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

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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Assembly of outer-membrane proteins in bacteria and mitochondria

Microbiology ◽

10.1099/mic.0.042689-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2587-2596 ◽

Cited By ~ 74

Author(s):

Jan Tommassen

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Cell Envelope ◽

Outer Membrane Proteins ◽

Basic Mechanism ◽

Eukaryotic Cell ◽

Mitochondrial Outer Membrane ◽

Central Component ◽

Cell Organelles ◽

C Terminus

The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobic α-helices, integral outer-membrane proteins (OMPs) form β-barrels. Similar β-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How these β-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly of β-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrial β-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.

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Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.6.1913-1922.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 1913-1922 ◽

Cited By ~ 50

Author(s):

Anindya S. Ghosh ◽

Kevin D. Young

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Outer Membrane ◽

Fluorescent Dyes ◽

Cell Envelope ◽

Outer Membrane Proteins ◽

E Coli ◽

Physiological Processes ◽

Side Walls

ABSTRACT In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.

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Bacterial Phage Receptors, Versatile Tools for Display of Polypeptides on the Cell Surface

Journal of Bacteriology ◽

10.1128/jb.183.23.6924-6935.2001 ◽

2001 ◽

Vol 183 (23) ◽

pp. 6924-6935 ◽

Cited By ~ 25

Author(s):

Hildegard Etz ◽

Duc Bui Minh ◽

Carola Schellack ◽

Eszter Nagy ◽

Andreas Meinke

Keyword(s):

Amino Acids ◽

Membrane Proteins ◽

Cell Surface ◽

Outer Membrane ◽

Cell Sorting ◽

Fusion Proteins ◽

Outer Membrane Proteins ◽

Wild Type ◽

E Coli ◽

Multiple Copies

ABSTRACT Four outer membrane proteins of Escherichia coli were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescence-activated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of E. coli as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B12 and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and λ, OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of E. coli for diverse applications.

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A bacterial secretosome for regulated envelope biogenesis and quality control

10.1101/2022.01.12.476021 ◽

2022 ◽

Author(s):

Daniel William Watkins ◽

Ian Collinson

Keyword(s):

Quality Control ◽

Outer Membrane ◽

Protein Delivery ◽

Cell Envelope ◽

Outer Membrane Proteins ◽

Inner Membrane ◽

Protein Insertion ◽

Membrane Protein Insertion ◽

Sec Translocon ◽

Complex Forms

As the first line of defence against antibiotics, the Gram-negative bacterial envelope and its biogenesis are of considerable interest to the microbiological and biomedical communities. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machinery. The Sec translocon, a ubiquitous integral inner-membrane (IM) complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed to the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm or delivered to the β-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core heterotrimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein delivery and quality control. Here, we conduct a biochemical exploration of this secretosome: a very large, versatile and inter-changeable assembly with the Sec-translocon at its core; featuring interactions that facilitate secretion (SecDF), inner- and outer-membrane protein insertion (respectively, YidC and BAM), protein folding and quality control (e.g. PpiD, YfgM and FtsH). We propose the dynamic interplay amongst these and other factors act to ensure efficient whole envelope biogenesis, regulated to accommodate the requirements of cell elongation and division. This organisation would be essential for cell wall biogenesis and remodelling and thus its perturbation would be a good strategy for the development of anti-microbials.

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Characterization of the Effects ofn-butanol on the cell envelope ofE. coli

10.1101/062547 ◽

2016 ◽

Author(s):

Eugene Fletcher ◽

Teuta Pilizota ◽

Philip R. Davies ◽

Alexander McVey ◽

Chris E. French

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Cell Envelope ◽

Membrane Protrusion ◽

Bacterial Strains ◽

External Concentration ◽

Inner Membrane ◽

E Coli ◽

Rational Engineering

ABSTRACTBiofuel alcohols have severe consequences on the microbial hosts used in their biosynthesis, which limits the productivity of the bioconversion. The cell envelope is one of the most strongly affected structures, in particular, as the external concentration of biofuels rises during biosynthesis. Damage to the cell envelope can have severe consequences, such as impairment of transport into and out of the cell; however the nature of butanol-induced envelope damage has not been well characterized. In the present study, the effects ofn-butanol on the cell envelope ofEscherichia coliwere investigated. Using enzyme and fluorescence-based assays, we observed that 1% v/v n-butanol resulted in release of lipopolysaccharides from the outer membrane ofE. coliand caused ‘leakiness’ in both outer and inner membranes. Higher concentrations ofn-butanol, within the range of 2% – 10% (v/v), resulted in inner membrane protrusion through the peptidoglycan observed by characteristic blebs. The findings suggest that strategies for rational engineering of butanol-tolerant bacterial strains should take into account all components of the cell envelope.

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Colicin translocation across the Escherichia coli outer membrane

Biochemical Society Transactions ◽

10.1042/bst20120255 ◽

2012 ◽

Vol 40 (6) ◽

pp. 1475-1479 ◽

Cited By ~ 18

Author(s):

Nicholas G. Housden ◽

Colin Kleanthous

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Protein Interactions ◽

Cell Envelope ◽

Protonmotive Force ◽

Protein Protein Interactions ◽

Inner Membrane ◽

E Coli ◽

Group A

We are investigating how protein bacteriocins import their toxic payload across the Gram-negative cell envelope, both as a means of understanding the translocation process itself and as a means of probing the organization of the cell envelope and the function of the protein machines within it. Our work focuses on the import mechanism of the group A endonuclease (DNase) colicin ColE9 into Escherichia coli, where we combine in vivo observations with structural, biochemical and biophysical approaches to dissect the molecular mechanism of colicin entry. ColE9 assembles a multiprotein ‘translocon’ complex at the E. coli outer membrane that triggers entry of the toxin across the outer membrane and the simultaneous jettisoning of its tightly bound immunity protein, Im9, in a step that is dependent on the protonmotive force. In the present paper, we focus on recent work where we have uncovered how ColE9 assembles its translocon complex, including isolation of the complex, and how this leads to subversion of a signal intrinsic to the Tol–Pal assembly within the periplasm and inner membrane. In this way, the externally located ColE9 is able to ‘connect’ to the inner membrane protonmotive force via a network of protein–protein interactions that spans the entirety of the E. coli cell envelope to drive dissociation of Im9 and initiate entry of the colicin into the cell.

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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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