016 ◽

Cited By ~ 26

Author(s):

Wallace A. Kaserer ◽

Xiaoxu Jiang ◽

Qiaobin Xiao ◽

Daniel C. Scott ◽

Matthew Bauler ◽

...

Keyword(s):

Amino Acids ◽

Outer Membrane ◽

Cell Envelope ◽

Outer Membrane Proteins ◽

Binding Motif ◽

Inner Membrane ◽

E Coli ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

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Structure of BamA, an essential factor in outer membrane protein biogenesis

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714007482 ◽

2014 ◽

Vol 70 (6) ◽

pp. 1779-1789 ◽

Cited By ~ 64

Author(s):

Reinhard Albrecht ◽

Monika Schütz ◽

Philipp Oberhettinger ◽

Michaela Faulstich ◽

Ivan Bermejo ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Cell Envelope ◽

Significant Degree ◽

State Function ◽

E Coli ◽

Functional Studies ◽

C Terminus ◽

Protein Biogenesis

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. InEscherichia colithis function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of theE. coliBamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA fromHaemophilus ducreyiandNeisseria gonorrhoeaeand are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation,E. coliBamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer.E. coliBamA is characterized by a discontinuous β-barrel with impaired β1–β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

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High-resolution gold labeling

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147491 ◽

1993 ◽

Vol 51 ◽

pp. 330-331

Author(s):

James F. Hainfeld ◽

Frederic R. Furuya ◽

Kyra Carbone ◽

Martha Simon ◽

Beth Lin ◽

...

Keyword(s):

Dihydrofolate Reductase ◽

Polypeptide Chain ◽

External Surface ◽

Gold Cluster ◽

Macromolecular Complex ◽

Gold Compound ◽

Central Cavity ◽

Chain Folding ◽

E Coli ◽

Chaperonin Groel

A recently developed 1.4 nm gold cluster has been found to be useful in labeling macromolecular sites to 1-3 nm resolution. The gold compound is organically derivatized to contain a monofunctional arm for covalent linking to biomolecules. This may be used to mark a specific site on a structure, or to first label a component and then reassemble a multicomponent macromolecular complex. Two examples are given here: the chaperonin groEL and ribosomes.Chaperonins are essential oligomeric complexes that mediate nascent polypeptide chain folding to produce active proteins. The E. coli chaperonin, groEL, has two stacked rings with a central hole ∽6 nm in diameter. The protein dihydrofolate reductase (DHFR) is a small protein that has been used in chain folding experiments, and serves as a model substrate for groEL. By labeling the DHFR with gold, its position with respect to the groEL complex can be followed. In particular, it was sought to determine if DHFR refolds on the external surface of the groEL complex, or whether it interacts in the central cavity.

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Faculty Opinions recommendation of The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1065743.524831 ◽

2007 ◽

Author(s):

Miguel Valvano

Keyword(s):

Cell Division ◽

Outer Membrane ◽

E Coli ◽

Membrane Invagination

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E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

Scientific Reports ◽

10.1038/s41598-019-51031-0 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Andrea Bogutzki ◽

Natalie Naue ◽

Lidia Litz ◽

Andreas Pich ◽

Ute Curth

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Protein Interactions ◽

Dna Polymerase Iii ◽

Protein Protein Interactions ◽

Single Stranded Dna ◽

E Coli ◽

Polymerase Iii ◽

C Terminus ◽

Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

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The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽

10.3390/catal11080926 ◽

2021 ◽

Vol 11 (8) ◽

pp. 926

Author(s):

Maria C. Martins ◽

Susana F. Fernandes ◽

Bruno A. Salgueiro ◽

Jéssica C. Soares ◽

Célia V. Romão ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Domain ◽

Reductase Activity ◽

Conserved Residues ◽

Mutant Proteins ◽

E Coli ◽

C Terminus ◽

Bona Fide ◽

Oxygen Reductase ◽

Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

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Function Trumps Form in Two Sugar Symporters, LacY and vSGLT

International Journal of Molecular Sciences ◽

10.3390/ijms22073572 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3572

Author(s):

Jeff Abramson ◽

Ernest M. Wright

Keyword(s):

Binding Site ◽

Sugar Transport ◽

Electrochemical Potential ◽

Inverted Repeats ◽

Side Chains ◽

Transmembrane Helices ◽

Central Cavity ◽

Potential Gradients ◽

Sodium Binding ◽

Major Facilitator

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1–5 and TM 6–10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.

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Conformational dynamics and membrane interactions of the E. coli outer membrane protein FecA: A molecular dynamics simulation study

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2012.08.021 ◽

2013 ◽

Vol 1828 (2) ◽

pp. 284-293 ◽

Cited By ~ 41

Author(s):

Thomas J. Piggot ◽

Daniel A. Holdbrook ◽

Syma Khalid

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Membrane Protein ◽

Outer Membrane ◽

Simulation Study ◽

Outer Membrane Protein ◽

Conformational Dynamics ◽

Dynamics Simulation ◽

Membrane Interactions ◽

E Coli

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Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Nanomaterials ◽

10.3390/nano11071661 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1661

Author(s):

Mei-Hsiu Chen ◽

Tse-Ying Liu ◽

Yu-Chiao Chen ◽

Ming-Hong Chen

Keyword(s):

Outer Membrane ◽

Membrane Vesicle ◽

Glioma Cells ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

E Coli ◽

Tumor Bearing ◽

Nano Gold ◽

Gl261 Cell

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.

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