Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 338-344
Author(s):  
J S Wolfson ◽  
D C Hooper ◽  
M N Swartz ◽  
G L McHugh
Keyword(s):  
Escherichia Coli ◽  
Dna Gyrase ◽  
B Subunit ◽  
Plasmid Loss ◽  
Escherichia Coli Cells ◽  
Bacterial Mutant ◽  
Bacterial Enzyme ◽  
Kinetics Of ◽  
Plasmid Elimination ◽  
Native Plasmid

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.

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

scholarly journals Kinetics of Acid-mediated Disassembly of the B Subunit Pentamer of Escherichia coli Heat-labile Enterotoxin

1995 ◽  
Vol 270 (50) ◽  
pp. 29953-29958 ◽  
Author(s):  
Lloyd W. Ruddock ◽  
Stephen P. Ruston ◽  
Sharon M. Kelly ◽  
Nicholas C. Price ◽  
Robert B. Freedman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
B Subunit ◽  
Heat Labile ◽  
Kinetics Of

scholarly journals Conjugative Transfer Facilitates Stable Maintenance of IncP-1 Plasmid pKJK5 in Escherichia coli Cells Colonizing the Gastrointestinal Tract of the Germfree Rat

2006 ◽  
Vol 73 (1) ◽  
pp. 341-343 ◽  
Author(s):  
Martin Iain Bahl ◽  
Lars Hestbjerg Hansen ◽  
Tine Rask Licht ◽  
Søren J. Sørensen
Keyword(s):  
Escherichia Coli ◽  
Flow Cytometry ◽  
Gastrointestinal Tract ◽  
Quantitative Determination ◽  
Conjugative Transfer ◽  
Plasmid Loss ◽  
Escherichia Coli Cells ◽  
Stable Maintenance ◽  
Germfree Rats

ABSTRACT Quantitative determination of IncP-1 plasmid loss from Escherichia coli cells colonizing the gastrointestinal tracts of germfree rats was achieved by flow cytometry. Results show that the plasmid's ability to conjugate counteracts plasmid loss and is thus an important mechanism for the stable maintenance of IncP-1 plasmids within the gastrointestinal environment.

A novel zinc-dependent D-serine dehydratase from Saccharomyces cerevisiae

2007 ◽  
Vol 409 (2) ◽  
pp. 399-406 ◽  
Author(s):  
Tomokazu Ito ◽  
Hisashi Hemmi ◽  
Kunishige Kataoka ◽  
Yukio Mukai ◽  
Tohru Yoshimura
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Alanine Racemase ◽  
Absorption Analysis ◽  
Edta Treatment ◽  
Putative Protein ◽  
Escherichia Coli Cells ◽  
Bacterial Enzyme ◽  
Serine Dehydratase ◽  
Pyridoxal Enzyme

YGL196W of Saccharomyces cerevisiae encodes a putative protein that is unidentified but is predicted to have a motif similar to that of the N-terminal domain of the bacterial alanine racemase. In the present study we found that YGL196W encodes a novel D-serine dehydratase, which belongs to a different protein family from that of the known bacterial enzyme. The yeast D-serine dehydratase purified from recombinant Escherichia coli cells depends on pyridoxal 5′-phosphate and zinc, and catalyses the conversion of D-serine into pyruvate and ammonia with the Km and kcat values of 0.39 mM and 13.1 s−1 respectively. D-Threonine and β-Cl-D-alanine also serve as substrates with catalytic efficiencies which are approx. 3 and 2% of D-serine respectively. L-Serine, L-threonine and β-Cl-L-alanine are inert as substrates. Atomic absorption analysis revealed that the enzyme contains one zinc atom per enzyme monomer. The enzyme activities toward D-serine and D-threonine were decreased by EDTA treatment and recovered by the addition of Zn2+. Little recovery was observed with Mg2+, Mn2+, Ca2+, Ni2+, Cu2+, K+ or Na+. In contrast, the activity towards β-Cl-D-alanine was retained after EDTA treatment. These results suggest that zinc is involved in the elimination of the hydroxy group of D-serine and D-threonine. D-Serine dehydratase of S. cerevisiae is probably the first example of a eukaryotic D-serine dehydratase and that of a specifically zinc-dependent pyridoxal enzyme as well.

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