scholarly journals Colicins, spermine and cephalosporins: a competitive interaction with the OmpF eyelet

2003 ◽  
Vol 376 (1) ◽  
pp. 245-252 ◽  
Author(s):  
Jérôme BREDIN ◽  
Valérie SIMONET ◽  
Ramkumar IYER ◽  
Anne H. DELCOUR ◽  
Jean-Marie PAGÈS
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Essential Role ◽  
Wild Type ◽  
Potassium Efflux ◽  
Channel Size ◽  
Escherichia Coli Cells ◽  
Epitope Structure ◽  
Resistant Strains ◽  
Kinetics Of

The L3 loop is an important feature of the OmpF porin structure, contributing to both channel size and electrostatic properties. Colicins A and N, spermine, and antibiotics that use OmpF to penetrate the cell, were used to investigate the structure–function relationships of L3. Spermine was found to protect efficiently cells expressing wild-type OmpF from colicin action. Among other solutes, sugars had minor effects on colicin A activity, whereas competitions between colicin A and antibiotic fluxes were observed. Among the antibiotics tested, cefepime appeared the most efficient. Escherichia coli cells expressing various OmpF proteins mutated in the eyelet were tested for their susceptibility to colicin A, and resistant strains were found only among L3 mutants. Mutations at residues 119 and 120 were the most effective at conferring resistance to colicin A, probably due to epitope structure alteration, as revealed by a specific antipeptide. More detailed information was obtained on mutants D113A and D121A, by focusing on the kinetics of colicin A and colicin N activities through measurements of potassium efflux. D113 appeared to play an essential role for colicin A activity, whereas colicin N activity was more dependent on D121 than on D113.

scholarly journals Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

1991 ◽  
Vol 276 (3) ◽  
pp. 637-641 ◽  
Author(s):  
F F Craig ◽  
A C Simmonds ◽  
D Watmore ◽  
F McCapra ◽  
M R H White
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Cos Cells ◽  
Escherichia Coli Cells ◽  
Luminescent Signal ◽  
The Difference ◽  
Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

Kinetics of mercuric reduction in intact and permeabilized Escherichia coli cells

1990 ◽  
Vol 12 (11) ◽  
pp. 854-859 ◽  
Author(s):  
George P. Philippidis ◽  
Janet L. Schottel ◽  
Wei-Shou Hu
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli Cells ◽  
Kinetics Of

The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3

2012 ◽  
Vol 97 (11) ◽  
pp. 4849-4858 ◽  
Author(s):  
Jasmina Nikodinovic-Runic ◽  
Lydie Coulombel ◽  
Djordje Francuski ◽  
Narain D. Sharma ◽  
Derek R. Boyd ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Wild Type ◽  
Styrene Monooxygenase ◽  
Escherichia Coli Cells

K+ Uptake by Fermenting Escherichia coli Cells: pH Dependent Mode of the TrkA System Operating

2000 ◽  
Vol 20 (4) ◽  
pp. 277-288 ◽  
Author(s):  
Armen Trchounian ◽  
Hiroshi Kobayashi
Keyword(s):  
Escherichia Coli ◽  
Membrane Vesicles ◽  
Wild Type ◽  
K Uptake ◽  
Logarithmic Growth Phase ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
K Concentration ◽  
System Operating ◽  
Logarithmic Growth

