scholarly journals Constructing a DNAzyme Delivery and Expression System for Escherichia coli: A Prototype for Phage Therapy

2021 ◽  
Vol 2 (2) ◽  
pp. 19-25
Author(s):  
Hugo V. C. Oliveira ◽  
Spartaco Astolfi-Filho ◽  
Edmar V. Andrade
Keyword(s):  
Escherichia Coli ◽  
Protein Delivery ◽  
Phage Therapy ◽  
Leukemia Virus ◽  
Expression System ◽  
Constitutive Expression ◽  
Primary Component ◽  
Morphological Pattern ◽  
Filamentous Form ◽  
E Coli

Antisense oligonucleotides exhibit high potential for use as therapeutic agents. '10-23' DNAzymes are antisense molecules with a high chemical stability and catalytic efficiency. In the present study, we developed a phagemid containing a DNAzyme expression system regulated by two promoters. One of these promoters, pA1, promotes constitutive expression of Moloney murine leukemia virus reverse transcriptase (MoMuLV-RT). The other promoter, plac, regulates transcription of the RNA substrate from which MoMuLV-RT produces the DNAzyme by reverse transcription. The ftsZ DNAzyme was used to validate this expression system in the phagemid, named pDESCP. ftsZ DNAzyme expression altered the morphological pattern of Escherichia coli from a bacillary to filamentous form. In E. coli FtsZ is the primary component of the cell division apparatus, forming a structure known as Z-ring, which is the place of division. It is suggested that the DNAzyme ftsZ is decreasing the translation of this protein. Delivery of pDESCP into F+ strain of E. coli cells, using VCSM13, and the possible insertion of other DNAzymes into the cassette makes this phagemid an important prototype for phage therapy.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Viability and survival capability of quinolone-resistant uropathogenic Escherichia coli

2010 ◽  
Vol 5 (6) ◽  
pp. 827-830
Author(s):  
Georgi Slavchev ◽  
Nadya Markova
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Expression System ◽  
Antibiotic Sensitivity ◽  
Uropathogenic Escherichia Coli ◽  
Resistant Bacteria ◽  
E Coli ◽  
Serum Bactericidal Assay ◽  
Tract Infections ◽  
Macrophage Killing

AbstractUropathogenic strains of E. coli isolated from urine of patients with urinary tract infections were tested for antibiotic sensitivity using bio-Merieux kits and ATB-UR 5 expression system. The virulence of strains was evaluated by serum bactericidal assay, macrophage “killing” and bacterial adhesive tests. Survival capability of strains was assessed under starvation in saline. The results showed that quinolone-resistant uropathogenic strains of E. coli exhibit significantly reduced adhesive potential but relatively high resistance to serum and macrophage bactericidity. In contrast to laboratory strains, the quinolone-resistant uropathogenic clinical isolate demonstrated increased viability during starvation in saline. Our study suggests that quinolone-resistant uropathogenic strains are highly adaptable clones of E. coli, which can exhibit compensatory viability potential under unfavorable conditions. The clinical occurrence of such phenotypes is likely to contribute to the survival, persistence and spread strategy of resistant bacteria.

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

Genetics and regulation of peptidase N in Escherichia coli K-12

1982 ◽  
Vol 152 (2) ◽  
pp. 848-854
Author(s):  
M T McCaman ◽  
A McPartland ◽  
M R Villarejo
Keyword(s):  
Escherichia Coli ◽  
Constitutive Expression ◽  
Growth Cycle ◽  
Growth Media ◽  
Chromosome Loss ◽  
E Coli ◽  
Cell Enzyme ◽  
Nutritional Needs ◽  
K 12 ◽  
Type Strains

Escherichia coli K-12 strains contain a cytoplasmic activity, peptidase N, capable of hydrolyzing alanine-p-nitroanilide. Mutations in the structural gene for the enzyme, pepN, were mapped, and the properties of mutant strains were examined. The pepN locus lay between ompF and asnS at approximately 20.8 min on the E. coli chromosome. Loss of peptidase N activity through mutation had no apparent effect on the growth rate or nutritional needs of the cell. Enzyme levels in wild-type strains were constant throughout the growth cycle and were constitutive in all of the growth media tested. Starvation for carbon, nitrogen, or phosphate also did not alter enzyme levels. Constitutive expression of peptidase N is consistent with the idea that the enzyme plays a significant role in the degradation of intracellularly generated peptides.

Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

1998 ◽  
Vol 64 (3) ◽  
pp. 1163-1165 ◽  
Author(s):  
Georges Feller ◽  
Olivier Le Bussy ◽  
Charles Gerday
Keyword(s):  
Escherichia Coli ◽  
Enzyme Stability ◽  
Expression System ◽  
Wild Strain ◽  
Culture Temperature ◽  
E Coli ◽  
System A ◽  
Irreversible Denaturation ◽  
The Antarctic ◽  
Folding State

ABSTRACT α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.

scholarly journals Constitutive soxR Mutations Contribute to Multiple-Antibiotic Resistance in Clinical Escherichia coli Isolates

2005 ◽  
Vol 49 (7) ◽  
pp. 2746-2752 ◽  
Author(s):  
Anastasia Koutsolioutsou ◽  
Samuel Peña-Llopis ◽  
Bruce Demple
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Dna Sequences ◽  
Single Point ◽  
Constitutive Expression ◽  
Clinical Isolates ◽  
Multiple Antibiotic Resistance ◽  
E Coli ◽  
First Time

ABSTRACT The soxRS regulon of Escherichia coli and Salmonella enterica is induced by redox-cycling compounds or nitric oxide and provides resistance to superoxide-generating agents, macrophage-generated nitric oxide, antibiotics, and organic solvents. We have previously shown that constitutive expression of soxRS can contribute to quinolone resistance in clinically relevant S. enterica. In this work, we have carried out an analysis of the mechanism of constitutive soxS expression and its role in antibiotic resistance in E. coli clinical isolates. We show that constitutive soxS expression in three out of six strains was caused by single point mutations in the soxR gene. The mutant SoxR proteins contributed to the multiple-antibiotic resistance phenotypes of the clinical strains and were sufficient to confer multiple-antibiotic resistance in a fresh genetic background. In the other three clinical isolates, we observed, for the first time, that elevated soxS expression was not due to mutations in soxR. The mechanism of such increased soxS expression remains unclear. The same E. coli clinical isolates harbored polymorphic soxR and soxS DNA sequences, also seen for the first time.

Oral Delivery Systems for Encapsulated Bacteriophages Targeted at Escherichia coli O157:H7 in Feedlot Cattle

2010 ◽  
Vol 73 (7) ◽  
pp. 1304-1312 ◽  
Author(s):  
K. STANFORD ◽  
T. A. McALLISTER ◽  
Y. D. NIU ◽  
T. P. STEPHENS ◽  
A. MAZZOCCO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oral Delivery ◽  
Phage Therapy ◽  
Feedlot Cattle ◽  
Low Ph ◽  
Complete Loss ◽  
Escherichia Coli O157 ◽  
Polymer Encapsulation ◽  
E Coli ◽  
Oral Delivery Systems

Bacteriophages are natural predators of bacteria and may mitigate Escherichia coli O157:H7 in cattle and their environment. As bacteriophages targeted to E. coli O157:H7 (phages) lose activity at low pH, protection from gastric acidity may enhance efficacy of orally administered phages. Polymer encapsulation of four phages, wV8, rV5, wV7, and wV11, and exposure to pH 3.0 for 20 min resulted in an average 13.6% recovery of phages after release from encapsulation at pH 7.2. In contrast, untreated phages under similar conditions had a complete loss of activity. Steers (n = 24) received 1011 CFU of naladixic acid–resistant E. coli O157:H7 on day 0 and were housed in six pens of four steers. Two pens were control (naladixic acid–resistant E. coli O157:H7 only), and the remaining pens received polymer-encapsulated phages (Ephage) on days −1, 1, 3, 6, and 8. Two pens received Ephage orally in gelatin capsules (bolus; 1010 PFU per steer per day), and the remaining two pens received Ephage top-dressed on their feed (feed; estimated 1011 PFU per steer per day). Shedding of E. coli O157:H7 was monitored for 10 weeks by collecting fecal grab and hide swab samples. Acceptable activity of mixed phages at delivery to steers was found for bolus and feed, averaging 1.82 and 1.13 × 109 PFU/g, respectively. However, Ephage did not reduce shedding of naladixic acid–resistant E. coli O157:H7, although duration of shedding was reduced by 14 days (P < 0.1) in bolus-fed steers as compared with control steers. Two successful systems for delivery of Ephage were developed, but a better understanding of phage–E. coli O157:H7 ecology is required to make phage therapy a viable strategy for mitigation of this organism in feedlot cattle.

scholarly journals Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

2003 ◽  
Vol 50 (1) ◽  
pp. 239-247 ◽  
Author(s):  
Anna-Maria Ochocka ◽  
Marzena Czyzewska ◽  
Tadeusz Pawełczyk
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Rho Gtpase ◽  
Expression System ◽  
Cloning And Expression ◽  
Soluble Form ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Step Procedure ◽  
Shock Proteins

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

Expression and Regulation of the Arsenic Resistance Operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli

1998 ◽  
Vol 64 (2) ◽  
pp. 411-418 ◽  
Author(s):  
Katsuhisa Suzuki ◽  
Norio Wakao ◽  
Tetsuya Kimura ◽  
Kazuo Sakka ◽  
Kunio Ohmiya
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Constitutive Expression ◽  
Amino Acid Sequences ◽  
Gene Products ◽  
Arsenic Resistance ◽  
E Coli ◽  
Molecular Weights ◽  
Rna Polymerase Promoter ◽  
Extension Analysis

ABSTRACT The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order: arsR, arsD, arsA,arsB, arsC. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coliplasmid R773 and IncN plasmid R46. The ars operon cloned from A. multivorum conferred resistance to arsenate and arsenite on E. coli. Expression of the arsgenes with the bacteriophage T7 RNA polymerase-promoter system allowedE. coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E. coli. Although the arsR gene of A. multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon.

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