The purification from Escherichia coli of a protein relaxing superhelical DNA

1976 ◽  
Vol 54 (4) ◽  
pp. 301-306 ◽  
Author(s):  
M. G. Burrington ◽  
A. R. Morgan
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Rapid Screening ◽  
Intact Protein ◽  
Winding Number ◽  
Circular Dna ◽  
E Coli ◽  
The Individual ◽  
Negative Supercoils

The Escherichia coli omega protein was first described by Wang (Wang, J.C.: J. Mol. Biol. 55, 523–533 (1971)) as having the ability to relax supercoiled covalently-closed circular DNA by changing the topological winding number, α. We have developed a rapid assay for omega activity which has allowed us to purify the protein to homogeneity. It appears to be an αβ-ype subunit protein with a molecular weight of the intact protein of about 80 000 (determined by gel filtration) and of the individual subunits of 56 000 and 31 000 (sodium dodecyl sulfate polyacrylamide gels). We have confirmed Wang's observation that it only partly relaxes negative supercoils, and is not active on positive supercoils. Its characteristics with respect to pH, salts, temperature and chromatography are described. A method for rapid screening of E. coli for omega mutants is described.

scholarly journals Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase

1980 ◽  
Vol 191 (1) ◽  
pp. 209-219 ◽  
Author(s):  
J Hughes ◽  
G Mellows
Keyword(s):  
Escherichia Coli ◽  
Exchange Reaction ◽  
Rat Liver ◽  
Equilibrium Dialysis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Trna Synthetase ◽  
Stable Complex ◽  
E Coli ◽  
Escherichia Coli B

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9′-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9′-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.

scholarly journals Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

1977 ◽  
Vol 165 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Dobrivoje V. Marinkovic ◽  
Jelka N. Marinkovic
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Galactosidase Activity ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Molecular Weights ◽  
Terminal Amino

Carboxymethylated β-galactosidase from Escherichia coli was dissociated at 100°C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore β-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ‘complementable fractions’. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the β-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented β-galactosidase can exist.

scholarly journals Characterization of Soluble Enzyme II Complexes of the Escherichia coli Phosphotransferase System

2004 ◽  
Vol 186 (24) ◽  
pp. 8453-8462 ◽  
Author(s):  
Mohammad Aboulwafa ◽  
Milton H. Saier
Keyword(s):  
Escherichia Coli ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Kinetic Properties ◽  
Phosphotransferase System ◽  
E Coli ◽  
Before And After ◽  
Oligomeric Form ◽  
Triton X

ABSTRACT Plasmid-encoded His-tagged glucose permease of Escherichia coli, the enzyme IIBCGlc (IIGlc), exists in two physical forms, a membrane-integrated oligomeric form and a soluble monomeric form, which separate from each other on a gel filtration column (peaks 1 and 2, respectively). Western blot analyses using anti-His tag monoclonal antibodies revealed that although IIGlc from the two fractions migrated similarly in sodium dodecyl sulfate gels, the two fractions migrated differently on native gels both before and after Triton X-100 treatment. Peak 1 IIGlc migrated much more slowly than peak 2 IIGlc. Both preparations exhibited both phosphoenolpyruvate-dependent sugar phosphorylation activity and sugar phosphate-dependent sugar transphosphorylation activity. The kinetics of the transphosphorylation reaction catalyzed by the two IIGlc fractions were different: peak 1 activity was subject to substrate inhibition, while peak 2 activity was not. Moreover, the pH optima for the phosphoenolpyruvate-dependent activities differed for the two fractions. The results provide direct evidence that the two forms of IIGlc differ with respect to their physical states and their catalytic activities. These general conclusions appear to be applicable to the His-tagged mannose permease of E. coli. Thus, both phosphoenolpyruvate-dependent phosphotransferase system enzymes exist in soluble and membrane-integrated forms that exhibit dissimilar physical and kinetic properties.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals The AIDA Autotransporter System Is Associated with F18 and Stx2e in Escherichia coli Isolates from Pigs Diagnosed with Edema Disease and Postweaning Diarrhea

2001 ◽  
Vol 8 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Ulla Niewerth ◽  
Andreas Frey ◽  
Thomas Voss ◽  
Chantal Le Bouguénec ◽  
Georg Baljer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Virulence Factors ◽  
Rapid Screening ◽  
Edema Disease ◽  
E Coli ◽  
Large Numbers ◽  
High Prevalence ◽  
Pcr Method ◽  
Postweaning Diarrhea

