Proteolytic digestion and labelling studies of the organization of the proteins in the outer membrane of Escherichia coli

1977 ◽  
Vol 55 (10) ◽  
pp. 1082-1090 ◽  
Author(s):  
Reinhart A. F. Reithmeier ◽  
Philip D. Bragg
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Hydrophobic Interactions ◽  
Proteolytic Digestion ◽  
Wild Type ◽  
Terminal Portion ◽  
Intact Cells ◽  
Protein Components ◽  
Pronase Treatment ◽  
Protein B

The arrangement of the proteins in the outer membrane of Escherichia coli was examined by treating intact cells and isolated membrane preparations with fluorescamine and with pronase. Intact wild-type cells, or those of a mutant in which the core region of the lipopolysaccharide was absent, were equally resistant to pronase treatment. The protein components of isolated outer membrane preparations varied in their rate of digestion and labelling with fluorescamine. The N-terminal portion of protein B was removed by pronase to yield a fragment (protein Bp) still embedded in the membrane. Protein Bp was not significantly enriched in nonpolar amino acids, suggesting that protein B may not be held in the membrane primarily by hydrophobic interactions. This was confirmed by reconstitution experiments in which protein B could be reassociated with itself, without lipopolysaccharide or phospholipid, in the presence of a divalent cation such that pronase digestion of the reassociated material gave protein Bp.

scholarly journals Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli.

1981 ◽  
Vol 77 (2) ◽  
pp. 121-135 ◽  
Author(s):  
H Nikaido ◽  
E Y Rosenberg
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Specific Class ◽  
Wild Type ◽  
Intact Cells ◽  
E Coli ◽  
Low Concentrations ◽  
Transmembrane Pores ◽  
Diffusion Rates ◽  
Type Strains

Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.

Involvement of outer membrane proteins in freeze–thaw resistance of Escherichia coli

1984 ◽  
Vol 30 (3) ◽  
pp. 339-344 ◽  
Author(s):  
Peter H. Calcott ◽  
Katherine N. Calcott
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Damage ◽  
Wild Type ◽  
Freezing And Thawing ◽  
Freeze Thaw ◽  
Porin Proteins ◽  
Protein Components ◽  
Type Strains

Two families of Escherichia coli mutants with altered outer membrane protein components were examined for sensitivity to freezing and thawing and other stresses. A mutant unable to make the lipoprotein (lpo) was extremely sensitive to freezing and thawing in water or saline and to challenge with detergent, while the mutant unable to make the porin proteins (ompB) was more resistant than the isogenic wild type; strains unable to make the tsx and ompA proteins were slightly more sensitive to the stresses. Similarly, the lpo deficient strain exhibited more and the ompB less wall and membrane damage than the wild-type strains. Little difference in the extent of wall damage, but more membrane damage, was seen for the two tsx and the ompA strains when compared with the wild-type strain. The roles of the specific proteins in determining sensitivity to freeze–thaw are discussed.

Extrusion of actin-positive strands from HEp-2 and Int 407 cells caused by outer membrane preparations of enteropathogenicEscherichia coliand specific attachment of wild type bacteria to the strands

2001 ◽  
Vol 47 (8) ◽  
pp. 727-734 ◽  
Author(s):  
Sukumaran Sunil Kumar ◽  
Vasantha Malladi ◽  
Krishnan Sankaran ◽  
Richard Haigh ◽  
Peter Williams ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Three Dimensional ◽  
Secretory Proteins ◽  
Enteropathogenic Escherichia Coli ◽  
Wild Type ◽  
Cytopathic Effects ◽  
Cytoskeletal Elements

Enteropathogenic Escherichia coli (EPEC) causes persistent infantile diarrhoea. This nontoxigenic E. coli exhibits a complicated pathogenic mechanism in which its outer membrane proteins and type III secretory proteins damage intestinal epithelium and cause diarrhoea. In accordance with this, our previous study using HEp-2 cells demonstrated cytopathic effects caused by cell-free outer membrane preparations of EPEC. In this study, we report the extrusion of actin-positive strands from HEp-2 and Int 407 cells when treated with outer membrane preparations. An interesting observation of this work, perhaps relevant to the characteristic localized three-dimensional colony formation of EPEC, is the attachment of a wild type EPEC strain to these actin-positive strands.Key words: enteropathogenic Escherichia coli, actin, outer membrane proteins, cytoskeletal elements.