Escherichia coli accumulates K+ by means of multiple transportsystems, of which TrkA is the most prominent at neutral and alkalinepH while Kup is major at acidic pH. In the present study, K+ uptakewas observed with cells grown under fermentative conditions at an initialpH of 9.0 and 7.3 (the medium pH decreased to 8.4 and 6.8, respectively, during the mid-logarithmic growth phase), washed with distilled water andresuspended in a K+ containing medium at pH 7.5 in the presence ofglucose. The kinetics for this K+ uptake and the amount of K+accumulated by the wild type and mutants having a functional TrkA or Kup could confirm that K+ uptake by E. coli grown either at pH 9.0or pH 7.3 occurs mainly through TrkA. The following results distinguishpH dependent mode of TrkA operating: (1) K+ uptake was inhibited byDCCD in cells grown either at pH 9.0 or pH 7.3, although the stoichiometryof K+ influx to DCCD-inhibited H+ efflux for bacteria grownat pH 9.0 varied with external K+ concentration, but remained constantfor cells grown at pH 7.3; (2) K+ uptake was observed with an atpDmutant grown at pH 9.0 but not at pH 7.3; (3) The DCCD-inhibited H+efflux was increased 8-fold less by 5 mM K+ added into a K+ freemedium for bacteria grown at pH 9.0 than that for cells grown at pH 7.3;(4) the DCCD-inhibited ATPase activity of membrane vesicles from bacteriagrown at pH 9.0 was reduced a little in the presence of 100 mM K+, but stimulated more than 2.4-fold at pH 7.3.

KINETICS OF THREONINE DEAMINASE OF ESCHERICHIA COLI K-12 AND A STREPTOMYCIN-DEPENDENT MUTANT

1967 ◽  
Vol 45 (1) ◽  
pp. 1-10 ◽  
Author(s):  
I. D. Desai ◽  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Selective Advantage ◽  
Kinetic Properties ◽  
Wild Type ◽  
Threonine Deaminase ◽  
E Coli ◽  
Type K ◽  
Hyperbolic Curve ◽  
K 12 ◽  
Kinetics Of

The relation between threonine deaminase activity and threonine concentration in sonic extracts of wild-type and streptomycin-dependent Escherichia coli K-12 was found to follow a hyperbolic curve. A similar relationship was obtained between enzyme activity and pyridoxal concentration. However, when serine was used as substrate, the activity–concentration curve was sigmoid, suggesting that serine may be a weaker effector of allosteric transition than threonine. The kinetic properties of the (derepressed) threonine deaminase of streptomycin-dependent E. coli K-12 were found to be similar to those of the enzyme of the wild-type K-12.It is postulated that derepression of threonine deaminase in streptomycin-dependent E. coli K-12 provides a selective advantage which permits exponential growth of this mutant in the presence of L-valine, which is an excretory product of streptomycin-dependent microorganisms.

scholarly journals Lysine Residue 117 of the FasG Adhesin of Enterotoxigenic Escherichia coli Is Essential for Binding of 987P Fimbriae to Sulfatide

1999 ◽  
Vol 67 (11) ◽  
pp. 5755-5761 ◽  
Author(s):  
Byung-Kwon Choi ◽  
Dieter M. Schifferli
Keyword(s):  
Escherichia Coli ◽  
Essential Role ◽  
Surface Display ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Enterotoxigenic Escherichia Coli ◽  
Wild Type ◽  
Binding Properties ◽  
Brush Borders ◽  
Positively Charged

ABSTRACT The FasG subunit of the 987P fimbriae of enterotoxigenic strains ofEscherichia coli was previously shown to mediate fimbrial binding to a glycoprotein and a sulfatide receptor on intestinal brush borders of piglets. Moreover, the 987P adhesin FasG is required for fimbrial expression, since fasG null mutants are nonfimbriated. In this study, fasG was modified by site-directed mutagenesis to study its sulfatide binding properties. Twenty single mutants were generated by replacing positively charged lysine (K) or arginine (R) residues with small, nonpolar alanine (A) residues. Reduced levels of binding to sulfatide-containing liposomes correlated with reduced fimbriation and FasG surface display in fourfasG mutants (R27A, R286A, R226A, and R368). Among the 16 remaining normally fimbriated mutants with wild-type levels of surface-exposed FasG, only one mutant (K117A) did not interact at all with sulfatide-containing liposomes. Four mutants (K117A, R116A, K118A, and R200A) demonstrated reduced binding to such liposomes. Since complete phenotypic dissociation between the structure and specific function of 987P was observed only with mutant K117A, this residue is proposed to play an essential role in the FasG-sulfatide interaction, possibly communicating with the sulfate group of sulfatide by hydrogen bonding and/or salt bridge formation. Residues K17, R116, K118, and R200 may stabilize this interaction.

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