ABSTRACT Pathogenic Escherichia coli strains are known to cause edema disease (ED) and postweaning diarrhea (PWD) in piglets. Although the exact mechanisms of pathogenicity that lead to ED-PWD remain to be elucidated, E. coli-borne Shiga-like toxin and adhesion-mediating virulence factors such as F18 adhesin or F4 fimbriae are believed to play a central role in ED-PWD. In light of these observations we investigated whether another E. coliadhesin, the plasmid-encoded AIDA (adhesin involved in diffuse adherence) might also be present in ED-PWD-causing E. coli isolates. For rapid screening for the AIDA system in large numbers of isolates, a multiplex PCR method along with a duplex Western blot procedure was developed. When screening 104 strains obtained from pigs with or without ED-PWD, we observed a high prevalence of the AIDA operon in porcine E. coli isolates, with over 25% of all strains being AIDA positive, and we could demonstrate a significant association of the intact AIDA gene (orfB) with ED-PWD, while defects in orfB were associated with the absence of disease. Although our data hint toward a contribution of AIDA to ED-PWD, further studies will be necessary since the presence of the AIDA genes was also associated with the presence of the Shiga-like toxin and F18 adhesin genes, two reported virulence factors for ED-PWD.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

scholarly journals Antimicrobial Interventions to Reduce Shiga Toxin-Producing Escherichia coli (STEC) Surrogate Populations on Beef Striploins Intended for Blade Tenderization

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
C. L. Thomas ◽  
H. Thippareddi ◽  
M. Rigdon ◽  
S. Kumar ◽  
R. W. McKee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Antimicrobial Treatment ◽  
Sodium Chlorite ◽  
Beef Industry ◽  
Anterior Portion ◽  
E Coli ◽  
Group Data ◽  
Blade Tenderization ◽  
Antimicrobial Interventions

ObjectivesBlade tenderization (BT) is used in the beef industry to improve tenderness of steaks prepared from subprimals but can translocate surface pathogens to the interior of meat. Application of antimicrobial solutions on the surface of subprimals prior to blade tenderization can reduce the risk of translocation of surface microorganisms. The objectives of this research were: 1) evaluate the efficacy of antimicrobial interventions applied to inoculated (surrogate Escherichia coli) beef striploins prior to blade tenderization; and 2) examine the transfer of E. coli from inoculated striploins to subsequent non-inoculated subprimals.Materials and MethodsThe anterior portion of whole muscle beef striploins (30.48 cm) were inoculated (lean side) across a 10 cm band with an approximately 8.00 log CFU/mL cocktail containing non-pathogenic, rifampicin-resistant surrogate STEC strains (BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431). The inoculated striploins were sprayed with (1) levulinic acid (5.0%) + sodium dodecyl sulfate (0.50%) (LVA+SDS), (2) peroxyacetic acid (2000 ppm; PAA; FCN 1666), (3) acidified sodium chlorite (1200 ppm; ASC), or (4) lactic acid (4.5%; LA) by passing through a spray cabinet and blade tenderized, along with an inoculated, non-sprayed control (CON). To evaluate the potential for cross-contamination of subsequent subprimals, an inoculated striploin (for each treatment) was blade tenderized followed by a non-inoculated beef striploin. For each striploin, surface and subsurface samples (2.54 cm wide) were collected from three different locations including the anterior, middle, and posterior end of each striploin. A total of 30 striploins across three replications were randomly assigned to treatment stratification. Sponge samples were also collected from the blade tenderizer (plate of the blade unit and blades) after each treatment group. Data were analyzed using Proc Mixed (SAS Inst., v.9.4; Cary, NC) as a completely randomized split-plot design. Microbial counts for all samples were log transformed and then analyzed for the main effects of antimicrobial treatment, location (anterior to posterior and surface or interior), and their interaction. Differences were considered significant at α ≤ 0.05.ResultsPAA was more effective in reducing E. coli populations (1.80 log CFU/g; P ≤ 0.05) and had lowest recovery of the microorganism from the striploin subsurface compared to other treatments, followed by LVA+SDS (1.00 log CFU/g). E. coli populations gradually decreased (P ≤ 0.05) on the surface and subsurface as sampling moved anterior to posterior. However, E. coli populations were similar (P > 0.05) on the posterior end of inoculated striploins and the anterior end of the subsequent, non-inoculated striploins, indicating transfer of microorganisms from one striploin to the following striploin. E. coli populations of 3.03 log CFU/cm2 and 2.47 log CFU/cm2 were recovered from the plate of the blade unit and the blades of the blade tenderizer. E. coli populations recovered from the plastic plate (3.46 log CFU/cm2) and blades (2.87 log CFU/cm2) of the blade tenderizer were the similar (P > 0.05) for all treatment groups except for PAA (1.41 log CFU/cm2 and 0.97 log CFU/cm2, respectively).ConclusionThese results showed that PAA and LVA+SDS can be used to improve the safety of blade tenderized beef.