Extrusion of actin-positive strands from HEp-2 and Int 407 cells caused by outer membrane preparations of enteropathogenic Escherichia coli and specific attachment of wild type bacteria to the strands

2001 ◽  
Vol 47 (8) ◽  
pp. 727-734 ◽  
Author(s):  
Sukumaran Sunil Kumar ◽  
Vasantha Malladi ◽  
Krishnan Sankaran ◽  
Richard Haigh ◽  
Peter Williams ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Wild Type ◽  
Specific Attachment

Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Essential Role ◽  
Biochemical Analysis ◽  
Hydrophobic Interactions ◽  
Outer Membrane Proteins ◽  
Structural Basis ◽  
Conserved Residues ◽  
Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

Stimulation of Escherichia coli adenylate cyclase by lactose in strains carrying mutations in lactose permease

1981 ◽  
Vol 1 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Alan Peterkofsky ◽  
Celia Gazdar
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Inhibitory Effect ◽  
Lactose Permease ◽  
Mutant Form ◽  
Wild Type ◽  
Camp Phosphodiesterase ◽  
Intact Cells

When a wild-type strain of Escherichia coli contains lactose permease, the accumulation of cyclic AMP (cAMP) by intact cells is inhibited by lactose. This inhibitory effect of lactose is observed in a strain with a mutant cAMP phosphodiesterase and therefore involves a regulation of adenylate cyctase activity. Some E. coli strains carrying mutations in lactose permease show an effect opposite to that of the wild-type strain; the accumulation of cAMP by intact cells is stimulated by lactose, but only when the mutant permease is present. Insertion of lactose permease into the membrane of ceils can produce a change in the specific activity of adenylate cycIase; induction of the wild-type transporter is correlated with a decrease in the specific activity, while implantation of a mutant form of lactose permease can lead to an increase in the specific activity. From these data, it is suggested that the state of the lactose transporter in the cell membrane influences the activity of adenytate cyclase.

scholarly journals Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport

2021 ◽  
Author(s):  
Thushani D. Nilaweera ◽  
David A. Nyenhuis ◽  
David S. Cafiso
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Pathogenic Bacteria ◽  
Structural Transition ◽  
Substrate Binding ◽  
Outer Membrane Proteins ◽  
Intact Cells ◽  
Pulse Epr ◽  
Inner Membrane Protein ◽  
Dependent Transport

AbstractOuter membrane TonB-dependent transporters facilitate the uptake of trace nutrients and carbohydrates in Gram negative bacteria and are essential for pathogenic bacteria and the health of the microbiome. Despite this, their mechanism of transport is still unknown. Here, pulse EPR measurements were made in intact cells on the Escherichia coli vitamin B12 transporter, BtuB. Substrate binding was found to alter the C-terminal region of the core and shift an extracellular substrate binding loop 2 nm towards the periplasm; moreover, this structural transition is regulated by an ionic lock that is broken upon binding of the inner membrane protein TonB. Significantly, this structural transition is not observed when BtuB is reconstituted into phospholipid bilayers. These measurements suggest an alternative to existing models of transport, where TonB binding alone is sufficient to produce allosteric rearrangements in the transporter. They also demonstrate the importance of studying outer membrane proteins in their native environment.

scholarly journals Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

2000 ◽  
Vol 346 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Richard GRIEßLER ◽  
Sabato D'AURIA ◽  
Reinhard SCHINZEL ◽  
Fabio TANFANI ◽  
Bernd NIDETZKY
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Active Site ◽  
Thermal Denaturation ◽  
Hydrophobic Interactions ◽  
Wild Type ◽  
Reversible Inactivation ◽  
The Stability ◽  
Maltodextrin Phosphorylase

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each ≈ 90-kDa subunit contains active-site pyridoxal 5ʹ-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133 → Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at ≈ 45 °C. They are manifested by slight increases in unordered structure and 1H/2H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at ≈ 45 °C without prior release into solution of pyridoxal 5ʹ-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 °C. Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx. 58 and 67 °C, respectively. Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a ≈ 15 °C higher value on the temperature scale. Apart from greater stability, the Asn-133 → Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme. A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP.

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