scholarly journals Purification and Characterization of an ATPase GsiA from Salmonella enterica

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Zhongshan Wang ◽  
Meng Zhang ◽  
Xiaodong Shi ◽  
Quanju Xiang
Keyword(s):  
Atpase Activity ◽  
Salmonella Enterica ◽  
Gene Deletion ◽  
Purification And Characterization ◽  
Uptake Mechanism ◽  
Oxidation Method ◽  
E Coli ◽  
Two Hybrid

The coding sequence of Salmonella enterica gsiA was cloned and expressed in E. coli. The protein was purified and ATPase activity was characterized by NADH oxidation method. GsiA exhibited optimum activity at 30°C and at pH 8 in Tris/HCl buffer. GsiA protein was stable at 20°C. 66% and 44% activity remained after incubation at 30°C and 40°C for 30 min. pH 7 and pH 9 incubation would obviously reduce the ATPase activity. In vivo functionality of gsiA was determined by constructing gene deletion strains. gsiA was shown to be essential for GSI mediated glutathione uptake and gsiA deletion could decrease the virulence of Salmonella enterica. Interactions of glutathione import proteins GsiA, GsiB, GsiC, and GsiD were investigated by using bacterial two-hybrid system. GsiA could interact with itself and inner membrane proteins GsiC and GsiD. This report provides the first description of gsiA functions in Salmonella enterica. The results could help elucidating the glutathione uptake mechanism and glutathione functions in bacteria.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

Characterization of in vitro and in vivo murine models of human enteropathogenic E. coli (EPEC) infection

2003 ◽  
Vol 124 (4) ◽  
pp. A558
Author(s):  
Suzana D. Savkovic ◽  
Farol L. Tomson ◽  
Michelle Muza ◽  
Gail Hecht
Keyword(s):  
Murine Models ◽  
E Coli

scholarly journals Dynamic regulation of extracellular ATP in Escherichia coli

2017 ◽  
Vol 474 (8) ◽  
pp. 1395-1416 ◽  
Author(s):  
Cora Lilia Alvarez ◽  
Gerardo Corradi ◽  
Natalia Lauri ◽  
Irene Marginedas-Freixa ◽  
María Florencia Leal Denis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Atp Release ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Extracellular Atp ◽  
Outer Membrane Vesicles ◽  
Dynamic Regulation ◽  
E Coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1–1 µM). Exposure to MST7 and MEL enhanced ATP release by 3–7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6–7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

scholarly journals First Report of a Foodborne Salmonella enterica Serovar Gloucester (4:i:l,w) ST34 Strain Harboring blaCTX–M–55 and qnrS Genes Located in IS26-Mediated Composite Transposon

2021 ◽  
Vol 12 ◽  
Author(s):  
Lili Li ◽  
Rikke Heidemann Olsen ◽  
Anhua Song ◽  
Jian Xiao ◽  
Chong Wang ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Structural Unit ◽  
Bacterial Species ◽  
Meat Products ◽  
Sequence Type ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Long Read ◽  
Phenotypic And Genotypic Characterization

Extended-spectrum β-lactamases (ESBLs) production and (fluoro)quinolone (FQ) resistance among Salmonella pose a public health threat. The objective of this study was the phenotypic and genotypic characterization of an ESBL-producing and nalidixic acid-resistant Salmonella enterica serovar Gloucester isolate (serotype 4:i:l,w) of sequence type 34 (ST34) from ready-to-eat (RTE) meat products in China. Whole-genome short and long read sequencing (HiSeq and MinION) results showed that it contained blaCTX–M–55, qnrS1, and tetB genes, with blaCTX–M–55 and qnrS1 located in chromosomal IS26-mediated composite transposon (IS26–qnrS1–IS3–Tn3–orf–blaCTX–M–55–ISEcp1–IS26). The same genetic structure was found in the chromosome of S. enterica subsp. enterica serovar Typhimurium strain and in several plasmids of Escherichia coli, indicating that the IS26-mediated composite transposon in the chromosome of S. Gloucester may originate from plasmids of E. coli and possess the ability to disseminate to Salmonella and other bacterial species. Besides, the structural unit qnrS1–IS3–Tn3–orf–blaCTX–M–55 was also observed to be linked with ISKpn19 in both the chromosomes and plasmids of various bacteria species, highlighting the contribution of the insertion sequences (IS26 and ISKpn19) to the co-dissemination of blaCTX–M–55 and qnrS1. To our knowledge, this is the first description of chromosomal blaCTX–M–55 and qnrS in S. Gloucester from RTE meat products. Our work expands the host range and provides additional evidence of the co-transfer of blaCTX–M–55 and qnrS1 among different species of Salmonella through the food chain.

scholarly journals Characterization of MazFSa, an Endoribonuclease from Staphylococcus aureus

2007 ◽  
Vol 189 (24) ◽  
pp. 8871-8879 ◽  
Author(s):  
Zhibiao Fu ◽  
Niles P. Donegan ◽  
Guido Memmi ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Environmental Stress Response ◽  
Binding Studies ◽  
Cell Growth Arrest ◽  
Consensus Sequences ◽  
E Coli ◽  
Endoribonuclease Activity

ABSTRACT The mazEF homologs of Staphylococcus aureus, designated mazEFsa , have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF Sa , as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF Sa leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF Sa is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF Sa cleaves single-strand RNA preferentially at the 5′ side of the first U or 3′ side of the second U residue within the consensus sequences VUUV′ (where V and V′ are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE Sa binds MazF Sa to form a complex to inhibit the endoribonuclease activity of MazF Sa . Contrary to the system in E. coli, exposure to selected antibiotics augmented mazEFsa transcription, akin to what one would anticipate from the environmental stress response of the sigB system. These data indicate that the mazEF system of S. aureus differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.

Expression, purification and characterization of human urodilatin in E. coli

2007 ◽  
Vol 55 (2) ◽  
pp. 312-318 ◽  
Author(s):  
Ziyong Sun ◽  
Wei Lu ◽  
Yanchun Tang ◽  
Jing Zhang ◽  
Junyong Chen ◽  
...  
Keyword(s):  
Purification And Characterization ◽  
E Coli
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