scholarly journals The Effect Ultrasound and Surfactants on Nanobubbles Efficacy against Listeria innocua and Escherichia coli O157:H7, in Cell Suspension and on Fresh Produce Surfaces

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2154
Author(s):  
Shamil Rafeeq ◽  
Reza Ovissipour
Keyword(s):  
Escherichia Coli ◽  
Cell Suspension ◽  
Sodium Dodecyl ◽  
Listeria Innocua ◽  
Wash Water ◽  
Tween 20 ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Bacterial Removal ◽  
The Impact

Removing foodborne pathogens from food surfaces and inactivating them in wash water are critical steps for reducing the number of foodborne illnesses. In this study we evaluated the impact of surfactants on enhancing nanobubbles’ efficacy on Escherichia coli O157:H7, and Listeria innocua removal from spinach leaves. We evaluated the synergistic impact of nanobubbles and ultrasound on these two pathogens inactivation in the cell suspension. The results indicated that nanobubbles or ultrasound alone could not significantly reduce bacteria in cell suspension after 15 min. However, a combination of nanobubbles and ultrasonication caused more than 6 log cfu/mL reduction after 15 min, and 7 log cfu/mL reduction after 10 min of L. innocua and E. coli, respectively. Nanobubbles also enhanced bacterial removal from spinach surface in combination with ultrasonication. Nanobubbles with ultrasound removed more than 2 and 4 log cfu/cm2 of L. innocua and E. coli, respectively, while ultrasound alone caused 0.5 and 1 log cfu/cm2 of L. innocua and E. coli reduction, respectively. No reduction was observed in the solutions with PBS and nanobubbles. Adding food-grade surfactants (0.1% Sodium dodecyl sulfate-SDS, and 0.1% Tween 20), did not significantly enhance nanobubbles efficacy on bacterial removal from spinach surface.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

scholarly journals A Comparative Study of the Adherent-Invasive Escherichia coli Population and Gut Microbiota of Healthy Vegans versus Omnivores

2020 ◽  
Vol 8 (8) ◽  
pp. 1165
Author(s):  
Rebecca Veca ◽  
Christian O’Dea ◽  
Jarred Burke ◽  
Eva Hatje ◽  
Anna Kuballa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gut Microbiota ◽  
Random Amplified Polymorphic Dna ◽  
Fecal Microbiota ◽  
Vegan Diet ◽  
Rrna Gene ◽  
E Coli ◽  
Abundance And Diversity ◽  
The Individual ◽  
Diversity Profiles

Adherent-invasive Escherichia coli (AIEC) strains carry virulence genes (VGs) which are rarely found in strains other than E. coli. These strains are abundantly found in gut mucosa of patients with inflammatory bowel disease (IBD); however, it is not clear whether their prevalence in the gut is affected by the diet of the individual. Therefore, in this study, we compared the population structure of E. coli and the prevalence of AIEC as well as the composition of gut microbiota in fecal samples of healthy participants (n = 61) on either a vegan (n = 34) or omnivore (n = 27) diet to determine whether diet is associated with the presence of AIEC. From each participant, 28 colonies of E. coli were typed using Random Amplified Polymorphic DNA (RAPD)–PCR. A representative of each common type within an individual was tested for the presence of six AIEC-associated VGs. Whole genomic DNA of the gut microbiota was also analyzed for its diversity profiles, utilizing the V5-V6 region of the16S rRNA gene sequence. There were no significant differences in the abundance and diversity of E. coli between the two diet groups. The occurrence of AIEC-associated VGs was also similar among the two groups. However, the diversity of fecal microbiota in vegans was generally higher than omnivores, with Prevotella and Bacteroides dominant in both groups. Whilst 88 microbial taxa were present in both diet groups, 28 taxa were unique to vegans, compared to seven unique taxa in the omnivores. Our results indicate that a vegan diet may not affect the number and diversity of E. coli populations and AIEC prevalence compared to omnivores. The dominance of Prevotella and Bacteroides among omnivores might be accounted for the effect of diet in these groups.

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