Single-Channel Water Permeabilities of Escherichia coli Aquaporins AqpZ and GlpF

Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin–fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of theEscherichia colimaltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathwayviathe activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.

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High-resolution, low-dose electron cryomicroscopy of negatively stained matrix porin, a transmembrane protein from Escherichia coli outer membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128250 ◽

1987 ◽

Vol 45 ◽

pp. 788-789

Author(s):

A. Massalski ◽

H. J. Sass ◽

F. Zemlin ◽

E. Beckmann ◽

M. van Heel ◽

...

Keyword(s):

Escherichia Coli ◽

Single Channel ◽

Transmembrane Protein ◽

Membrane Vesicles ◽

3D Image ◽

Resolution Limit ◽

Electron Microscopic ◽

3D Image Reconstruction ◽

Lack Of Information ◽

Microscopic Studies

Porin (ompF gene product), an integral channel-forming protein in the outer membrane of Escherichia coli, is regarded as a molecular sieve allowing non-specific passive diffusion for low-molecular-weight (Mr < 700d) hydrophilic solutes.Electron microscopic studies of the negatively stained membrane vesicles reconstituted with porin and phospholipids have shown the presence of the stain-filled triplet indentation. Subsequent 3D image reconstruction demonstrated that three channels on the outer surface of the cell merge into a single channel at the periplasmatic face. Both 2D and 3D image reconstructions, based on electron micrographs from negatively stained preparations, are very informative about the size and distribution of the transmembrane channels but they contain very little or no information on the protein trimers which form the channels. This lack of information about the trimer structure and location is not surprising since the diffraction resolution limit, in our previous studies, is around 22Å.

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Monitoring Single-channel Water Permeability in Polarized Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m111.291864 ◽

2011 ◽

Vol 286 (46) ◽

pp. 39926-39932 ◽

Cited By ~ 10

Author(s):

Liudmila Erokhova ◽

Andreas Horner ◽

Philipp Kügler ◽

Peter Pohl

Keyword(s):

Water Permeability ◽

Single Channel ◽

Channel Water ◽

Polarized Cells

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Permeation of Antibiotics through Escherichia coli OmpF and OmpC Porins

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109357791 ◽

2010 ◽

Vol 15 (3) ◽

pp. 302-307 ◽

Cited By ~ 59

Author(s):

Kozhinjampara R. Mahendran ◽

Mohamed Kreir ◽

Helge Weingart ◽

Niels Fertig ◽

Mathias Winterhalter

Keyword(s):

Escherichia Coli ◽

Single Molecule ◽

Single Channel ◽

Patch Clamp Technique ◽

Single Molecule Level ◽

Solution Exchange ◽

Automated Patch Clamp ◽

Low Capacitance ◽

Bacterial Porins

A chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo–giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics—cephalosporins and fluoroquinolones—through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. The chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level.

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Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.185.18.5491-5499.2003 ◽

2003 ◽

Vol 185 (18) ◽

pp. 5491-5499 ◽

Cited By ~ 40

Author(s):

Sun Nyunt Wai ◽

Marie Westermark ◽

Jan Oscarsson ◽

Jana Jass ◽

Elke Maier ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Interactions ◽

Single Channel ◽

Dominant Negative ◽

Amino Acid Residues ◽

Wild Type ◽

Protein Protein Interactions ◽

Mutant Proteins ◽

Negative Feature

ABSTRACT We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.

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The channel-tunnel HI1462 of Haemophilus influenzae reveals differences to Escherichia coli TolC

Microbiology ◽

10.1099/mic.0.28805-0 ◽

2006 ◽

Vol 152 (6) ◽

pp. 1639-1647 ◽

Cited By ~ 5

Author(s):

Georg Polleichtner ◽

Christian Andersen

Keyword(s):

Escherichia Coli ◽

Single Channel ◽

Efflux Pump ◽

Wild Type ◽

Multidrug Efflux ◽

Channel Conductance ◽

Single Channel Conductance ◽

Multidrug Efflux Pump ◽

E Coli ◽

Tunnel Entrance

Efflux pumps play a major role in multidrug resistance of pathogenic bacteria. The TolC homologue HI1462 was identified as the single channel-tunnel in Haemophilus influenzae required to form a functional multidrug efflux pump. The outer-membrane protein was expressed in Escherichia coli, purified and reconstituted in black lipid membranes. It exhibited a comparatively small single-channel conductance of 43 pS in 1 M KCl and is the first known TolC homologue which is anion-selective. The HI1462 structure was modelled and an arginine residue lining the tunnel entrance was identified. The channel-tunnel of a mutant with the arginine substituted by an alanine residue was cation-selective and had a sevenfold higher single-channel conductance compared to wild-type. These results confirm that the arginine is responsible for anion selectivity and forms a salt bridge with a glutamate residue of the adjacent monomer, establishing a circular network, which keeps the tunnel entrance in a tightly closed conformation. In in vivo experiments, both the wild-type HI1462 and the mutant were able to substitute for E. coli TolC in the haemolysin secretion system, but not in the AcrAB/TolC multidrug efflux pump. The structure–function relationship of HI1462 is discussed in the context of the well-studied TolC channel-tunnel of E. coli.

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