scholarly journals Escherichia coli amino acid auxotrophic expression host strains for investigating protein structure-function relationships

2020 ◽  
Author(s):  
Toshio Iwasaki ◽  
Yoshiharu Miyajima-Nakano ◽  
Risako Fukazawa ◽  
Myat T Lin ◽  
Shin-Ichi Matsushita ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Structure ◽  
Amino Acid ◽  
Structure Function ◽  
Growth Medium ◽  
Isotope Labeling ◽  
Water Soluble ◽  
Expression Host ◽  
Host Strains

Abstract A set of C43(DE3) and BL21(DE3) Escherichia coli host strains that are auxotrophic for various amino acids is briefly reviewed. These strains require the addition of a defined set of one or more amino acids in the growth medium, and have been specifically designed for overproduction of membrane or water-soluble proteins selectively labeled with stable isotopes such as 2H, 13C and 15N. The strains described here are available for use and have been deposited into public strain banks. Although they cannot fully eliminate the possibility of isotope dilution and mixing, metabolic scrambling of the different amino acid types can be minimized through a careful consideration of the bacterial metabolic pathways. The use of a suitable auxotrophic expression host strain with an appropriately isotopically labeled growth medium ensures high levels of isotope labeling efficiency as well as selectivity for providing deeper insight into protein structure-function relationships.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

Physiologische Bedeutung der „Stringent Control“ bei Escherichia coli unter extremen Hungerbedingungen / Stringent Control and Starvation Survival in Escherichia coli

1989 ◽  
Vol 44 (9-10) ◽  
pp. 838-844 ◽  
Author(s):  
H. Mach ◽  
M. Hecker ◽  
I. Hill ◽  
A. Schroeter ◽  
F. Mach
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Protein Turnover ◽  
Stringent Response ◽  
Phosphate Starvation ◽  
Pleiotropic Effects ◽  
Prolonged Starvation ◽  
Stringent Control ◽  
Starvation Survival

The viability of three isogenic relA+/relA strain pairs of Escherichia coli (CP78/CP79; NF 161/ NF162; CP 107/CP 143) was studied during prolonged starvation for amino acids, glucose or phosphate. After amino acid limitation we found a prolonged viability of all relA+ strains which synthesized ppGpp. We suggest that some ppGpp-mediated pleiotropic effects of the stringent response (e.g. glykogen accumulation, enhanced protein turnover) might be involved in this prolongation of survival. After glucose or phosphate starvation there was no difference in the relA+/relA strains either in the ppGpp content or in the survival.

scholarly journals Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Im [corrected] associated with a sulI-type integron.

1997 ◽  
Vol 41 (2) ◽  
pp. 314-318 ◽  
Author(s):  
E Hannecart-Pokorni ◽  
F Depuydt ◽  
L de wit ◽  
E van Bossuyt ◽  
J Content ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Resistance Gene ◽  
Citrobacter Freundii ◽  
Gram Negative ◽  
Gene Environment ◽  
Insert Region ◽  
Klebsiella Spp

The amikacin resistance gene aac(6')-Im [corrected] from Citrobacter freundii Cf155 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 521 bp located down-stream from the 5' conserved segment of an integron. The sequence of this aac(6')-Im [corrected] gene corresponded to a protein of 173 amino acids which possessed 64.2% identity in a 165-amino-acid overlap with the aac(6')-Ia gene product (F.C. Tenover, D. Filpula, K.L. Phillips, and J. J. Plorde, J. Bacteriol. 170:471-473, 1988). By using PCR, the aac(6')-Im [corrected] gene could be detected in 8 of 86 gram-negative clinical isolates from two Belgian hospitals, including isolates of Citrobacter, Klebsiella spp., and Escherichia coli. PCR mapping of the aac(6')-Im [corrected] gene environment in these isolates indicated that the gene was located within a sulI-type integron; the insert region is 1,700 bases long and includes two genes cassettes, the second being ant (3")-Ib.

THE OXIDATION OF ESTRONE-16-C14BY PEROXIDASE

1962 ◽  
Vol 40 (1) ◽  
pp. 459-469 ◽  
Author(s):  
P. H. Jellinck ◽  
Louise Irwin
Keyword(s):  
Amino Acids ◽  
Hydrogen Peroxide ◽  
Amino Acid ◽  
Serum Albumin ◽  
Oxidase Activity ◽  
Water Soluble ◽  
The Other ◽  
Aerobic Incubation ◽  
Nitrogenous Compounds ◽  
Initial Generation

Aerobic incubation of estrone-16-C14with peroxidase in the presence of serum albumin and other proteins resulted in the formation of water-soluble, ether-insoluble metabolites in high percentage yields. Similar products were formed when protein was replaced by cysteine or tryptophan but none of the other amino acids tested had any effect. The evidence points to an initial generation of hydrogen peroxide from these nitrogenous compounds by the enzyme acting as an aerobic oxidase, and the subsequent peroxidation of estrone to highly reactive products. These then combine with the protein or amino acid or else undergo alternative reactions. A strong chemical bond is formed with albumin and attempts to release the estrone metabolites from it were unsuccessful. Uterine homogenates from estrogen-treated rats showing high DPNH oxidase activity contained no "peroxidase" as measured by the formation of water-soluble products from estrone in the presence of protein.

scholarly journals Amino Acid Toxicities of Escherichia coli That Are Prevented by Leucyl-tRNA Synthetase Amino Acid Editing

2007 ◽  
Vol 189 (23) ◽  
pp. 8765-8768 ◽  
Author(s):  
Vrajesh A. Karkhanis ◽  
Anjali P. Mascarenhas ◽  
Susan A. Martinis
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Trna Synthetase ◽  
Amino Acid Editing

ABSTRACT Leucyl-tRNA synthetase (LeuRS) has evolved an editing function to clear misactivated amino acids. An Escherichia coli-based assay was established to identify amino acids that compromise the fidelity of LeuRS and translation. Multiple nonstandard as well as standard amino acids were toxic to the cell when LeuRS editing was inactivated.

scholarly journals Mutation in the DNA Gyrase A Gene ofEscherichia coli That Expands the Quinolone Resistance-Determining Region

2001 ◽  
Vol 45 (8) ◽  
pp. 2378-2380 ◽  
Author(s):  
S. Marvin Friedman ◽  
Tao Lu ◽  
Karl Drlica
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Dna Gyrase ◽  
Ofescherichia Coli ◽  
Quinolone Resistance ◽  
Gyrase A

ABSTRACT In three Escherichia coli mutants, a change (Ala-51 to Val) in the gyrase A protein outside the standard quinolone resistance-determining region (QRDR) lowered the level of quinolone susceptibility more than changes at amino acids 67, 82, 84, and 106 did. Revision of the QRDR to include amino acid 51 is indicated.

One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains

2012 ◽  
Vol 426 (2) ◽  
pp. 126-128 ◽  
Author(s):  
Christopher O’Grady ◽  
Benjamin L. Rempel ◽  
Akosiererem Sokaribo ◽  
Sergiy Nokhrin ◽  
Oleg Y. Dmitriev
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Isotope Labeling ◽  
Selective Isotope Labeling ◽  
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